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MICROBIOLOGY LABORATORY GUIDEBOOK

UNITED STATES DEPARTMENT OF AGRICULTUREFOOD SAFETY AND INSPECTION SERVICEOFFICE OF PUBLIC HEALTH AND SCIENCE

MICROBIOLOGY DIVISION

B. P. DEY, DVM, MS, MPH, Ph.D., EditorC. P. LATTUADA, Ph.D., Co-Editor

Edi t or i a l Boar d

A. M. McNAMARA, Sc.D., R. P. MAGEAU., Ph.D. and S. S. GREEN., Ph.D.

3RD EDITION, 1998 VOLUMES 1 & 2

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USDA/FSIS Microbiology Laboratory Guidebook 3rd Edition/1998

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FOREWORD

The 1993 Es cher i c hi a col i O157:H7 outbreak in the Pacific Northwestfocused national attention on food safety. Since then, the number

of requests for reprints on analytical methods used by the Microbiology Division, Office of Public Health and Science, Food Safety and Inspection Service, United States Department of Agriculture, has increased dramatically. Scientists within theDivision have responded to these requests by completely revisingand updating our Microbiology Laboratory Guidebook (MLG) for publication.

This MLG is our laboratory guidebook for the microbiologicalanalysis of meat, poultry, and egg products that fall under thejurisdiction of USDA. It contains methods that FSIS prefers to usefor the analysis of these foods. Since USDA does not endorse orapprove methods for use by the food industry, inclusion of a

particular method in the MLG should not be construed in this manner. Similarly, the mention of specific brand or trade namesfor a product, medium, chemical or reagent associated with methodscontained herein does not constitute endorsement or selectivity bythe authors or USDA over similar products that might also besuitable.

The use of the MLG comes with several caveats. This guidebook was written for microbiologists, and its interpretation and use should only be undertaken by trained microbiologists. FSIS assumes noresponsibility for any economic, personal injury or other damagethat may occur to individuals or organizations because of the useof methods contained in this guidebook. Users should note and pay

particular attention to the safety caution symbol (†) and written warnings associated with certain hazardous chemicals or dangerous biological materials used in some of the methods. Users must actin a responsible manner at all times to protect themselves and theenvironment during performance of these methods. This guidebook must be supplemented with quality assurance and quality control programs as well as chemical, biological, and employee safetyhazards management programs in order to operate a microbiologylaboratory. These programs are beyond the scope of this guidebookand are the sole responsibility of the user to develop and implement.

This guidebook contains protocols for analytical tests that arerequired by FSIS regulatory activities. Some protocols, such asthe Bioassay procedure for antibiotic residue detection and quantitation, may not be of value to commercial laboratories nor do we expect others to try to commercialize them. They are included here primarily as informational material since they are part of ourcurrent analytical methods.

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The 1998, 3rd edition MLG publication consists of two separatevolumes with a newly revised format utilizing a loose-leaf binder.This format should make the updating of chapters easier byallowing the substitution of a single chapter or page versusreprinting of the entire MLG. Because we anticipate the addition

of new materials, the chapter numbers between volumes are notcontinuous in order to accommodate all changes. Publishing this new 3rd edition MLG replaces all previous MLGversions and supersedes all Laboratory Communications, which should be discarded.

Finally, to produce a work of this magnitude requires a team ofdedicated scientists and support staff. I would like to thank thefollowing people for their efforts: Larry H. Dillard, Joseph Y.Chiu and James G. Eye for coordinating the FSIS Technical SupportLaboratory reviews of the manual; Microbiology Division staff members Bhabani P. Dey, Stanley S. Green, Charles P. Lattuada,

Bonnie E. Rose, Richard P. Mageau, and Gerri M. Ransom forcomposing, editing and proofreading many chapters; and Julie M.Hall for providing secretarial support in typing most of thechapters under trying conditions and meeting the demands of adiverse group of scientists.

Ann Marie McNamara, Sc.D. January 1998Director Microbiology DivisionOffice of Public Health and ScienceEditorial Board, MLG

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GENERAL CONSIDERATIONS

Before any analyst attempts to perform the microbiological methodscontained within this Microbiology Laboratory Guidebook (MLG), it

might be helpful to call attention to the following generalconsiderations in the use of this guidebook.

In order to maximize the achievement of successful results whenusing the various methods in this MLG, it should be clearlyunderstood that all methods and procedures should be performed atall times in a manner as close as possible to the prescribed directions. Particular attention should be paid to all details provided in a given analytical procedure. Changes or shortcutsshould not be attempted in a method simply to accommodate factors,for example, such as processing a large number of similar samplesthrough the method at the same time.

All chemicals, media, immunoreagents and commercial test kitsshould be within current shelf expiration dates and be subjected toquality control and quality assurance procedures to insure their proper performance for their intended purpose and use within the methods presented in this MLG. All instrumentation should besubjected to continuous maintenance and appropriate quality control procedures to insure unquestionably correct performance during usein all methods. The use of positive and negative test controls atall times, as specified for a given procedure, should beimplemented. Adequate documentation and record keeping should beemployed for all analytical results, test controls, qualityassurance and quality control procedures, instrument maintenance

programs, and any observed laboratory deviations to the above or in methods performance.

Although all of the methods described in this guidebook have exactnumerical values given for performance parameters such as weightand volume measures, pH, time and temperature to achieve optimum results, it should be clearly understood that an acceptable rangeexists within which optimum results can still be expected to beachieved without compromising the integrity of the method. For anygiven method, unless otherwise clearly stated within the text ofthis MLG, the following allowable ranges for the given parametersare considered to be acceptable and are applicable:

Weight and volume measures: ± 1% pH: ± 0.2 unitsTime: hours ± 1 hour; minutes ± 1%Temperature: ± 1.0

oC

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CHAPTER 1. SAMPLE PREPARATION FOR MEAT, POULTRY AND PASTEURIZEDEGG PRODUCTS

Charles P. Lattuada and B. P. Dey

1.1 Introduction

The purpose for the microbiological examinations of meat and poultry products is to obtain information. This informationgathering may follow a qualitative or quantitative analyticalformat. The format followed is called the sampling plan. Many microorganisms are present in very low numbers and require one or more enrichment steps. If cell injury is anticipated, a non-selective enrichment frequently is used to resuscitate cells,followed by a more selective enrichment.

The analyst must study all records and correspondence before

examining the sample. Care must be exercised in maintaining and handling the sample to insure that it is the same one that wascollected, that it has not been tampered with, and that itscondition is the same as it was at collection. The reserve sample must be stored properly to maintain its integrity in caseadditional analyses are required.

An analyst must be keenly aware that during all steps of theanalysis, it is important to minimize the growth of non-critical microorganisms and to prevent entrance of environmentalcontaminants. The organism(s) isolated must come from the testsample and not from an outside source. These facts cannot beover-emphasized and can be accomplished only if strict attention is paid to the following rules:

The sampling operation must be well organized, with allsupplies and equipment properly positioned before starting.

Ideally, sampling should be done in an area free of aircurrents following good aseptic procedures.

All work surfaces must be clean and sanitized.

Implements used for sampling must be sterile before use and protected from outside contamination during use.

The outside of the immediate container must be thoroughlysanitized.

Any laboratory person processing samples must be very familiar withaseptic techniques and the principles of sterilization,sanitization and disinfection. The person assigned to the samplingtask should know the sampling protocol to be used and have a

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reference copy at hand in case questions arise.

1.2 Sanitizing the Work Area

The work area must be clean and free from dust; detergent

sanitizers are satisfactory for cleaning. Before work begins, the work area should be cleaned and a sanitizer/disinfectant applied liberally and given time to act. Quaternary ammonium compounds,sodium hypochlorite and phenolic compounds all are suitable forthis purpose. The manufacturer's instructions regarding theconcentration needed and the time required for the compound to actshould be followed.

1.3 Sterilization of Instruments

a. All instruments and containers to be used in the sampleanalysis must be sterile. Any sterilization procedure may be used that is compatible with the material to be

sterilized. Sterilization implies the total destructionof all viable organisms as measured by an appropriateculturing method.

b. An exception can be made, if necessary, when the numberof instruments is limited (ie. chisels) and the testing protocol does not include sporeforming microorganisms.In which case, the instruments first are washed withsoap and water, rinsed and inspected to be sure there isno organic matter in crevices or hinges, then they may be steamed for 30 minutes in an instrument sterilizer or placed in boiling water for two minutes.

c. Do not dip instruments into alcohol and flame them as asubstitute for heat sterilization. It is not asubstitution for the methods given above.

1.4 Disinfection of Outer Surface of the Immediate Container

a. The outside covering of the intact immediate container must be decontaminated to the greatest extent possibleand particularly in the area where an opening will be made to expose the contents.

b. Hydrogen peroxide, tincture of iodine or 2500 ppm sodium

hypochlorite solution may be used for this purpose. Allow time for the disinfectant to act before openingthe container. Aseptically remove any residualdisinfectant to prevent its entering the container whenan opening is made.

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1.5 Cutting and Weighing Samples

a. The sample should never be touched with bare hands.During the process of sanitizing the immediatecontainer, the analyst should put on a pair of sterile

gloves for handling samples.

b. Sterile instruments should be used for cutting, removingand manipulating all samples.

c. The sample must be taken aseptically according to thesampling protocol and placed in the proper sterilecontainer for the next processing step. The remainderof the sample must be secured with an appropriatesterile closure that will preserve the sterility and integrity of the sample reserve. The sample reserve must be held according to the sampling protocol.

d. If the sample is to be weighed, the balance on whichsamples are weighed must be placed in an area that isclean and free of strong air currents.

e. If at all possible, the product should be weighed directly into the sterile container that will be used for dilution, mixing, blending and/or stomaching.

f. When weighing is complete, clean and disinfect the area with the same product used initially for disinfectingthe work area. All instruments, containers, gloves and other materials that may have been in contact with the product must be incinerated or terminally sterilized before cleaning or disposal.

1.6 Selected References

Block, S. S. (ed.). 1984. Disinfection, Sterilization and Preservation, 3rd Edition. Lea & Febiger, Philadelphia, PA.

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CHAPTER 2. PHYSICAL EXAMINATION OF MEAT AND POULTRY PRODUCTS

Charles P. Lattuada and B. P. Dey

2.1 Introduction

Microorganisms associated with meat and poultry products can be placed in three categories, beneficial, spoilage and pathogenic.Each product has a characteristic microbial profile called its"normal flora". Frequently information on changes in the "normalflora" can be obtained rapidly by simple observations. Theseobservations can be grouped into a category called organolepticobservations. The term "organoleptic" refers to the use of thesenses in determining the acceptability of a product. This would also include a direct microscopic examination.

Organoleptic analyses are of particular importance during

investigations of certain food production problems such asdetecting deleterious pre- or post-processing changes of canned products. Changes brought about by abusive handling and storagealso may be detected by organoleptic observation.

In order to make a valid judgment, based upon one or moreorganoleptic observations, the analyst must know the physicalcharacteristics of a "normal" product. This knowledge can begained by experience and specialized training. Each laboratoryshould have Standard Operating Procedures (SOPs) describing theorganoleptic standards for the acceptance or rejection of samples.

When judging a product to be abnormal, if possible, the decision

should be based on a comparison of the suspect product with onethat is normal, if readily available. This minimizes thesubjectivity of the decision that a product has an "off odor", "offcolor", or other sensory abnormality. Tasting products as part ofa microbiological examination is a dangerous practice and should beavoided. When the question to be answered is related to spoilage,odor is of primary importance; chemical and/or bacteriologicalresults are corroborative and substantiating.

2.2 Examination

The following guideline establishes a standardized inter-laboratory procedure for characterizing samples.

a. Appearance: Changes in color; degradation of fat; presence of foreign materials such as metal, hair,feathers, sand, charcoal, etc.

b. Texture: Change in consistency; development of slime; breakdown of structure (proteolysis), etc.

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c. Odor: Examples of words used to describe off-odors are:sour (acidic), moldy, musty, fishy, rancid, fruity,yeasty (beer-like) and putrid. However, if the analystcannot decide how to classify an odor it is acceptableand appropriate to say simply: "off-odor" or "taint".

Notations as to whether the off-odor is strong or slightare also in order.

2.21 Odor Examination By a Panel

In some cases results of odor examinations are equivocal and anodor detection panel, consisting of at least three members must beformed. The purpose of this panel is to evaluate aroma only, and its judgement must not be swayed by appearances. Only people witha good sense of smell can be assigned to it. The coordinator, whois not a panel member, will prepare the samples and ensure that thefollowing procedures are followed:

a. The test must be conducted in a well-ventilated areafree of strong odors.

b. At least 15 - 20% of the samples in the test groupshould be normal, wholesome, product-counterparts of thesamples being examined. The normal controls should beas similar to the test product as possible with respectto ingredients, processing, packaging, size, age and handling procedures.

c. All samples should be presented to the smell panel insequentially coded glass jars or polyethylene bags ofthe same size and shape, similar in weight and at the

same temperature (usually 35°°C). Both the normal and questionable products should be presented in a random order with a rest between samples. Do not decontaminatecans by flaming since heating and/or burning thecontents could alter or mask any other odors that might be present.

d. Before beginning the examination, the panel membersshould smell and discuss the characteristic aroma of anormal product. They should be made aware that it isfor general reference only, since normal products mayvary slightly in odor and intensity. They then should

rest until the samples are presented to allow recoveryof the sense of smell which tires easily.

e. During the actual sample analysis, each panel membershould remove the jar lid or open the bag, sniff thecontents without glancing at them, replace the lid/closethe bag and return the container to the panelcoordinator. The panelist's sensory perceptions should

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be entered on a score pad containing a list ofappropriate terms with notations about whether the odor was strong or weak.

f. During the examination the panel members must not

comment, exclaim or use body language that conveys theirimpression of the odors to other members of the panel.

Caution: It is not to be assumed that a smell panel composed of laboratory personnel will have the degree of skill attained by professional odor analysts. The purpose of a panel oflaboratory personnel is to detect the odors of decompositionor product contamination with an odorous compound.

2.3 Determination of pH in Meat and Poultry Products

Potentiometric measurements should be used to determine the pH of afood product. The accuracy of most pH meters is approximately 0.1

pH units and reproducibility should be approximately ± 0.005 pHunits. Both the glass and reference electrode are usually housed in a single tube, called the combination electrode. To obtainaccurate results the same temperature should be used forstandardization with the buffers and the sample. Measurements

should be taken within the temperature range of 20 to 30°°C.

2.31 Equipment and Reagents

a. Blender b. Beaker, 100 mlc. Separatory funneld. pH meter, suitable for reading pH from 0 to 14 in 0.1

unit increments. A rugged, designated combinationelectrode should be used for pH measurement of meats and poultry. A flat combination electrode works well fordetermining the surface pH of canned foods.

e. Distilled waterf. Certified buffer solutions of pH 7.00, and either pH

4.00 or 10.00. The buffers chosen should bracket thedesired pH.

2.32 Procedure

a. Calibrate the pH meter, according to manufacturer's

instructions, using certified buffers pH 7.00 and either pH 4.00 or 10.00.

b. Most products will be solid and require blending. A 1:5or 1:10 dilution should be made with distilled water ina clean blender jar. Blend to a thin uniform consistency and perform the pH measurement. If fat oroil causes fouling of the electrode, transfer a portion

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of the homogenate to a separatory funnel and draw off a portion of the aqueous phase. On certain productscentrifugation may be required in order to recover a measurable aqueous phase.

c. Adjust the temperature control on the pH meter to thatof the sample (ideally 25°°C) and immerse the pH electrodeinto the liquid phase.

d. A surface electrode may be used with certain low fat products that present a flat, solid core surface. If asurface measurement is taken, ensure that the electrodehas good contact with the product surface.

e. Record pH to the nearest 0.1 unit.

2.4 Determination of Water Activity (A w) of Meat and PoultryProducts

The free moisture level in food is called water activity (a w). Thisis the water available to support microbiological growth in thefood. It can be lowered by dehydration or by the addition of binding agents such as salt or sugar. The growth of differenttypes and genera of microorganisms is controlled by the wateractivity level in a specific product. Much information exists onthe water activity limits of growth for microorganisms. Forexample, the limit of growth for Cl os t r i di um bo t ul i num occurs

between an a w of 0.935 and 0.945. Canned foods with an a w of ≤≤0.85are exempt by the FDA from the canned food regulations and cured

meats without nitrates must have an a w of ≤≤0.92. It is important,

therefore, that the a w in foods be measured very accurately. A detailed list of growth limiting a w values can be found in Chapter8 of the Compendium of Methods for the Microbiological Examinationof Foods.

Measurement of the a w in a food sample is affected by both time and temperature. It is dependent upon allowing enough time for the water vapor of the sample to reach equilibrium with the air spacein a closed container, such as a closed jar, at a constanttemperature. When incubation is required for equilibration, it isabsolutely necessary to maintain accurate temperature control ofthe food samples inside the incubator used for a w. It is equallyimportant to allow ample time for the humidity of the air space

above the sample to reach equilibrium with the food sample.

2.41 Decagon

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The Decagon CX-2 will measure a w in less than 5 minutes. Theinstrument has rapid vapor equilibration, does not requiretemperature equilibration and requires only a small sample(approximately 5 grams of food). The instrument does not have to be calibrated, but quality control samples, consisting of deionized

water and various salt slushes, must be included in an analysis. When a very wet sample and a very dry one follow one another, twointerim readings should be taken of the second sample beforecollecting data with the third reading. When a reading iscompleted,the instrument will "beep" continuously. The onlyreported material to interfere with a Decagon reading is propyleneglycol. Foods containing propylene glycol should not be analyzed by this method.

2.42 Equipment and Materials

a. Decagon, Model CX-2 manufactured by Decagon Devices,Inc., Pullman, WA 99163-0835.

b. Blender and blending jarsc. Transfer pipettes

2.43 Procedure

a. In order to obtain a representative sample,approximately 100-200 grams of food should be blended.

b. Remove at least two samples, approximately 5 grams each,for a w determination; the cup should never be filled above the fill level line molded into the side of the plastic cup.

c. Follow the manufacturer's directions contained in theDecagon Manual very carefully when performing thisanalysis.

d. Saturated salt solutions should be used for referencecontrols. The following saturated salt mixes and theirexpected a w at 25

oC normally are used:

NaCl ---------0.755KBr ----------0.811KCl ----------0.845(NH4)H2PO4-----0.934

Note: Never leave a sample in the instrument after areading has been taken.

2.44 American Instrument Electronic Hydrometer

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Another method for determining a w is the American InstrumentElectronic Hydrometer. Reportedly, it is an accurate instrumentfor measurement of the a w in food products, provided the manufacturer's directions are followed carefully. The instrument measures the changes in electrical resistance of specially coated

lithium chloride sensors. The electronic part of the instrument isvery rugged and needs no special care. The sensors, like pHelectrodes, are very sensitive and can be affected permanently by water condensation, desiccation, corrosive chemicals such as mercury vapor, unstable hydrocarbons such as ketones; halogengases; and sulfur compounds such as hydrogen sulfide and sulfurdioxide. Sensors can be affected reversibly by polar vapors suchas ammonia, amines, alcohols, glycols and glycerols. The responseof sensors will return to normal, from slightly higher readings, ifthe polar vapors are removed by aeration.


a. American Instrument Electronic Hydrometer (Model No.30-87 or equivalent) manufactured by Newport Scientific,Inc., 8246E Sandy Court, Jessup, MD 20794.

b. Sensors, Color Code-Gray, (Cat-No. 4822W) for the aboveinstrument, available from the same manufacturer. TheCompany makes different types of sensors for differentranges of humidities. This sensor is the one mostcommonly used in meat and poultry product analyses. Theyhave an a w range of about 0.81 to 0.99. Each sensor isunique and comes with its own factory calibration curve.When purchasing gray sensors specify that the a wreadings between 0.90 - 0.94 be inside the linear portion of the calibration curve. Also request that the

correction factor of each sensor at 30°°C (86°°F) beincorporated into each calibration curve.

c. Sensor lids and 8-gang switch box. These socket typelids normally fit into the rims of standard pint sizecanning jars. The 8-gang switch box allows measurementof eight samples at a time. The sensor connectorsshould be labeled 1 to 8 to correspond to the switch position.

d. A forced-air incubator should be used to hold the

samples at 30 ± 0.5°°C. If necessary, cut a 1.5" diameterhole in the incubator to introduce the electrical leadsfor the eight sensors into the incubator. Be sure to

fill the hole with sealant.e. Clean and dry standard pint-size glass canning jars,

without chips or cracks on the rims, for the samples.f. Pipettes

g. Preparation of a saturated ammonium phosphate, monobasic, [(NH4)H2PO4] slush

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(NH4)H2PO4, reagent grade 200 g Merthiolate 25 mgGlass distilled water

Place the ammonium phosphate and merthiolate in a

new or clean pint-size jar, slowly add glass-distilled water (approximately 2-3 ml at atime), and stir vigorously with a spoon untilapproximately one half of the crystals aredissolved. Care must be taken to avoid splashingthe salts onto the sides and rims of the jar.

Incubate the salt slushes at 30°°C for 2-3 days toestablish equilibrium.

h. Preparation of saturated potassium dichromate (K 2CrO4)slush

Use the same procedure as above. Omit the merthiolate.

i. Store the salt slushes indefinitely in a 30°°C incubatorat all times except to install or remove sensors.

j. The a w of the salt slushes should be (measured with acalibrated gray sensor):

(NH4)H2PO4 slush 0.929 at 30°°C

K 2CrO4 slush 0.865 at 30°°C

2.46 Procedure

a. Follow the manufacturer's directions very carefully whenusing this method.

b. Test each sensor first in (NH4)H2PO4 and then in K 2CrO4salt slush and record the results on the analysis sheet.The sample test results will be recorded on the samesheet. Do not use sensors that differ from the expected value of the salt slush by more than a w 0.01 unit.

c. If the a w is going to be measured in other than the rangespecified for the grey sensor, be sure to use theappropriate sensor and prepare salt slushes appropriatefor the expected range. A table of other salt slushescan be found in Chapter 8, "Measurement of wateractivity (a w) and acidity", in the Compendium of Methodsfor the Microbiological Examination of Foods.


Greenspan, L. 1977. Humidity fixed points of binary saturated aqueous solutions. J. Res. Nat. Bur. Stand. 81A:89-96.

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Prior, B. A. 1979. Measurement of water activity in foods: A review. J. Food Prot. 42:668-674.

Troller, J. A., and V. N Scott. 1992. Measurement of water

activity (a w) and acidity, p. 135-151.I n

C. Vanderzant and D. F. Splittstoesser (ed.), Compendium of Methods for the Microbiological Examination of Foods. 3rd Edition. Amer. Publ.Hlth. Assoc. Washington, D.C.

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CHAPTER 3. EXAMINATION OF FRESH, REFRIGERATED AND FROZEN PREPARED MEAT, POULTRY AND PASTEURIZED EGG PRODUCTS

Charles P. Lattuada, Larry H. Dillard and Bonnie E. Rose

3.1 Introduction

The laboratory methods contained in this section of the Guidebookare used to detect and, when desired, quantitate selected microorganisms in samples collected in federally inspected meat, poultry and egg processing establishments. They generally followthe Compendium of Methods for the Microbiological Examination ofFoods and AOAC International's Official Methods of Analysis. The methods presented in this section may be used to analyze samplesof:

a. fresh, frozen, smoked, cured or dehydrated meat and poultry products;

b. prepared/ready-to-eat products such as pot pies,luncheon meats, dinners, battered or breaded meat and poultry products;

c. refrigerated meat or poultry salads;

d. dehydrated soups and sauces containing the requisiteamount of meat or poultry;

e. meat snacks, hors d'oeuvres, pizza and specialty items;

f. various ingredients incorporated with meat and poultry products such as spices, vegetables, breading material, milk powder, dried egg, vegetable proteins;

g. pasteurized egg products;

h. environmental samples from areas in which any of theabove are processed or manufactured.

The quantity and types of mesophilic microorganisms present in oron any of these products offer a means of evaluating the degree ofsanitation used during the process. If the results obtained forcoliforms, Es cher i c hi a c ol i , and St aphy l ococcus aur eus areunusually high, they might result in some type of officialfollow-up action. Any such follow-up analysis will use theappropriate Final Action Method found in the latest edition ofOfficial Methods of Analysis of AOAC International or any of itssupplements. Pertinent sections in the 16th Edition are:

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♦♦ Aerobic Plate Count (APC): 966.23

♦♦ Coliform Group and E. c ol i : 966.24

♦♦ S. aur eus : 987.09

3.11 Comparison With the AOAC Method

The procedures in the following sections of this Chapter are eitherthe same as those published by the AOAC or generally follow an AOAC method. The following is a listing of deviations:

a. The procedure for determining numbers of coliform and E .col i differ from the AOAC procedure as follows:

i. Use a single tube of laurel sulfate tryptose broth(LST) per dilution, rather than three tubes perdilution.

ii. Incubate inoculated LST and EC broths for 24 ± 2 h.

iii. Consider the presence of gas in LST and EC brothsas positive for coliform and E. c ol i respectively, with no further testing required.

b. The procedure for the enumeration of S. aur eus differsfrom the AOAC procedure in that only one tube, instead of three, per dilution is used to determine theestimated count.

3.12 General Guidelines for Testing Fresh or Prepared Foods

a. Do not combine the components of composite items such asfrozen dinners into a single sample. To the greatest

extent possible, examine as separate samples thevegetable or non-meat portion(s) and the meat portion.

b. The quantity, condition and suitability of the sampleare very important.

i. The quantity should be sufficient to perform theanalysis and have a reasonable amount in reservefor repeat testing.

ii. The condition of receipt should be in keeping withgood microbiological practices for the analysis(es)requested.

iii. The sample should be, to the greatest extent

possible, representative of the whole of theoriginal product at the time the sample was taken.

iv. When appropriate and if possible, samples should bereceived at the laboratory in their originalunopened package(s) (intact sample).

3.13 Tests Covered in This Section

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a. Aerobic plate count b. Coliform and E. c ol i quantitative estimatesc. S. aur eus


a. Balance, capacity ≥≥2 kg, sensitivity ± 0.1 g b. Blender and sterile blender jars

c. Stomacher™™ and sterile stomacher bags

d. Incubators at 35 ± 1.0°°C, and 20 ± 1.0oC

e. Water bath at 45.5 ± 0.05°°C

f. Water bath at 37 ± 1.0°°Cg. Manual or Automatic colony counter and tally registerh. Sterile, disposable/reusable dishes, pans or trays for

sample cuttingi. Sterile forceps, spoon, knife, scissors and other

sterile sampling equipment

j. Sterile 1, 5 and 10 ml pipettesk. Sterile 100 x 15 mm petri dishesl. Transfer loop, 3 mm m. Microscope and clean slidesn. Refrigerated centrifugeo. Refrigerator p. pH meter

3.21 Media

a. Plate count agar (PCA) in containers suitable for making pour plates

b. Laurel sulfate tryptose (LST) broth with fermentationtubes

c. EC broth with fermentation tubesd. Surface dried Baird-Parker plates (egg tellurite glycine

pyruvate agar, ETGPA)e. Brain heart infusion (BHI) brothf. Trypticase soy broth with 10% sodium chloride and 1%

sodium pyruvate (PTSBS)g. Toluidine blue DNA agar

3.22 Reagents

a. Butterfield's phosphate diluent

b. Gram stain reagentsc. Desiccated rabbit plasma (coagulase) EDTA d. Tris Buffere. Ammonium sulfate [(NH4)2SO4], reagent gradef. Triton X-100g. 3M trichloroacetic acid solutionh. 1N HCl solution

3.3 Preparation and Dilution of Samples

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See Section 1.3 - 1.5 (Sterilization of Instruments,Disinfection of Containers, and Cutting and Weighing Samples)

3.31 Food Homogenates

a. Using sterile spoons, forceps, scissors, etc.,aseptically weigh 50 ± 0.1 g of the sample into asterile blender jar or stomacher bag.

b. If the sample is frozen, remove portions, whenever possible, without thawing the larger sample and weigh 50± 0.1 g of the sample into a sterile blender jar orstomacher bag. It is well known that freeze/thaw cyclesare damaging to bacteria. This is particularlyimportant when a re-examination of the product may be

necessary. Otherwise, partially thaw the sample at 2-5°°Cfor about 18 h, or by placing the sample in a watertight

container and immersing it in cold water for 1-2 h.

c. Add 450 ml sterile Butterfield's phosphate diluent and stomach for 2 minutes, or blend at high speed for two minutes. The total volume in the blender jar mustcompletely cover the blades. This becomes the 1:10dilution.

d. Permit the foam to settle; then pipet 10 ml of the blended 1:10 dilution into a 90 ml dilution blank to make the 1:100 dilution. Repeat this procedure to prepare serial dilutions of 10

-3, 10

-4, etc. Shake all

dilutions 25 times in a one foot arc. Use a separate 10 ml pipette to prepare each dilution. Pipettes mustdeliver accurately the required volumes. Do not deliverless than 10% of a pipette's volume. For example, todeliver one ml, do not use a pipette of more than 10 mlvolume.

e. The analyst should strive to minimize the time from whenthe sample is stomached or blended until all thedilutions have been placed in or on the appropriate medium; ideally this time should not exceed 15 minutes whenever possible.

f. If the sample consists of less than 50 g, weigh abouthalf the sample, and add the amount of diluent required to make a 1:10 dilution (nine times the weight of the portion of sample used) and proceed as above.

g. Hold reserves of each sample at or below -15°°C (5°°F),unless the product is stored normally at ambient

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3-5

temperature or unless a specific protocol specifiesotherwise. Samples should be held until a determinationis made that a repeat test is not necessary or for thelength of time designated by the testing protocol.

3.32 Whole Bird Rinse

a. Since there are differences between sample types and sizes (eg. chicken vs. turkey carcasses), be sure tocheck the specific program protocol before using this procedure.

b. Aseptically transfer the carcass to a sterile Stomacher3500 bag (or equivalent), draining as much excess fluid as possible during the transfer.

Note: Larger (24 x 30-36 in.) bags will have to be used with turkeys.

c. Add 400 ml (chickens) or 600 ml (turkeys) ofButterfield's Phosphate Diluent (BPD) to the carcass inthe bag. Pour approximately one half the volume intothe interior cavity of the bird and the other half overthe skin. Note: If Sa l mone l l a is the ONLY targetanalyte, Buffered Peptone Water (BPW) may be substituted for the BPD.

d. Rinse the bird, inside and out, with a rocking motionfor 1 min at a rate of approximately 35 forward and backswings per minute. This is done by grasping the carcassin the bag with one hand and the closed top of the bag

with the other. Rock with a reciprocal motion in an 18-24 inch arc, assuring that all surfaces (interior and exterior) are rinsed.

e. Aseptically remove the carcass from the bag, drainingexcess rinsed liquid into the bag, dispose of thecarcass, and culture the bird rinse liquid according to protocol directions.

3.33 Egg Products

a. Liquid eggs must be held at 4.4°°C (40oF) or below for

valid analysis.

b. Frozen samples must be thawed as rapidly as possible in

a water bath at 45°°C.

c. Exposed or leaking samples should not be analyzed.

d. Mix the sample with a sterile spoon, spatula, or by

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3-6

shaking.

e. Aseptically weigh a minimum of 100 g of egg sample intoa sterile blender jar or sealable bag containing 900 mlof the appropriate enrichment or buffer. If a specific

protocol requires a sample size greater than 100 g, the1:10 ratio must be maintained in the same enrichment or buffer.

f. Mix the 1:10 sample enrichment/buffer well by shaking,stomaching, or blending.

g. Dried egg samples should be rehydrated slowly bygradually adding the enrichment/diluent to the sample.This is done by adding a small portion of liquid to thesample and mixing aseptically to obtain a homogeneoussuspension. Repeat this procedure three times and thenadd the remainder of the liquid. Mix until a lump-free

suspension is obtained.

h. Incubate or transfer to the appropriate enrichment medium and incubate according to the protocol(s) beingused.

3.4 Aerobic Plate Count (APC)

a. Pour Plates (Reference AOAC 966.23 C)

i. Using the dilutions prepared in section 3.3, pipet1 ml from the 10

-1, 10

-2, 10

-3, 10

-4etc. dilutions

into each of four petri dishes, two for each

incubation temperature. Plate additional dilutions when expecting higher bacterial levels.

ii. Use separate sterile pipettes for each dilution.

iii. Add molten Plate Count Agar cooled in a water bath

to 45 ± 1°°C. Uniformly mix the agar and theinoculum by gently swirling or tilting each plate,taking care not to generate bubbles.

iv. Allow the agar to harden and then place one series

of duplicate plates in a 35 ± 1°°C incubator for

48 h. Incubate the other series at 20 ± 1°°C forfour or five days.

v. Use a colony counter and count colonies on theduplicate plates in a suitable range (30-300colonies per plate). If plates do not contain

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3-7

30-300 colonies, record the dilution counted and the number of colonies found. Average the countsobtained from duplicate plates, multiply by thedilution factor and report this number as theaerobic plate count per gram or milliliter at the

incubation temperature used.

b. Alternate Methods - AOAC

i. Aerobic Plate Count in Foods: Hydrophobic Grid Membrane Filter Method

*(AOAC 986.32)

ii. Dry Rehydratable Film (Petrifilm Aerobic Plate™™) Method

*(AOAC 990.12)

iii. Spiral Plate Method *(AOAC 977.27)

*Since these methods are available commercially, the manufacturer's directions should be followed.

3.5 Coliform Group and Es cher i c hi a c ol i

a. Estimated Count Procedure (Reference AOAC 966.24)


-1, 10

-2, 10

-3etc. dilutions into LST

broth, one tube per dilution. Inoculate additionaldilutions when expecting higher bacterial levels.The highest dilution of sample must be sufficientlyhigh to yield a negative end point.


iii. Incubate the tubes of LST broth at 35°°C for24 ± 2 h.

iv. Examine each tube for gas formation as evidenced bydisplacement of fluid in the inverted tubes or byeffervescence when tubes are shaken gently.

v. Consider any tube of LST broth displaying gas ascoliform positive, and report the number ofcoliform per gram in accordance with the highestdilution with gas. When a "skip" occurs, report byusing the missing estimate (for example: If the

10-1

, 10-2

, and 10-4

dilutions produce gas but the10

-3dilution tube is non-gassing, report "1,000

coliforms per gram.")

b. Fecal Coliform (E. c ol i ) Estimated Count Procedure(Reference AOAC 966.24)

i. Use a 3 mm calibrated loop to transfer one loopful

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3-8

from every gas-positive LST broth tube to acorrespondingly marked tube of EC broth.

ii. Incubate the EC tubes in a 45.5 ± 0.05°°C covered water bath for 24 ± 2 h. Submerge the EC tubes in

the bath so that the water level is above the levelof medium in the tubes.

iii. Record every tube producing gas, as evidenced bydisplacement of liquid in the inverted tube or byeffervescence when tubes are shaken gently.

iv. Report the number of E. c ol i per gram in accordance with the highest dilution displaying gas. When a"skip" occurs, report by using the missing estimate(for example: If the 10

-1, 10

-2, and 10

-4dilutions

produce gas but the 10-3

dilution tube isnon-gassing, report "1,000 E. c ol i per gram.")

c. Alternate Methods - AOAC

i. Coliform and Es cher i c hi a c ol i Counts in Foods:Hydrophobic Grid Membrane Filter/MUG Method

*

ii. Coliform and Es cher i c hi a c ol i Counts in Foods: DryRehydratable Film

*

*Since these methods are available commercially, the manufacturers's directions should be followed.

3.6 St aphy l ococcus aur eus

a. Estimated Count Procedure (Reference AOAC 987.09)


-1, 10

-2, 10

-3etc. dilutions into

tubes containing 10 ml of Trypticase (tryptic) SoyBroth with 10% sodium chloride and 1% sodium pyruvate (PTSBS), one tube per dilution. Inoculateadditional dilutions when expecting higher

bacterial levels. The highest dilution of sample must be sufficiently high to yield a negative end point.


iii. Incubate the PTSBS tubes at 35°°C for 48 h.iv. Using a 3 mm calibrated loop, transfer a loopful

from each growth-positive tube as well as from thetube of the next highest dilution to previously prepared plates of Baird-Parker agar. Streak in a manner to produce well-isolated colonies.

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3-9

v. Incubate the Baird-Parker plates at 35°°C for 48 h.

vi. Typical S. aur eus colonies appear as circular,convex, smooth, grey-black to jet-black colonies on

uncrowded plates and frequently have an off-white margin surrounded by a zone of precipitation(turbidity) followed by a clear zone. The coloniesusually have a buttery to gummy consistency.

vii. Test two or more isolates, from each useable plate meeting the above description (3.6,vi), forcoagulase as in Section 3.6 (c).

b. Direct Plating

i. If S. aur eus counts of 100 cfu per gram or more areexpected, direct plating can be done using

Baird-Parker agar.

ii. Pipet 0.1 ml from each dilution on previously prepared and dried Baird-Parker agar plates. Useseparate accurate pipettes for each dilution.

iii Distribute the inoculum evenly over the surface ofthe plates using separate, sterile, fire polished, bent-glass rods ("hockey sticks") for each plate. Mark plates according to the dilution used.

iv. Invert plates and incubate at 35°°C for 48 h.

v. Select plates containing approximately 20 or more well-isolated typical S. aur eus colonies. Count plates containing 20-200 colonies. Typicalcolonies are circular, convex, smooth, grey-blackto jet-black and frequently have an off-white margin surrounded by a zone of precipitation(turbidity) followed by a clear zone. The coloniesusually have a buttery to gummy consistency.

vi. Select 10 colonies from those counted and inoculate

each into separate 13 x 100 millimeter tubescontaining 0.2 ml of BHI broth for coagulase

testing. Test for coagulase as in 3.6 (c).vii. Calculate the total number of colonies represented

by coagulase positive cultures and multiply by theappropriate sample dilution factor to record thenumber of coagulase positive staphylococci pergram.

c. Coagulase Test for St aphy l ococcus aur eus

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3-10

i. Use an inoculating needle to obtain a small amountof growth from each suspect colony and place itinto 13 X 100 mm tubes containing 0.2 ml of BHIBroth.

ii. A known coagulase positive and a known negativeculture should be inoculated into BHI broth at thesame time as the samples.

iii. Incubate each tube at 35°°C for 18-24 h.

iv. Add 0.5 ml of rabbit plasma with EDTA,reconstituted according to the manufacturer'sdirections, to the BHI cultures.

v. Mix thoroughly and place the tubes in a 35-37°°C. water bath.

vi. Examine these tubes each hour, from one through sixhours, for clot formation. Any degree of clottingshould be interpreted as a positive reaction.

3.61 Special Sampling Procedure for Fermented Sausage Products

a. Introduction

During the early stages of sausage fermentation,staphylococci can grow extensively if the starterculture is not added or fermentation fails with no

concomitant production of lactic acid and drop in pH.Failure can be caused by poor quality starter culturesor the improper use of starter cultures or "backinoculation". S. aur eus growth is aerobic and usuallyconfined to the outer 1/8 inch of the sausage.Enterotoxin may be formed as a result of this growth.

Coagulase-positive staphylococcal counts on large sticksof salami have been noted to vary widely. On largesticks, some areas may have very few staphylococci whileother areas may have levels in excess of 10

6/g. Whenever

possible, obtain 1-2 pounds of the suspect sausage. Inorder to obtain a representative sample, portions should

be taken from several different areas and composited fortesting.

b. Procedure

i. If the sausage is moldy, wipe the mold off thesausage casing with a piece of sterile tissue paperand proceed.

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3-11

ii. To collect a sample, use a sterile, sharp knife and cut several thick slices from the sausage near theends as well as in the middle. Aseptically trim and save the outer 1/8 to 1/4 inch portion of the

sausage and label it "shell portion". Even if theamount of sample is limited, do not cut deeper than1/4 inch.

iii. Working aseptically, blend 25-50 g of the shell portion for enterotoxin testing; the same blended sample can be used to test for viablecoagulase-positive S. aur eus as described insection 3.6.

iv. Analyze the sample by either of the following procedures.

3.62 The (Presumptive) Staphylococcal Enterotoxin Reverse PassiveLatex Agglutination Test

The procedure for this test is given in (15.20) and usually isthe method of choice.

3.63 Thermonuclease Assay

a. Introduction

This procedure is based on the detection of a heatstable DNase which is produced by most strains of S.aur eus , including 98.3% of the enterotoxigenic strains.

This heat stable DNase is produced in detectable amountsunder all conditions which permit the growth of S.aur eus and the production of enterotoxin. The DNase isable to survive processing conditions which would destroy viable S. aur eus .

This method can be used to screen large sausages or alarge number of samples to identify "hot spots".It has been shown (Tatini, 1981) that the detection ofDNase with this procedure is indicative of S. aur eus

populations of ≥≥105per gram.

b. Procedure:

i. Blend 20 g of shell, 10 g (NH4)2SO4, and 2 ml TritonX-100 in 40 ml of distilled water.

ii. Adjust the pH of this slurry to 4.5-4.8 with 1NHCl.

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3-12

iii. Centrifuge under refrigeration at 7-10,000 RPM for15 min.

iv. Decant and discard the supernatant and add 0.05 mlcold 3M trichloroacetic acid for each ml of the

original slurry, mix and centrifuge a second timeas above.

v. Decant and discard the supernatant. Re-suspend the precipitate in 1 ml of Tris buffer, adjusted to pH8.5, and then adjust the volume to 2 ml with Tris buffer.

vi. Boil the solution for ≥≥15 but ≤≤90 min, cool and store under refrigeration until needed.

vii. Cut 2 mm diameter wells into air dried ToluidineBlue DNA Agar.

viii. Dispense the food extract into one or more wellsusing a Pasteur pipette. Do not overfill the well.

ix. Incubate these plates, agar side down, at 37°°C for 4to 24 h.

x. Any pink halo, extending 1 mm beyond the well isconsidered positive for thermonuclease.

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3-13


Cunniff, P. (ed.). 1995. Official Methods of Analysis of AOACInternational, 16th Edition. AOAC International Inc.,Gaithersburg, MD 20877.

Emswiler-Rose, B. S., R. W. Johnston, M. E. Harris, and W. H.Lee. 1980. Rapid detection of staphylococcal thermonucleaseon casings of naturally contaminated fermented sausages. Appl. Environ. Microbiol. 440:13-18.

Lancette, G. A., and S. R. Tatini. 1992. St aphy l o coccus aur eus , p. 533-550. I n C. Vanderzant and D. F. Splittstoesser(ed.), Compendium of Methods for the MicrobiologicalExamination of Foods. Amer. Publ. Hlth. Assoc., Washington,D.C. 20005.

Tatini, S. R. 1981. Thermonuclease as an indicator of

staphylococcal enterotoxins in food, p. 53-75. I n R. L. Ory(ed.), Antinutrients and Natural Toxicants in Foods. Food and Nutrition Press, Inc., Westport, CT.

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The methods described in this guidebook are for use by the FSIS laboratories.

FSIS does not specifically endorse any of the mentioned test products and

acknowledges that equivalent products may be available for laboratory use._________________________________________________________________________________

United States

Department of

Agriculture

Food Safety

and Inspection

Service

Office of

Public Health

Science

Laboratory QA/QC Division

950 College Station Road

Athens, GA 30605

_______________________________________________________________________________________________

Laboratory Guidebook

Notice of Change

Chapter new, revised, or archived: MLG 4.04

Title: Isolation and Identification of Salmonella from Meat, Poultry, and Egg

Products

Effective Date: 2/4/08

Description and purpose of change(s):

An alternative test for performing the flagellar (H) antigen agglutination test was

validated. A section describing sample pooling was removed.

QD-F-Micro-0004.03 Issuing Authority: Laboratory Quality Assurance Division (LQAD)

Page 1 of 1 Effective: 5/29/07

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United States Department of Agriculture

Food Safety And Inspection Service, Office of Public Health Science

MLG 4.04 Page 1 of 14

Title: Isolation And Identification of Salmonella From Meat, Poultry And Egg Products

Revision:04 Replaces: MLG 4.03 Effective: 2/4/08

Issuing Authority: Laboratory Quality Assurance Division (LQAD)

Procedure Outline

4.1 Introduction

4.1.1 General

4.1.2 Limits of Detection

4.2 Safety Precautions4.3 Quality Control Procedures

4.3.1 Method Controls

4.3.2 Specific Procedure Controls

4.4 Equipment, Reagents, Media and Test Kits4.4.1 Equipment4.4.2 Reagents

4.4.3 Media

4.4.4 Cultures4.5 Isolation Procedures

4.5.1 Breading Mixes, Dehydrated Sauces and Dried Milk

4.5.2 Ready-to-Eat Foods4.5.3 Fermented Product

4.5.4 Raw Meat

4.5.5 Carcass Sponge Samples

4.5.6 Whole Bird Rinses4.5.7 Liquid, Frozen, Cooked or Dried Egg Samples

4.5.8 Sanitation Series Food Homogenates

4.5.9 Most Probable Numbers (MPN) Determination4.6 Examination of and Picking Colonies from Plating Media

4.6.1 Picking colonies

4.6.2 Screening Media4.7 Biochemical Procedures

4.8 Serological Tests

4.8.1 Somatic (O) Antigen Agglutination Tests

4.8.2 Flagellar (H) Antigen Agglutination Tests

4.9 Storage of Cultures4.10 References

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4.1 Introduction

4.1.1 General

This method describes the analysis of various meats, meat products, sponge and rinse

samples, eggs, and egg products for Salmonella. It is not intended for the isolation and identification of Salmonella Typhi.

Success in isolating Salmonella from any food can be related to a number of factors

including food preparation procedures, the number of organisms present, sample handlingafter collection, etc. With raw meat samples the competitive flora may be the mostimportant factor. It varies from sample to sample and from one kind of meat to another.

Another consideration is whether the examination is for routine monitoring or epidemiological purposes. The analyst may choose to augment the method for

epidemiological purposes with additional enrichment procedures and culture media, two

temperatures of incubation, intensified picking of colonies from plates, and/or rapid screening methods.

All isolates must be identified as Salmonella biochemically and serologically.

Unless otherwise stated all measurements cited in this method have a tolerance range of

± 2%.


The Salmonella detection limit for this method has been determined to be less than

1 colony forming unit (cfu)/g in a 25 g sample.

4.2 Safety Precautions

Salmonella are generally categorized as BioSafety Level 2 pathogens. CDC guidelines for manipulating Biosafety Level 2 pathogens should be followed whenever live cultures of Salmonella are used. A Class II laminar flow biosafety cabinet is recommended for procedures in

which infectious aerosols or splashes may be created. All available Material Safety Data Sheets

(MSDS) must be obtained from the manufacturer for the media, chemicals, reagents and microorganisms used in the analysis. The personnel who will handle the material should read all

MSDS sheets.

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4.3 Quality Control Procedures

4.3.1 Method Controls

Include at least three method controls in all analyses. These controls must include a

Salmonella spp. H2S-negative culture, a Salmonella spp. H2S-positive culture and anuninoculated media control. To facilitate identification of control isolates, the laboratory

may use strains of uncommonly found serogroups. S. Abaetetuba, serogroup F, is suggested

as a readily available, H2S-positive culture that is not commonly found in meats or meat

products. Salmonella serotype Choleraesuis is typically negative for H2S production. Thesecultures may be obtained from ATCC. Other serotypes may be found that have aberrantH2S-negative strains. The control cultures should be inoculated into either a meat matrix or

the matrix that is being analyzed. Incubate the controls along with the samples, and analyze

them in the same manner as the samples. Confirm at least one isolate from each positivecontrol sample. In the absence of a positive test sample, control cultures may be

terminated at the same point as the sample analyses.

4.3.2 Specific Procedure Controls

The biochemical and serological tests used for confirmation of the sample isolates require

the use of appropriate controls to verify that the results are valid. Salmonella ‘O’ antiserashould be tested with QC control sera before initial use, and with a saline control for each

test. Biochemical kit and rapid test manufacturers may specify control cultures for use with

their products. If not specified, quality control procedures for biochemical tests and testmedia should include cultures that will demonstrate pertinent characteristics of the product.

4.4 Equipment, Reagents, Media and Test Kits

All of the materials listed below may not be needed. Media and reagents specific to the biochemical

test method that is used will be needed in addition to the materials listed below. See Section 4.7.

4.4.1 Equipment

a. Sterile tablespoons, scissors, forceps, knives, glass stirring rods, pipettes, petri dishes, test tubes, bent glass rods ("hockey sticks") as needed

b. Blending/mixing equipment: Sterile Osterizer-type blender with sterilized

cutting assemblies, and blender jars or Mason jars and adapters for use with

Mason jars; or a Stomacher ™ (Tekmar or equivalent) with sterile Stomacher

bags

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c. Sterile Stomacher ™ 3500 bags, plain, clear polypropylene autoclave bags

(ca. 24" x 30 - 36"), or Whirl-Pak ™ bags (or equivalent)

d. Incubator, 35 ± 2°C

e. Incubator or water bath, 42 ± 0.5oC

f. Water bath, 48-50°C

g. Glass slides, glass plate marked off in one-inch squares or agglutination ringslides

h. Balance, 2000 g capacity, sensitivity of 0.1 g

i. Inoculating needles and loops

j. Vortex mixer k. VITEK

®system

4.4.2 Reagents

a. Crystal violet dye, 1% aqueous solution, steamed b. Butterfield's phosphate diluent

c. Saline, 0.85%

d. Saline, 0.85% with 0.6% formalin for flagellar antigen testse. Calcium carbonate, sterile

f. Salmonella polyvalent O antiserum

g. Salmonella polyvalent H antiserum, Slide Agglutination H Antisera fromStatens Serum Institut (SSI), or equivalent.

h. Salmonella individual O grouping sera for groups A-I (antisera for further O

groups are optional)

i. (Optional) Oxoid Salmonella Latex Test (Unipath Company, Oxoid Division, Ogdensburg, NY) or equivalent

j. Additional reagents as needed for biochemical tests: GNI cards for VITEK ®

system

4.4.3 Media

a. Buffered peptone water (BPW) b. TT broth (Hajna)

c. Modified Rappaport Vassiliadis (mRV) broth, Rappaport-Vassiliadis R10 broth, or Rappaport-Vassiliadis Soya Peptone Broth (RVS)

d. Brilliant green sulfa agar (BGS; contains 0.1% sodium sulfapyridine)

e. Xylose lysine Tergitol™ 4 agar (XLT4) or Double modified lysine iron agar

(DMLIA)f. Triple sugar iron agar (TSI)

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g. Lysine iron agar (LIA)

h. Trypticase soy broth (TSB) or Tryptose brothi. Trypticase soy agar (TSA)

j. Nutrient agar slants

k. Nutrient broth, semi-solid

l. Tryptic soy agar with 5% sheep blood agar m. Additional media as needed for biochemical tests

4.4.4 Cultures

At least one H2S-positive strain of Salmonella and one H2S-negative strain of Salmonella are required for method controls.

4.5 Isolation Procedures

4.5.1 Breading Mixes, Dehydrated Sauces and Dried Milk

For dehydrated sauces, dried milk, and breading mixes add BPW as described for

powdered egg in Section 4.5.8.

4.5.2 Ready-to-Eat Foods

Follow program requirements for preparing sample and sub-sample composites. Outbreak

investigation requirements may differ; in which case, follow the client specifications for those samples.

a. Weigh 325 g of the composite sample into a Stomacher bag (or sterile blender jar if required by the client or sample type).

b. Add approximately one third to half of 2925 ml of ambient temperature sterile

buffered peptone water. Blend or stomach approximately 2 minutes, and then add

the remainder of the 2925 ml of BPW.

c. Incubate at 35 ± 2°C for 20-24 h.

d. Transfer 0.5 ± 0.05 ml of incubated broth into 10 ml TT and 0.1 ± 0.02 ml into 10

ml of mRV broth.

e. Incubate the enrichment broths at 42 ± 0.5°C for 22-24 h.

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f. Streak above enrichments on BGS and either DMLIA or XLT4 agar plates. Use one10-microliter loopful for each plate. Do not subdivide plates for streaking multiple

samples; streak the entire agar plate with a single sample enrichment.

g. Incubate at 35 ± 2°C.

h. Examine in 18-24 h. Select colonies. Refer to Section 4.6 et seq.

i. Re-incubate all plates for an additional 18-24 h. Reexamine initially negative platesand pick colonies as above. Reserve, under refrigeration, all plates from whichcolonies were picked. If suspect Salmonella colonies do not confirm, reexamine the

plates from which they were picked, and if appropriate, re-pick colonies for confirmation. See Section 4.6.1.b.

4.5.3 Fermented Products

Follow the procedure for ready-to-eat foods (Section 4.5.3) except:

a. Blend/stomach the sample with 10 g of sterilized calcium carbonate.

b. Use buffered peptone water that contains 1 ml of a 1% aqueous solution of crystal

violet per liter.

4.5.4 Raw Meat

If the sample is not already ground, in some cases it may be best to mince it with scissors or

leave it whole (e.g. chicken wings) to avoid jamming blender blades with skin or connective

tissue. Whirl-Pak ™ bags can be used in culturing these samples.

a. Weigh 25 ± 0.5g of meat into a sterile blender jar, other sterile jar or a Whirl-Pak ™

or Stomacher ™ bag. HACCP program samples collected using a sampling ring areallowed a weight range of 25 ± 2.5 g.

b. Add 225 ml of BPW. Stomach or blend, as required, for approximately two minutes

or shake thoroughly. Alternatively, ground beef samples may be briefly stomached

or hand mixed to disperse clumps.

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c. Incubate at 35 ± 2°C for 20-24 h.

d. Transfer 0.5 ± 0.05 ml into 10 ml TT broth and 0.1 ± 0.02 ml into 10 ml mRV broth.

e. Incubate at 42 ± 0.5°C for 22-24 h or in a water bath at 42 ± 0.5°C for 18-24 h.

f. Streak on DMLIA or XLT4 and BGS agar plates. Use one loopful of inoculum for

each plate. Do not subdivide plates for streaking multiple samples; streak the entire

agar plate with a single sample enrichment.

g. Incubate at 35 ± 2°C.

h. Examine in 18-24 h. Select colonies. See Section 4.6 et seq.

i. Re-incubate all plates for an additional 18-24 h. Reexamine initially negative plates

and pick colonies as above. Reserve, under refrigeration, all plates from whichcolonies were picked. If suspect Salmonella colonies do not confirm, reexamine the

plates from which they were picked, and if appropriate, re-pick colonies for

confirmation. See Section 4.6.1.b.

4.5.5 Carcass Sponge Samples

a. Add 50 ml of BPW to the sample bag containing the moistened sponge to bring thetotal volume to 60 ml. Mix well.

b. Incubate at 35 ± 2°C for 20-24 h.

c. Follow the procedures in Section 4.5.5, d-i.

4.5.6 Whole Bird Rinses

Due to differences between sample types/sizes (e.g. chicken vs. turkey carcasses), followinstructions given in the specific program protocol. For chicken carcasses, aseptically drain

excess fluid from the carcass and transfer the carcass to a sterile Stomacher ™ 3500 bag, a

plain, clear polypropylene bag (ca. 24" x 30-36"), or equivalent. Pour 400 ml (or other

volume specified in program protocol) of Buffered Peptone Water (BPW) into the cavity of

the carcass contained in the bag. Rinse the bird inside and out with a rocking motion for oneminute (ca. 35 RPM). This is done by grasping the broiler carcass in the bag with one hand

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and the closed top of the bag with the other. Rock with a reciprocal motion in about an

18-24 inch arc, assuring that all surfaces (interior and exterior of the carcass) are rinsed.Transfer the sample rinse fluid to a sterile container.

Use 30 ml of the sample rinse fluid obtained above for Salmonella analysis. Add 30 ml of

sterile BPW, and mix well. Incubate at 35 ± 2°C for 20-24 h, and then proceed according to

4.5.5 (d-i).

NOTE: If analyses other than Salmonella are to be performed, the carcass may be rinsed

in Butterfield's Phosphate Diluent instead of BPW. In this case, add 30 ml of 2X BPW to30 ml of carcass-rinse fluid, mix well, and continue as above.

4.5.7 Liquid, Frozen, Cooked or Dried Egg Samples

a. Mix the sample with a sterile spoon, spatula, or by shaking.

b. Aseptically weigh a minimum of 100 g of egg sample into a sterile blender jar, other

sterile jar, or a Whirl-Pak ™ or Stomacher ™ bag containing 900 ml of sterile BPW.If a special sample or specification requires a sample size other than 100 g, the ratio

of egg sample to BPW is to be maintained at 1:10.

c. Mix the inoculated BPW well by shaking, stomaching, or blending.

d. With dried egg samples, gradually add BPW to the sample. Add a small portion of

sterile BPW and mix to obtain a homogeneous suspension. Add the remainder of the BPW. Mix until a lump-free suspension is obtained.

e. Incubate at 35 ± 2°C for 20-24 h, and then proceed according to 4.5.5 (d-i).

4.5.8 Sanitation Series Food Homogenates (optional)

To isolate salmonellae from food samples homogenized as outlined in MLG 3 Section 3.3.1,as part of a sanitation test series, use the 10

-1food homogenate dilution (See also this

chapter, Section 4.5.1 Sample Pooling).

a. Weigh 250 g of food homogenate into a sterile jar (this contains 25 g of product).

b. Add 25 ml of 10x BPW (broth made to ten time’s normal strength).

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c. Incubate 24-26 h at 35 ± 2°C.

d. Transfer 0.5 ± 0.05 ml into 10 ml of TT broth and 0.1 ± 0.02 ml into 10 ml of mRV broth.

e. Incubate at 42 ± 0.5°C for 22-24 h.

f. Streak on DMLIA or XLT4 and BGS agar plates. Use one loopful for each plate.

Do not subdivide plates for streaking multiple samples; streak the entire agar platewith a single sample enrichment.

g. Incubate 18-24 h at 35 ± 2°C.

h. Select colonies. See Section 4.6 et seq.

i. Re-incubate all plates for an additional 18-24 h. Reexamine initially negative platesand pick colonies per Section 4.6. Reserve all plates from which colonies were

picked. If suspect Salmonella colonies do not confirm, reexamine the plates from

which they were picked, and if appropriate, re-pick colonies for confirmation. SeeSection 4.6.1.b.

4.5.9 Most Probable Numbers (MPN) Determination

Follow MPN instructions given in the specific program protocol or see MLG Appendix 2,

Most Probable Number Procedure and Tables.

4.6 Examination of and Picking Colonies from Plating Media

4.6.1 Picking Colonies

a. After the recommended incubation interval, examine the selective-differential agar

plates for the presence of colonies meeting the description for suspect Salmonella

colonies. Pick well-isolated colonies.

• BGS. Select colonies that are pink and opaque with a smooth appearance and

entire edge surrounded by a red color in the medium. On very crowded plates,look for colonies that give a tan appearance against a green background.

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• XLT4. Select black colonies or red colonies with or without black centers. Therim of the colony may still be yellow in 24 h; later it should turn red.

• DMLIA. Select purple colonies with or without black centers. Since

salmonellae typically decarboxylate lysine and ferment neither lactose nor sucrose, the color of the medium reverts to purple.

b. Pick up to three colonies from each plate, if available. (NOTE: Before any sample isreported as Salmonella-negative, a total of three typical colonies, if available, from

each selective agar plate must be examined). Pick only from the surface and center

of the colony. Avoid touching the agar because these highly selective mediasuppress growth of many organisms that may be viable.

If there are typical colonies on a plate, that are not well isolated, pick from the

typical colonies and re-streak directly to selective agar plates. Alternatively, place aloopful of growth into a tube of TT or mRV broth and incubate overnight, then re-

streak to selective agars.

4.6.2 Screening Media

a. Inoculate TSI and LIA slants in tandem with a single pick from a colony by stabbing

the butts and streaking the slants in one operation. If screw cap tubes are used, thecaps must be loosened. Incubate at 35 ± 2°C for 24 ± 2 h.

b. Examine TSI and LIA slants as sets. Note the colors of butts and slants, blackening

of the media and presence of gas as indicated by gas pockets or cracking of the agar.

Note also the appearance of the growth on the slants along the line of streak.Discard, or re-streak for isolation, any sets that show "swarming" from the original

site of inoculation. Discard sets that show a reddish slant in lysine iron agar.

Isolates giving typical Salmonella spp. reactions and isolates which are suggestive, but not typical of Salmonella spp. should be confirmed by a combination of

biochemical and serological procedures. Refer to Table 1 for a summary of TSI-

LIA reactions. The motility testing in the last column of the table is optional.

c. (Optional: for some biochemical test kits) Streak a TSA + 5% sheep blood agar

plate from either the TSI or LIA slant. Incubate 18-24 h at 35 ± 2°C.

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4.7 Biochemical Procedures

Commercially available biochemical test kits, including automated systems may be used for

biochemical identification. If the VITEK ®

system is used, the cytochrome oxidase and gram stain

tests are optional. Alternatively, use traditional methods of biochemical identification. Refer to

AOAC Official Method 967.27 or "Edwards and Ewing's Identification of Enterobacteriaceae", 4thEdition, for biochemical reactions of Enterobacteriaceae and for fermentation media and test

procedures.

Table 1.

Triple Sugar

Iron Agar

Lysine Iron Agar Polyvalent

Sera

Disposal

Butt Slant H2S Butt H2S O H

Y R + P + + + B. & M. T.

Y R + P + + - B. & M. T.

Y R - P - B. & M. T.

Y R - Y - + + * B. & M. T.

Y R - Y - - - Discard

Y R + Y +/- B. & M. T.

Y Y - Y or P - Discard

Y Y + P + ** B. & M. T.

NC NC Discard

Y = Yellow; R = Red; P = Purple; B. & M. T. = Biochemical and motility tests; NC = No change in color fruninoculated medium.

* Salmonella Typhisuis (found seldom in swine in U.S.)

** Salmonella enterica subsp. arizonae or S. enterica subsp. diarizonae

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4.8 Serological Tests

4.8.1 Somatic (O) Antigen Agglutination Tests

At a minimum, isolates should be tested with polyvalent O antiserum reactive withserogroups A through I. Following a positive reaction with polyvalent O antiserum, it is

necessary to type the isolate using individual Salmonella antisera for O groups A through I.

Testing for O groups A through I should encompass the majority of the Salmonella

serotypes commonly recovered from meat and poultry products. Occasionally, however, anisolate will be recovered which is typical of Salmonella biochemically and is serologically poly H-positive, but is non-reactive with antisera to groups A through I. Such an isolate

should be reported as "Salmonella non A-I" or "Salmonella O group beyond I".

Use growth from either the TSI or LIA slant. Test first with polyvalent O antiserum.

Include a saline control with each isolate. If there is agglutination with the saline control

alone (autoagglutination), identify such a culture by biochemical reactions only. If thesaline control does not agglutinate and the polyvalent serum does, test the culture with

Salmonella O grouping antisera. Record positive results and proceed to H agglutination

tests.

4.8.2 Flagellar (H) Antigen Agglutination Tests

Inoculate trypticase soy broth or tryptose broth. Incubate at 35 ± 2°C overnight or until

growth has an approximate density of three on the McFarland scale. Add an equal amount

of saline containing 0.6% formalin and let sit one hour. Remove one ml to each of two 13 x100 mm test tubes. To one of the tubes, add Salmonella polyvalent H serum in an amount

indicated by the serum titer or according to the manufacturer's instructions. The other tube

serves as an autoagglutination control. Incubate both tubes at 48-50°C in a water bath for up

to 1 h. Record presence or absence of agglutination.

If desired, use Spicer-Edwards pooled serum or H typing serum. Find details in "Edwardsand Ewing's Identification of Enterobacteriaceae" (Ewing, 1986).

The Oxoid Salmonella Latex Test, SSI H Antisera for Slide Agglutination, or equivalent,may be used as an optional method for H antigen agglutination testing. Follow the

manufacturer's instructions. If a suspect Salmonella isolate is negative by the agglutination

test, perform the poly H tube agglutination test described above.

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4.9 Storage of Cultures

Do not store cultures on TSI agar because this tends to cause roughness of O antigens. For short-

term (2-3 months) storage, inoculate a nutrient agar slant, incubate at 35 ± 2°C overnight and then

store at 2-8°C.

Store "working" Salmonella stock cultures on nutrient agar slants. Transfer stocks monthly onto

duplicate nutrient agar slants, incubate overnight at 35 ± 2°C, and then store them at 2-8°C. Use

one of the slants as the working culture. Use the other slant for sub-culturing to reduce theopportunity for contamination. Cultures may be subcultured up to 5 times. After this period the

culture must be re-confirmed biochemically or a new culture initiated.

For long term storage freeze cultures using cryo-beads, i.e. Cryostor™ or equivalent, lyophilize or

use the procedure that follows. Subculture Salmonella isolates by picking a colony with an

inoculating needle and stabbing it into semi-solid nutrient broth (0.75% agar). Incubate at 35 ± 2°C

overnight, and then seal with hot, paraffin-soaked corks. Household wax is better than embedding paraffin because it stays relatively soft at room temperature making the corks easy to remove. Store

the cultures in the dark at room temperature. Such cultures will remain viable for several years.


Bailey, J. S., J. Y. Chiu, N. A. Cox, and R. W. Johnston. 1988. Improved selective procedure for

detection of salmonellae from poultry and sausage products. J. Food Prot. 51:391-396.

Centers for Disease Control and Prevention and National Institutes of Health (CDC/NIH). 2007.

BioSafety in Microbiological and Biomedical Laboratories, 5th

ed. U.S. Government PrintingOffice, Washington, D.C. (internet site: http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm)

Horowitz, William. (ed.). 2000. Official methods of analysis of AOAC International, 17th Edition.AOAC International Inc., Gaithersburg, MD 20877.

Ewing, W. H. 1986. Edwards and Ewing's Identification of Enterobacteriaceae, 4th Edition.Elsevier Science Publishing Co., Inc., New York.

Federal Register, Vol. 61, No. 144, Thursday, July 25, 1996, Appendix E, pp. 38917 – 38925.

Miller, R. G., C. R. Tate, and E. T. Mallinson. 1994. Improved XLT4 agar: small addition of

peptone to promote stronger production of hydrogen-sulfide by Salmonellae. J. Food Prot. 57:854-

858.

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Vassiliadis, P., D. Trichopoulos,. A. Kalandidi, and E. Xirouchaki. 1978. Isolation of salmonellaefrom sewage with a new procedure of enrichment. J. Appl. Bacteriol. 66:523-528.

Insert No. 15983: Salmonella Antisera for in vitro diagnostic use, Statens Serum Institut, Denmark,

December 2005

Vassiliadis, P. 1983. The Rappaport-Vassiliadis (RV) enrichment medium for the isolation of

Salmonellas: an overview. J. Appl. Bacteriol. 54:69-76.

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United States Food Safety Office of Laboratory QA/QC Division

Department of and Inspection Public Health 950 College Station Road

Agriculture Service Science Athens, GA 30605

_______________________________________________________________________________________________


Notice of Change

Chapter new, revised, or archived: MLG 4C.02

Title: FSIS Procedure for the Use of a Polymerase Chain Reaction (PCR) Assay for

Screening Salmonella in Raw Meat, Carcass Sponge Samples, Whole Bird

Rinses, Ready-to-Eat Meat and Poultry Products and Pasteurized Egg Products



The chapter title was changed. The procedure to follow when a PCR indeterminate or

signal-error occurs was clarified in Section 4C.7 Interpretation of Results.



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MLG 4C.02 Page 1 of 4

Title: FSIS Procedure for the Use of a Polymerase Chain Reaction (PCR) Assay for Screening Salmonella in Raw

Meat, Carcass Sponge Samples, Whole Bird Rinses, Ready-to-Eat Meat and Poultry Products and Pasteurized

Egg Products

Revision: 02 Replaces: 01 Effective: 7/13/07

Approved by Laboratory Quality Assurance Division (LQAD)

Procedure Outline

4C.1 Introduction

4C.1.1 General4C.1.2 Limits of Detection

4C.2 Safety Precautions

4C.3 Quality Control Procedures

4C.3.1 Culture Controls4C.3.2 Sterility Control

4C.4 Equipment, Reagents and Media4C.5 Sample Preparation and Primary Enrichment

4C.5.1 Raw Meat, Carcass Sponge Samples, Whole Bird Rinses

4C.5.2 Ready-To-Eat Meat and Poultry Products and Pasteurized Egg Products

4C.6 The BAX®

System for Screening Salmonella Test Procedure

4C.7 Interpretation of Results

4C.8 Completion of Testing if BAX®

Unavailable

4C.9 Selected References

4C.1 Introduction

4C.1.1 General

This method describes the use of a commercial PCR-based screening procedure as

described in MLG 4, Section 4.4.5 to screen test Ready-to-Eat meat and poultry products,

pasteurized egg products, raw meat, carcass sponge samples, and whole bird rinses for Salmonella. All samples identified as potentially positive for the presence of Salmonella by this test are subject to cultural confirmation as described in MLG 4.

4C.1.2 Limits of Detection

For this method, Salmonella detection limits are determined to be better than 1 cfu/g in a

25g sample.

4C.2 Safety Precautions

CDC guidelines for the handling of BioSafety Level 2 organisms should be followed whenever

live cultures of Salmonella are used. All available Material Safety Data Sheets (MSDS) must be

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Egg Products



obtained from the manufacturer for the media, chemicals, reagents, and microorganisms used in

the analysis. The personnel who will handle the material should read all MSDS sheets.

4C.3 Quality Control Procedures

4C.3.1 Culture Controls

See MLG 4, Section 4.3.1 for a description of the culture controls.

4C.3.2 Sterility Control

Additionally, always prepare at least one “blank” (incubated but un-inoculated pre-enrichment/ enrichment broth) to provide a sterility control for the process.

4C.4 Equipment, Reagents, and Media

In addition to equipment, reagents and media used in analysis of samples as described in MLG 4,

the following materials will be needed.

a. PCR tube holder (Qualicon)

b. Cell lysis tube cooling block (Qualicon) held at 5 ± 3°Cc. Techne DB-2A, or equivalent, heating block set at 37 ± 2°C

d. Techne DB-2A, or equivalent, heating block set at 95 ± 3°C

e. Repeating pipettor to deliver 200 ± 20 μl, and sterile tips

f. Pipettor to deliver 5 ± l μl, and sterile disposable filtered tips

g. Pipettor to deliver 150 ± 15 μl, and sterile disposable filtered tips

h. Eight-channel pipettor to deliver 50 ± 5 μl, and sterile disposable tips

i. 12 X 75 mm Falcon 352063, or equivalent, tubes

j. Cell lysis tubes and caps, cell lysis tube rack and box (Genemate 8 strip tubes,

ISC Bioexpress, T-3120-5)k. Pipettor and 5 ml pipettes

l. BAX® System PCR Assay for Screening Salmonella kit (Qualicon # 17710608)

held at 5 ± 3°C

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Egg Products



4C.5 Sample Preparation and Primary Enrichment

4C.5.1 Raw Meat, Carcass Sponge Samples, Whole Bird Rinses

Perform sample preparation and pre-enrichment as described in MLG 4, Sections 4.5.5

through 4.5.7.

4C.5.2 Ready-To-Eat Meat and Poultry Products and Pasteurized Egg Products

Perform sample preparation and pre-enrichment as described in MLG 4, Sections 4.5.3,4.5.4, 4.5.8 and 4.5.9 with the exception of incubation time in BPW. The incubation time

in BPW for BAX®

System PCR Assay for Screening Salmonella analysis of Ready-to-Eat meat and poultry products and pasteurized egg products is 18-24h.

4C.6 The BAX®

System for Screening Salmonella Test Procedure

Follow the current BAX®

User’s Guide for preparing reagents, performing the test, and reading

the results. The equipment must be set up, operated, and all records documented according to

laboratory work instructions.

4C.7 Interpretation of Results

a. Samples that test BAX®

-negative will be reported as negative. Cultural analysis

will continue as per MLG 4, Section 4.5.3.d-i, of a sample BPW pre-enrichment that

tests BAX®

-positive, BAX®

-indeterminate, or has a BAX®

signal-error result. Or based on the findings of a cause analysis, the laboratory may analyze the

indeterminate or signal-error result samples by:

• repeating the BAX®

analysis from the rack loading step or

• preparing new BAX®

tubes and repeating the analysis.

b. In analytical runs where both positive controls test negative, the reserve sampleswill be retested beginning with sample preparation and enrichment. In analytical

runs where one of the positive controls tests negative, the laboratory shall growthe control culture and continue cultural analysis of all samples by proceeding

with isolation and purification steps as per MLG 4, Section 4.5.3.d-i.

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Egg Products



4C.8 Completion of Testing if BAX®

Unavailable

If circumstances (e.g. a power outage or equipment failure) do not allow testing using the BAX®

system, the laboratory shall, if possible, continue cultural analysis of all samples by proceeding

with isolation and purification steps as per MLG 4, Section 4.5.3.d-i.

4C.9 Selected References

Centers for Disease Control and Prevention and National Institutes of Health (CDC/NIH). 1999.BioSafety in Microbiological and Biomedical Laboratories, 4

thed. U.S. Government Printing

Office, Washington, D.C. also found on the internet at:

http://www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4toc.htm

BAX®

System PCR Automated Detection for Bacterial Screening User Guide, Dupont Qualicon.



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United States

Department of

Agriculture

Food Safety

and Inspection

Service

Office of

Public Health

Science



Athens, GA 30605

______________________________________________________________________________________________


Notice of Change

Chapter new, revised, or archived: MLG 5A.01

Title: FSIS Procedure for the Use of Escherichia coli O157:H7 Screening Tests



FSIS has extended the use of a commercial PCR based screening procedure to include

environmental swabs. Clarification for storing PCR cooling blocks in a freezer and an

updated limit of detection has also been included in this chapter. All samples

identified as potentially positive for Escherichia coli O157:H7 by either primary or

alternative tests are subject to cultural confirmation as described in this chapter and

MLG 5 Detection, Isolation, and Identification of Escherichia coli O157:H7 from

Meat Products.



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MLG 5A.01 Page 1 of 5




Procedure Outline

5A.1 Introduction5A.1.1 General

5A1.2 Limits of Detection

5A.2 Safety Precautions

5A.3 Quality Control Procedures5A.4 Equipment, Reagents and Media

5A.5 Sample Preparation and Primary Enrichment

5A.6 The BAX®

System and BAX®

E. coli O157:H7 MP Test for Screening Escherichia coli

O157:H75A.6.1 Procedure5A.6.2 Interpretation of Results

5A.7 Use of Lateral Flow Devices for Screening Escherichia coli O157:H75A.8 Selected References

5A.1 Introduction

5A.1.1 General

This method describes the use of a commercial PCR-based screening procedure to

screen test enrichment cultures of ground beef, fermented sausage, cooked meat patties, beef trim products, and environmental swabs for Escherichia coli O157:H7

( E. coli O157:H7). In the event that the BAX®

system equipment or BAX®

E. coli

O157:H7 MP test kits are unavailable, this method describes the use of lateral flow

devices that FSIS may use to screen for E. coli O157:H7.

Samples identified as potentially positive for the presence of E. coli O157:H7 by any

of these tests are subject to cultural confirmation as described in MLG 5.

Unless otherwise stated all measurements cited in this method have a tolerance of ± 2%.

5A1.2 Limits of Detection

This test has been shown to detect 0.23 colony forming units (cfu)/g in a 25 g sample of

75/25 (lean/fat) ground beef.

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E. coli O157:H7 is a human pathogen with a low infectious dose. (Ingestion of 100 cells can

cause disease.) The use of gloves and eye protection is highly recommended and all work

surfaces shall be disinfected prior to and immediately after use. Laboratory personnel must

abide by CDC guidelines for manipulating Biosafety Class II pathogens. A Class II laminar flow biosafety cabinet is recommended for activities with potential for producing aerosols of

pathogens. All available Material Safety Data Sheets (MSDS) should be obtained from the

manufacturer for the media, chemicals, reagents and microorganisms used in the analysis.

The personnel who will handle the materials should read all MSDS sheets.

5A.3 Quality Control Procedures

a. All media must be pre-warmed to 18-35ºC prior to use.

b. E. coli O157:H7 strain 465-97, or equivalent, shall be used as the positive control.

See MLG 5, Section 5.3c for the positive culture control procedures.

c. E. coli ATCC strain 25922, or equivalent, shall be used as the negative control for

bead capture and screen tests.

d. Prepare at least one “blank” (incubated but un-inoculated pre-enrichment/ enrichment

broth) to provide a sterility control for the process.

5A.4 Equipment, Reagents and Media

In addition to equipment, reagents and media used in analysis of samples as described inMLG 5, the following materials will be needed.

a. PCR tube holder (Qualicon or equivalent) b. Cell lysis tube cooling block (Qualicon or equivalent) held at 5 ± 3ºC

c. PCR cooling block (Qualicon or equivalent) held at < -10ºCd. Heating block set at 37 ± 2ºC

e. Heating block set at 95 ± 3ºCf. Repeating pipettor to deliver 200 ± 20 μL, and sterile tips

g. Pipettor to deliver 5 ± 1 μL, and sterile disposable filtered tips

h. Pipettor to deliver 150 ± 15 μL, and sterile disposable filtered tipsi. Eight-channel pipettor to deliver 50 ± 5 μL, and sterile disposable tips

j. 12 X 75 mm Falcon 352063, or equivalent, tubes

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k. Cell lysis tubes and caps, cell lysis tube rack and box (Genemate 8 strip tubes, ISC

Bioexpress, T-3120-5 or equivalent)l. Pipettor or pipettes to deliver 5 mL

m. BAX®

System PCR Assay for Screening E. coli O157:H7 MP kit (Qualicon #

17720673) held at 5 ± 3ºC

n. Optional for Lateral Flow Device Testing:

i. Transia™

Card E. coli O157:H7 (Diffchamb # ECO157), RapidChek ®

Pathogen Screening Test Kit (Strategic Diagnostics, Inc. # 7000160), or equivalent

ii. Polypropylene tubes and sterile pipetsiii. Heating block set at 97-100

oC, boiling water bath, or autoclave (isotherm

cycle)


Perform sample preparation and pre-enrichment as described in MLG 5, Section 5.5.

5A.6 The BAX®

System and BAX®

E. coli O157:H7 MP Test for Screening Escherichia coli

O157:H7

5A.6.1 Procedure

Follow the current BAX®

System User’s Guide for preparing reagents, performing

the test, and reading the results. The equipment must be set up, operated, and allrecords documented according to laboratory work instructions.

5A.6.2 Interpretation of Results

a. Samples that test BAX® -negative shall be reported as negative. Cultural

analysis shall continue as per MLG 5, Sections 5.6 and 5.7 for sample pre-

enrichments that test BAX® - positive, indeterminate, or have an invalid result.

Or based on the findings of a cause analysis, the laboratory may analyze theindeterminate or invalid result samples by:


analysis from the rack loading step


tubes and repeating the analysis or

• screen testing with a lateral flow device (LFD) described in Section 5A.7.

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If a LFD is utilized, the LFD result shall be reported.

b. In analytical runs where the positive control tests BAX® -negative,

indeterminate, or has a signal-error result, the entire batch of samples isaffected and a cause analysis shall be performed. Based on the findings the

laboratory may analyze the samples by:


analysis from the rack loading step


tubes and repeating the analysis

• screen testing with a lateral flow device (LFD) described in Section 5A.7 or

• preparing fresh samples from reserve tissues

The results of the chosen test shall be reported.

5A.7 Use of Lateral Flow Devices for Screening Escherichia coli O157:H7

In the event that the BAX®

system equipment fails or BAX®

E. coli O157:H7 MP test kits are

unavailable, the following lateral flow devices may be used until the BAX®

system is

available to screen for Escherichia coli O157:H7. The laboratory should follow the

manufacturer’s guidelines or other validated laboratory procedures. RapidChek

®

andTransia™

lateral flow devices have been validated for use in FSIS laboratories.

5A.7.1.1 Test Procedure

a. Remove the appropriate number of test devices from storage.

b. Transfer 1-3 ml of the enrichment broth (from each Stomacher bag)into a polypropylene tube with a sterile pipette. For use with a heating

block, use ~ 1 ml. Heat the capped tubes at 97-100ºC for the time

specified by the manufacturer.c. Follow the current manufacturers’ instructions for performing the test.

5A.7.1.2 Interpretation of Results

Follow the current manufacturer’s instructions for reading the test device

results.

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5A.8 Selected References

Centers for Disease Control and Prevention and National Institutes of Health (CDC/NIH).

2007. BioSafety in Microbiological and Biomedical Laboratories, 5th

ed. U.S. Government

Printing Office, Washington, D.C. also found at the CDC internet site.

BAX®

System PCR Assay for Screening E. coli O157:H7 MP User Guide Supplement andAutomated Detection for Bacterial Screening User Guide, Dupont Qualicon.

RapidChek ® E. coli O157: (including H7) Test User Guide, Strategic Diagnostics Inc.

Transia™

Card E. coli O157 Manufacturer’s Insert, Diffchamb

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United States

Department of

Agriculture

Food Safety

and Inspection

Service

Office of

Public Health

Science



Athens, GA 30605

__________________________________________________________________________________________________________

Laboratory GuidebookNotice of Change


Title: Detection, Isolation and Identification of Escherichia coli O157:H7 from Meat

Products



FSIS validated the use of a replacement enrichment broth, modified Tryptone Soya or

Trypticase Soy Broth with novobiocin plus casaminoacids (mTSB+n) to replace

modified EC broth with novobiocin (mEC+n). Based on validation study results, the

time and temperature range of the incubation was modified. Additional changes to this

method include a revised limit of detection, instructions for preparing beef

trim/ground beef component subsamples and environmental sponge samples for

analysis, and an expanded description of how FSIS confirms and reports E. coli

O157:H7.



acknowledges that equivalent products may be available for laboratory use._________________________________________________________________________________QD-F-Micro-0004.03 Issuing Authority: Laboratory Quality Assurance Division (LQAD)Page 1 of 1 Effective: 5/29/07

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Food Safety And Inspection Service, Office of Public Health and Science


Title: Detection, Isolation and Identification of Escherichia coli O157:H7 from Meat Products

Revision: 04 Replaces: MLG 5.03 Effective: 1/28/08


Procedure Outline

5.1 Introduction

5.1.1 General

5.1.2 Limits of Detection5.2 Safety

5.3 Quality Control Practices

5.4 Equipment, Materials, Media, Reagents and Test Kits5.4.1 Equipment

5.4.2 Media, Reagents and Cultures

5.4.3 Test Kits

5.5 Detection Procedure5.6 Isolation Procedure

5.7 Identification and Confirmation

5.8 Storage of Cultures5.9 Selected References

5.1 Introduction

5.1.1 General

The following method is used for the analysis of raw and ready-to-eat meat products, and

laboratory environmental sponge samples for Escherichia coli O157:H7. The method is based on enrichment in a selective broth medium, application of a rapid screening test,immunomagnetic separation (IMS) in paramagnetic columns, and plating on a highly selective

medium.

Unless otherwise stated all measurements cited in this method have a tolerance of ± 2%.


This test method has been shown to detect 0.23 colony forming units (cfu)/g in a 25 g sample of

75/25 (lean/fat) ground beef.

5.2 Safety

E. coli O157:H7 is a human pathogen with a low infectious dose. (Ingestion of 100 cells cancause disease.) The use of gloves and eye protection is mandatory and all work surfaces must be

disinfected prior to and immediately after use. Laboratory personnel must abide by CDC

guidelines for manipulating Biosafety Class II pathogens. A Class II laminar flow biosafetycabinet is recommended for activities with potential for producing aerosols of pathogens. All

available Material Safety Data Sheets (MSDS) should be obtained from the manufacturer for

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the media, chemicals, reagents and microorganisms used in the analysis. The personnel who

will handle the materials should read all MSDS sheets.


a. Rainbow

Agar plates have a shelf life of 2 weeks.

b. All media and E-Buffer must be pre-warmed to 18-35ºC prior to use.

c. The fluorescent strain of E. coli O157:H7 is used in this procedure to monitor for cross

contamination. The protocol for the use of the fluorescent strain of E. coli O157:H7 as a positive control follows:

Wild-type strains of E. coli O157:H7 transformed with pGFP produce a green

fluorescent protein. As a result of this transformation, fluorescent strains of E. coli

O157:H7 possess the unique property of expressing bright green fluorescence visible inthe dark when illuminated by long-wave UV light. This property, which sets them apart

from typical E. coli O157:H7, makes them useful positive controls for analyses of meat

samples for E. coli O157:H7. At different steps in the procedure, both test samples and (fluorescent) positive controls can be tested for the bright green fluorescence as a

Quality Control measure to make sure that positive sample isolates actually came from

the test sample and not from accidental contamination by the positive control cultures.

Results of studies done at the FSIS Beltsville Microbial Pathogens Laboratory showed that these fluorescent cultures can be subjected to E. coli O157:H7 isolation and

identification procedures without losing their fluorescent properties. These strains retain

their fluorescent properties when grown in SOB media with added ampicillin (SOB +A). These cultures must be transferred every 7 days to fresh SOB + A media, according

to the protocol outlined below. The fluorescent colonies are ready to be used as positive

controls on day 3 of the following protocol, and for the next 6 consecutive days without

losing their fluorescent properties. If these cultures are not needed on a continuous basis, they can be stored at refrigeration temperatures on SOB + A agar plates in zip-

lock bags or sealed with parafilm for 1 month and then transferred, or started up again2 days before needed. Strict adherence to the protocol described below is essential, in

order to ensure that the fluorescent strains do not lose their ability to express green

fluorescence.

i. Test the fluorescent E. coli O157:H7 strain (FSIS culture # EC 465-97 or the

currently designated control strain) on SOB + A agar plate for fluorescence byilluminating colonies under long-wave UV light in the dark.

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ii. Select only fluorescent colonies and inoculate into 10 ml of SOB + A broth

in a tube. Incubate at 35 ± 2°C overnight.

iii. Streak the culture from the SOB + A broth onto a SOB + A agar plate.

Incubate at 35 ± 2°C overnight.

iv. Examine colonies on the plate for fluorescence. The fluorescent colonies areready to be inoculated into modified TSB broth + novobiocin (mTSB+n) at this

stage. These cultures on SOB + A agar plates can be stored refrigerated and be

used as positive controls for 6 more days. Incubate the inoculated mTSB+n

positive control culture at 42 ± 1°C overnight, along with the test samples.

v. Continue analysis per Sections 5.5-5.7 and test the Blood Agar Plates of the

fluorescent positive controls and any positive sample cultures for fluorescence.

5.4 Equipment, Materials, Media, Reagents and Test Kits

5.4.1 Equipment

a. Balance, sensitivity of 0.1 g

b. Stomacher ™ 400 or 3500 with appropriate sizes of sterile Stomacher ™ bags, with or without mesh. (Tekmar Co., Cincinnati, Ohio), or equivalent bag mixer and bags

c. Incubators, static 42 ± 1°C, 35 ± 2°C, and 22 ± 2°Cd. Micropipettors to deliver 15-1000 µl with sterile disposable filtered micropipet tips

e. Mechanical Pipettor with 1.0 ml, 5.0 ml, 10.0 ml sterile pipettes

f. Inoculating loops, “hockey sticks” or spreaders, and needlesg. UV light (long-wave, e.g. VWR # 36553-124, or equivalent)

h. Filter unit, 0.2 µm, nylon, sterilei. Infrared thermometer

j. LabQuake®

Agitator (or equivalent) with clips to hold microcentrifuge tubesk. Sterile disposable 12 x 75 mm polypropylene tubes (e.g. Fisher # 14-956-1B, or

equivalent)

l. Microcentrifuge and sterile 1.5 ml microcentrifuge tubes

m. Sterile 50 ml conical tubes (e.g. Falcon

# 2070, or equivalent) or sterile bottles

n. Sterile 40 µm Cell Strainer (Falcon

# 2340, or equivalent)

o. MACS

Large Cell Separation Columns (Miltenyi Biotec # 422-02, or equivalent)

p. OctoMACS

Separation Magnet (Miltenyi Biotec # 421-09, or equivalent)

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q. Multistand to support OctoMACS

Separation Magnet (Miltenyi Biotec # 423-03, or

equivalent)

r. Tray, autoclavable, approximately 130 mm x 83 mm (e.g. VWR # 62663-222, or

equivalent) for use with the OctoMACS

s. VITEK ®

system


a. Modified TSB broth with novobiocin plus casaminoacids (mTSB+n) Oxoid, or

equivalent

b. Rainbow® Agar O157 (Biolog Inc., Hayward California, 94545) containing 10 mg/L

novobiocin plus 0.8 mg/L potassium tellurite, or equivalent selective medium

c. Tryptic soy agar with 5% sheep blood d. SOB + A Medium

e. E Buffer, approximately 7 ml per sample (Buffered Peptone Water, Bovine Albumin

Sigma # A7906-500G, or equivalent and Tween-20

, or equivalent)

f. Disinfectant (Lysol

I. C., 2.0%, or equivalent)

g. Dynal

# 710.04 anti- E. coli O157 antibody-coated paramagnetic beads (Dynal Inc.,

Lake Success, NY 11042), or equivalent

h. E. coli O157:H7 strain 465-97 (positive control used throughout method)i. E. coli ATCC strain 25922 (negative control for bead capture and screen tests)

5.4.3 Test Kits

a. E. coli O157:H7 latex agglutination test kit (RIM® E. coli O157:H7 Latex Test Kit,

REMEL, 12076 Santa Fe Drive, Lenexa, KS 66215), or equivalent

b. Biochemical test kit and system, GNI and GNI Plus cards (VITEK

system, bioMerieuxVitek, Inc., 595 Anglum Drive, Hazelwood, MO 63042-2395), or equivalent

c. Shiga toxin test kit [Premier

EHEC, cat. # 608096 (Meridian Diagnostics, Inc., 3471

River Hills Dr., Cincinnati, OH, 45244), or equivalent

5.5 Detection Procedure

a. Sample Preparation

Note: Disinfect the intact sample package(s) surface prior to opening.

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i. Raw ground beef microbiological testing programs.

Randomly collect five 65 ± 2 g sub-samples (total of 325 ± 10 g) that are

representative of the entire sample. Place each 65 ± 2 g sub-sample in a sterile

Strainer Stomacher ™

bag. Add 585 ml mTSB+n broth and pummel for two

minutes in a Stomacher ™

.

ii. Cooked meat patties and semi-dry and dry fermented sausages. Randomly

prepare five 65 ± 2 g sub-samples (total of 325 ± 10 g) that are representative of the entire sample. When appropriate, sample representative portions from both

the outer surface (shell) and inner section (core) of RTE products, especially

semi-dry and dry fermented sausages. Place each 65 ± 2 g sub-sample in a sterile

Strainer Stomacher ™

bag. Add 585 ml mTSB+n broth and pummel for two

minutes in a Stomacher ™

.

iii. Outbreak-related samples. Randomly collect thirteen 25± 1 g sub-samples (total

of 325 ± 13 g) that are representative of the entire sample. When possible avoid

the exterior surfaces of non-intact samples. Place each 25 ± 1 g sub-sample in

a sterile Strainer Stomacher ™

bag and add 225 ml of mTSB+n broth. Pummel

for 2 minutes in a Stomacher ™

.

iv. Beef trim/trim components. Prepare a representative sample of the product bytaking approximately equal portions from a variety of locations. For non-

comminuted components, FSIS inspectors submit samples that generously

represent the surface of trimmings. For intact chunk beef or cuts, favor sampling of the surface to mimic submitted beef trim product and to provide

the best opportunity for detection.

v. Environmental sponge samples. Add 50 ± 5 ml of mTSB+n broth to each bagged sponge sample and stomach 2 minutes.

vi. Most Probable Numbers (MPN) Determination. Follow MPN instructions given

in the specific program protocol or see MLG Appendix 2, Most Probable Number Procedure and Tables.

b. Incubate all bags (static) with their contents for 15 to 22 h at 42 ± 1°C. Include a positive, negative, and uninoculated medium control for each group of samples tested.

Use the fluorescent E. coli O157:H7 strain (FSIS culture # EC 465-97) as a positive

control and E. coli ATCC strain 25922 as the negative control.

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c. From the enrichment cultures in the Stomacher ™

bags, perform the screening test for E.

coli O157:H7 following the manufacturer's instructions. The enrichment culture may be

analyzed immediately upon removal from the incubator without waiting for temperingto room temperature. To prevent clogging the pipette tip, be sure to collect the

appropriate size sample from the enrichment culture outside the inner strainer bag.

d. Samples negative by the screening test can be reported as negative for E. coli O157:H7

and discarded.

e. Samples positive by the screening test should be reported as potential positives. Begin

isolation procedures from the enrichment culture in the Stomacher ™ bag.

5.6 Isolation Procedure

Note: Steps a.-l. may be performed in a sequence that is convenient to the laboratory personnel.

a. Prepare E Buffer by mixing 0.5 g Bovine Albumin and 50 µl Tween-20

into 100 mlBuffered Peptone Water (BPW). Filter sterilize (0.2 µm) and store at 2-8

oC.

b. Remove Rainbow®

Agar plates from 2-8oC storage, allowing 3 plates for each

screen-positive culture and each control. Be sure that plates have no visible surface

moisture at the time of use. If necessary, dry plates (e.g. for up to 30 minutes in alaminar flow hood with the lids removed) prior to use. Dried plates that are not used

should be labeled "dried", placed in bags and returned to 2-8oC.

c. Remove a bottle of E Buffer from 2-8oC storage. Decant 7 ml of

E Buffer for each culture and each control into a sterile tube or bottle and allow it towarm to at least 18

oC. (Return the stock E Buffer to 2-8

oC.)

d. For each positive control, negative control and screen-positive culture to beanalyzed, order and label 50 ml conical centrifuge tubes so that the positive control is

first, followed by the negative control, then all cultures. Maintain this order for

subsequent steps.

e. For each positive control, negative control, and screen-positive culture, label two

sterile 1.5 ml microcentrifuge tubes (for step g and step s), one 50 ml conical

centrifuge tube (for step h.) and two 12 x 75 mm capped tubes (one for step p.). For each pair of 12 x 75 mm tubes, label one tube and add 0.9 ml E Buffer (for step q.).

f. Prepare the Dynal #710.04 E. coli O157:H7 immunomagnetic bead suspension

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by following Table 1 below. Be sure to include the positive and negative controls in

the total number of cultures. Use the bead suspension immediately (step g), or hold at

2-8oC. Return the stock vial of Dynal #710.04 E. coli O157:H7 immunomagnetic

beads to 2-8oC.

g. Vortex the bead solution briefly (2-3 seconds), then add 50 µl to a labeled

microcentrifuge tube (from step e), one for each control and screen-positive culture.Use immediately or hold these tubes at 2-8

oC.

h. Place a 40 µm Cell Strainer on a labeled 50-ml conical centrifuge tube (from step e.).

Pipet 5 ± 1 ml of each control and enrichment culture into the respective Cell Strainer and collect at least 1.0 ml of filtrate.

i. Do not proceed with more than the number of tubes that the OctoMacs®

magnet(s)

will hold. Transfer 1.0 ml of a filtrate (step h.) to the corresponding microcentrifugetube containing the immunomagnetic bead suspension (step g.) and place in the clips

of the LabQuake®

tube agitator. Rotate the tubes for 10-15 min at 18-30°C.

j. Attach the OctoMACS®

Magnet to the Multistand.

k. Position a tray on the base of the Multistand so that it will collect the filtrate

passing through the columns. Add approximately 300 ml of 2% Lysol I. C. (or

equivalent) disinfectant to cover the bottom of the tray.

l. Label and place the appropriate number of Large Cell Separation columns on the

OctoMACS®

Magnet. Insert columns from the front making sure the column tips donot touch any surfaces. Leave the plungers in the bags at this time to maintain

sterility.

m. Transfer at least 0.5ml E Buffer to the top of each column and let the buffer

run through.

n. Resuspend, then transfer each culture and control from step i. to its corresponding

column.

o. After a culture or control has drained through, wash the column by applying 1.0

ml of E Buffer to each column and allow to drain. Repeat 3 more times for a total of

4 washes.

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p. After the last wash has drained, remove the column from the OctoMACS®

Magnet

and insert the tip into an empty labeled 12 x 75 mm tube (from step e.). Apply 1.0 ml

of E Buffer to the column, and using the plunger supplied with the column,immediately flush out the beads into the tube. Use a smooth, steady motion to avoid

splattering. Cap the tubes. Repeat this for each column. If the OctoMACS®

magnetis to be used for a second set of cultures, it must be decontaminated as described in

step u, below. Repeat steps j.-s. for the additional cultures.

q. Vortex the tubes from step p. briefly to resuspend the beads. Make a 1:10 dilution

of each treated bead suspension by adding 0.1 ml of the bead suspension to a 12 x 75

mm labeled tube containing 0.9 ml E Buffer (from step e.).

r. Vortex briefly to maintain beads in suspension and plate 0.1 ml from each tube

(from step p. and step q.) onto a labeled Rainbow®

Agar plate. Use a hockey stick or

spreader to spread plate the beads, being careful not to spread the beads against the

edge of the plate.

s. Vortex the tubes containing undiluted beads (from step p.) and transfer to a labeled

microfuge tube (from step e.) and centrifuge at least one minute using a bench-top

microcentrifuge to concentrate the beads. Withdraw and discard the supernatantwithout disturbing the beads. Add 0.1 ml of E Buffer to the beads, resuspend the

beads and transfer the beads to a labeled Rainbow® Agar plate. Spread plate the beads as described in step r.

t. As soon as there is no visible moisture on the agar surface, invert plates and

incubate for 24-26 h at 35 ± 2°C.

u. Decontaminate the OctoMACS®

Magnet by applying 2% Lysol

I. C. (or equivalent)disinfectant directly to the surface. After approximately ten minutes, rinse with

deionized or tap water. Allow the unit to air-dry or use absorbent paper towels to dry

the unit.

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Table 1. # of Cultures ul of Beads* ul of E-Buffer # of Cultures ul of Beads* ul of E-Buffer

1 15 135 26 145 1305

2 20 180 27 150 1350

3 25 225 28 155 1395

4 30 270 29 160 1440

5 35 315 30 165 1485

6 40 360 31 175 15757 45 405 32 180 1620

8 50 450 33 185 1665

9 55 495 34 190 1710

10 60 540 35 195 1755

11 65 585 36 200 1800

12 70 630 37 205 1845

13 75 675 38 210 1890

14 80 720 39 215 1935

15 85 765 40 220 1980

16 90 810 41 230 2070

17 95 855 42 235 2115

18 100 900 43 240 2160

19 105 945 44 245 2205

20 110 990 45 250 2250

21 120 1080 46 255 2295

22 125 1125 47 260 2340

23 130 1170 48 265 2385

24 135 1215 49 270 2430

25 140 1260 50 275 2475

* Dynal® anti- E. coli O157:H7 antibody-coated paramagnetic beads (vortex briefly before use)

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5.7 Identification and Confirmation

a. After incubation, E. coli O157:H7 colonies have black or gray coloration on

Rainbow®

Agar. When E. coli O157:H7 colonies are surrounded by pink or magenta

colonies, they may have a bluish hue. Mark colonies typical of E. coli O157:H7 and

perform latex agglutination assays for O157, following manufacturer’s instructions.Streak all latex positive colonies, up to a total of five from each sample (one per sub-

sample, if possible) onto Blood Agar plates. Incubate Blood Agar plates for 16-24 h

at 35 ± 2oC.

Note: If no typical colonies are present, hold the original Rainbow® plates at 20-24oC

for an additional 6-24 h then re-examine for typical colonies.

b. After incubation, examine the Blood Agar plates for purity under visible light, and

evidence of cross contamination with the positive control by using long wave UV light.

Only the positive control culture, E. coli O157:H7 strain 465-97, should fluoresce. If the

Blood Agar plates appear pure and uncontaminated, perform the following confirmatorytests:

i. Biochemical confirmation.

Inoculate VITEK-GNI or GNI Plus cards (if using VITEK

system), or an

equivalent biochemical identification testing system. The cytochrome oxidaseand gram stain tests are optional.

ii. O157 and H7 confirmation.To confirm the absence or presence of O157 and H7 antigens, use an E. coli

O157:H7 latex test agglutination kit (RIM

E. coli O157:H7 Latex Test Kit, or

equivalent). Use growth from the Blood Agar plate (from step b). For

inconclusive results, genetic testing (e.g. PCR) may be necessary.

iii. Shiga toxin/toxin genes confirmation.

The presence of Shiga toxin(s) in a culture isolate is confirmed by the use of a

toxin assay, e.g., Meridian Premier EHEC Kit, or equivalent. When Shiga

toxin(s) is (are) not demonstrated, detection of one or more toxin genes by PCR

is used for confirmation. NOTE: The positive control culture, E. coli O157:H7(FSIS culture # EC 465-97), is toxin-negative.

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c. FSIS uses the following definitions for reporting E. coli O157:H7:

• Potential Positive - a sample that causes a positive reaction with the screen test.

• Presumptive Positive - a sample that has typical colonies, observed on RainbowAgar, and reacts specifically with O157 antiserum.

• Confirmed Positive – a biochemically identified Escherichia coli isolate that isserologically or genetically determined to be “O157” that meets at least one of the

following criteria:1) Positive for Shiga toxin (ST) production

2) Positive for Shiga toxin gene(s) (stx)

3) Genetically determined to be “H7”

If an FSIS laboratory’s confirmatory test results are insufficient to allow

identification, then the isolate is transferred to the Outbreaks Section of the Eastern

Laboratory Microbiology Branch (OSEL), or current FSIS reference laboratory, for further testing prior to reporting.

For example, if a presumptive positive isolate is tested in a FSIS laboratory for Shigatoxin and it is Shiga toxin negative, then the isolate is transferred to OSEL for further

testing.

A Shiga toxin gene PCR test is performed in OSEL. If the isolate fails to express

Shiga toxin (toxin negative result), but has the genes necessary to express Shigatoxin, then it is considered by the agency to be E. coli O157:H7 confirmed positive.

If the isolate was H7 negative and found to be Shiga toxin and gene negative (byEHEC test and genetic test for Shiga toxin genes) additional PCR test(s) for H7

gene(s) are performed. If the H7 PCR test is positive, the isolate is considered E. coli

O157:H7 confirmed positive.

If the isolate is E. coli O157 presumptive positive, but additional tests show it to be

H7 negative (by latex agglutination and PCR) and Shiga toxin negative (by EHEC

test and genetic test for Shiga toxin genes), then the isolate would be reported as E.

coli O157:H7 negative.

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5.8 Storage of Cultures

For storage requirements of the fluorescent E. coli O157:H7 strain (FSIS culture # EC 465-97 or

the currently designated control strain), refer to Section 5.3.c. of this chapter.

Store other "working" E. coli stock cultures on nutrient agar slants. Transfer stocks monthly

onto duplicate nutrient agar slants, incubate overnight at 35 ± 2°C, and then store them at 2-8°C.

Use one of the slants as the working culture. Use the other slant for sub-culturing to reduce the

opportunity for contamination. Cultures may be subcultured up to 5 times. After this period theculture must be re-confirmed biochemically or a new culture initiated.

For long term storage freeze cultures using cryo-beads i.e. Cryostor™ or lyophilize.


Deng, M.Y., P.M. Fratamico, M.A. Jensen, and G. Tice. 1997. DNA amplification usingtableted PCR reagents for identification of Escherichia coli O157:H7 isolated from foods.

Journal of Rapid Methods and Automation in Microbiology. 5:61-74.

Ewing, W. H. 1986. Edwards and Ewing's Identification of Enterobacteriaceae, 4th Edition.

Elsevier Science Publishing Co., Inc., New York.

Feng P., S. D. Weagant, and M. A. Grant. FDA Bacteriological Analytical Manual Online,

Chapter 4: Enumeration of Escherichia coli and the Coliform Bacteria.

Fratamico, P. M., M. Y. Deng, T. P. Strobaugh, and S. A. Palumbo. 1997. Construction and

characterization of Escherichia coli O157:H7 strains expressing firefly luciferase and greenfluorescent protein and their use in survival studies. J. Food Prot. 60:1167-1173.

Harrison, B. and D. Warburton. 1997. Identification of Escherichia coli verotoxins by the

Meridian Premier EHEC kit®

. Laboratory procedure MFLP-93 In The Compendium of Analytical Methods, Vol. 3. Health Protection Branch, Health Canada, Ottawa, Canada.

Hitchins, A. D., P. A. Hartman, and E. C. D. Todd. 1992. Coliforms- Escherichia coli and itstoxins, p. 325-369. In C. Vanderzant and D. F. Splittstoesser (ed.), Compendium of Methods

for the Microbiological Examination of Foods. 3rd Edition. Amer. Publ. Hlth. Assoc.,

Washington, D.C. 20005.

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Park, C. H., K. M. Gates, N. M. Vandl, and D. L. Hixon. 1996. Isolation of Shiga-like toxin

producing Escherichia coli (O157 and non-O157) in a community hospital. Diagn.

Microbiol. Infect. Dis. 26:69-72.

Richmond, J.Y. and R.W. McKinney (ed.). 2007. Biosafety in Microbiological and

Biomedical Laboratories, 5th

ed. U.S. Government Printing Office, Washington, D.C.

Taormina, P. J., M. Rocelle, S. Clavero, and L. R. Beuchat. 1998. Comparison of selective

agar media and enrichment broths for recovering heat-stressed Escherichia coli O157:H7

from ground beef. Food Microbiol. 15:631-638.

Weagant, S. D., J. L. Bryant, and K. G. Jinneman. 1995. An improved rapid technique for

isolation of Escherichia coli from foods. J. Food Prot. 58:7-12.

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6-1

CHAPTER 6. ISOLATION, IDENTIFICATION, AND ENUMERATION OF CAMPYLOBACTER JEJUNI / COLI FROM MEAT AND POULTRYPRODUCTS

Gerri M. Ransom and Bonnie E. Rose

6.1 Introduction

Procedures for the recovery of Campy l obac t er spp. from foods areevolving and no single method can be recommended for testing a widevariety of foods. Isolation of Campy l obac t er j ej un i and Campy l obac t er col i is achieved both with and without selective brothenrichment. The procedures outlined below are among the most promising for the isolation and enumeration of these bacteria from raw/cooked meat and poultry products.

Campylobacters are sensitive to freezing and die off at room

temperature. Samples intended for Campy l obac t er examination should be transported and held at 4

oC. Sample analysis should begin as

soon as possible since campylobacters can be overgrown bycontaminating psychrotrophic bacteria. If freezing of samplescannot be avoided, cryoprotective agents should be used. Stern and Kotula, 1982, reported improved recovery of C. j ej un i from ground beef stored frozen in 10% dimethyl sulfoxide or glycerol.Blankenship et al., 1983, found that brucella broth supplemented with 10% polyvinyl pyrrolidine was suitable for transporting frozenswab samples (from freshly processed poultry carcasses) to a centrallaboratory for analysis.

Campylobacters are microaerophilic and certain environmentalstresses such as exposure to air, drying, low pH, and prolonged storage can be detrimental to their survival. Use of oxygen-quenching agents, a microaerobic atmosphere, and antibiotics thatsuppress competitors, significantly improve Campy l obac t er recovery.

6.2 Equipment, Reagents, and Media

6.21 Equipment

a. Phase-contrast microscope with 100X oil immersionobjective

b. Agitating incubator(s)/water bath(s) at 37 ± 1.0°°C

and 42 ± 1.0o

Cc. 42 ± 1.0

oC incubator (static)

d. Balance, sensitivity of 0.1 ge. Quart-size Qwik Seal® bags (Reynolds Metals Co.,

Richmond, VA; # RS78)f. Anaerobic jars (vented or non-vented)

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6-2

g. CampyPak Plus™™ (BBL 71045)or

Gas Generating Kits for Campy l obac t er (Oxoid BR56 for3.0-3.5 liter jars, or BR60 for 2.5-3.0 liter jars)

h. Vacuum pump and gauge with appropriate tubing and

connectors for evacuation of vented anaerobic jarsi. Gas cylinder containing a mixture of 5% O2, 10% CO2, and

85% N2 with appropriate tubing and connectors for gassingvented anaerobic jars and Qwik Seal® bags

j. Regulator for gas cylinder compatible with Compressed GasAssociation (CGA) connection on cylinder

k. Filter paper (for glycerol humectant and oxidase test)l. Petri dishes (100 x 15 mm disposable) m. Platinum or sterile plastic inoculating loops and needlesn. Microscope slides, cover slips, and immersion oil

o. 0.2 µµ m sterile membrane filters p. 16 x 150 mm and 16 x 125 mm screw-cap test tubes

q. 250-ml screw-cap bottlesr. Sterile swabs or bent glass rods ("hockey sticks")s. Sterile forceps and scissorst. Sterile pipettesu. Large sterile plastic bags

v. Stomacher™™ 400, and Stamacher™™ 400 bags w. Centrifuge, rotor, and 250-ml sterile centrifuge bottlesx. Sterile cheesecloth-lined funnels

6.22 Reagents

a. Glycerolb. 3% Hydrogen peroxide solution

c. Cephalothin antibiotic susceptibility discs (30 µµg)

d. Nalidixic acid antibiotic susceptibility discs (30 µµg)e. Oxidase reagent (1% Tetramethyl-p-phenylenediamine

dihydrochloride solution)f. Campy l obac t er latex test kit (optional

presumptive identification)

6.23 Media

a. Hunt Enrichment Broth (HEB)b. 0.1% peptone waterc. Modified Campy l obac t er Charcoal Differential

Agar (MCCDA)d. Brucella-FBP (BFBP) Brothe. Semisolid Brucella Glucose Medium f. Brucella-FBP (BFBP) Agarg. Enriched Semisolid Brucella Medium (optional)

6.3 Isolation and Enumeration

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6-3

a. Place 25 g meat or swab samples into 100 ml of HEB in aReynolds quart-size Qwik Seal® bag. Place the Qwik Seal®

bag inside a Stomacher™™ 400 bag for reinforcement and stomach for 2 minutes. Flatten the Qwik Seal® bag

against the lab bench edge to remove as much air as possible without spilling the contents, then seal the bag, leaving a 1/2 inch opening at one end. Asepticallyinsert the tip-end of a sterile 10 ml pipette (orequivalent) into the bag through this opening. Be surethat the mouth-end of the pipette contains a sterilecotton filter. Connect the mouth-end of the pipette tothe microaerobic Campy gas mixture (5% O2, 10% CO2, and 85% N2) with sterile rubber tubing equipped with a sterilefilter (a sterile filter can be made out of anautoclaved, shortened 25 ml volumetric pipette stuffed with glass wool). Slowly inflate the bag to capacity with the Campy gas mixture and continue to fill untilexcess gas flows from the bag. Then allow a small amountof gas to escape to provide for expansion, beforesecuring the remainder of the seal. Proceed to step d.

b. Place a raw whole chicken carcass or meat pieces (up to 3

lb) in a large sterile plastic bag such as a Stomacher™™3500 bag, and add 200 ml 0.1% peptone water. Twist bagto seal and shake contents for 2 minutes. Tilt the bagand hold back the meat pieces, allowing the rinse liquid to flow to one corner. Sanitize bag corner with 1000 ppm hypochlorite solution or 70% ethanol, then rinse insterile distilled water. Aseptically cut the corner of

the bag and pour the rinse through a sterile cheesecloth-lined funnel into a sterile 250 ml centrifuge bottle.Centrifuge at 16,000 x g for 15 minutes. Discard thesupernatant and suspend the pellet in 10 ml 0.1% peptone water. For detection, inoculate 1 ml of rinseconcentrate into 100 ml HEB in a Qwik Seal® bag. Thenfollow gassing steps as outlined, beginning with thethird sentence of step a. above.

c. If enumeration is desired, prepare a three tube MPN

series using HEB. Choose test dilutions and HEB volumes based on the expected numbers of campylobacters in the meat species being tested. For example, for poultry

rinse samples (prior to centrifuging) begin by addingthree 10 ml portions of the rinse to three 90 ml bottlesof HEB. (Alternatively, Qwik Seal® bags may be used here[see step a. above]). Then add 1 ml portions of therinse to each of three 9 ml tubes of HEB. Prepare serialdilutions of the rinse in 0.1% peptone water. Preparesubsequent MPN tubes by transferring 1 ml portions of the

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6-4

decimal dilutions into 9 ml tubes of HEB in triplicate.Place all bottles and tubes in anaerobic jars. See stepg. for jar gassing methods. Follow incubation steps beginning with step d. below. Use tubes or bottles found to contain confirmed Campy l obac t er to calculate MPN

(refer to appropriate tables).

d. Incubate gassed Qwik Seal® bags or anaerobic jarscontaining bottles or tubes at 37 ± 1.0

oC, shaking at 100

rpm for 4 h.

e. After the 4 h incubation at 37 ± 1.0oC, aseptically add

additional sterile cefoperazone solution to bring thefinal concentration in each enrichment vessel to 30 mg/L.Reestablish the microaerobic atmosphere and increase the

temperature to 42 ± 1.0oC. Continue the incubation for 20

h shaking at 100 rpm.

f. Swab/streak enrichments directly and at a 1:100dilution onto MCCDA plates (for cooked products, a 1:50dilution may be plated). Prepare the dilution byswirling a swab in the broth and twisting it against theside of the vessel to remove excess liquid. Break offthe swab tip into a tube containing 9.9 ml of 0.1% peptone water and vortex. Inoculate the plates by placing a swab into the enrichment or dilution and removing excess liquid as above. Swab approximately 40%of the MCCDA plate, then streak from the swabbed area toyield isolated colonies. Alternatively, 0.1 ml portionsof the enrichments or dilutions may be plated byspreading with a sterile bent glass rod. This platingtechnique may be used provided isolated colonies result.

g. Incubate the MCCDA plates at 42 ± 1.0oC for 24 h in an

anaerobic jar under microaerobic conditions. Add about 4drops of a humectant such as glycerol to a filter paperand place it in the jar to diminish typical confluent and swarming growth of Campy l obac t er . If no growth isachieved after 24 h, reincubate the plates for anadditional 24 to 48 h to attempt recovery. The microaerobic conditions can be achieved in the jar byeither of the following methods:

i. Evacuate the air from a vented anaerobic jar to a partial vacuum of 20 inches of Hg and fill the jar with a gas mixture of 5% O2, 10% CO2, and 85% N2.Repeat the evacuation-replacement procedure a totalof three times to assure proper atmosphericconditions.

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ii. CampyPak Plus™™ (BBL) or Gas Generating Kits forCampy l obac t er (Oxoid). Follow the manufacturer'sinstructions on use and disposal of the kit materials. Keep jars away from flames when opening.

NOTE: Gas generator envelopes should be used if non-vented anaerobic jars are the only type available.Evacuation-replacement gassing of vented anaerobic jars isvery economical.

To facilitate lid removal from a vented anaerobic jar, firstrelease pressure by opening clamped tubing on port or bydepressing the valve stem.

6.4 Identification of Campy l obac t er Campy l obac t er colonies on MCCDA are smooth, shiny, and convex with adefined edge, or flat, transparent or translucent, and spreading with an irregular edge; colorless to grayish or light cream; and usually 1 to 2 mm in diameter but may be pinpoint to several mm indiameter. Plates of Campy l obac t er colonies may be stored up to48 h refrigerated under microaerobic conditions if isolates cannot be picked immediately.

Use a platinum or plastic needle to pick three suspect Campy l obac t er colonies for each sample from the MCCDA plates and transfer each to10 ml of brucella-FBP (BFBP) broth. Since campylobacters can varygreatly in colonial morphology, it is advisable to similarly cultureat least one or all colony types present on the plates to assure thetarget is not overlooked. Alternatively, direct screening of

colonies by phase-contrast microscopy can be done prior to pickingisolates. To culture isolates, incubate the BFBP tubes with capsloosened for 24 to 48 h at 42 ± 1.0

oC in an atmosphere of 5% O2,

10% CO2, and 85% N2. Do not vortex culture tubes of Campy l obac t er ,this will introduce oxygen into the media.

Perform the following identification tests on each BFBP brothculture:

a. Examine a wet-mount preparation of the BFBP brothculture with a phase-contrast microscope using a 100X oilimmersion objective. Young cells of Campy l obac t er appear

as narrow curved rods (0.2 to 0.8 µµ m wide by 1.5 to 5 µµ m

long). The organisms show rapid movement with darting orcorkscrew-like motility. Pairs of cells can resemble thesilhouette of a gull's wing span or the letter S. Longerchains can appear helically curved, and multispiralled filamentous elongated forms may exist. Cells grown for more than 72 h may become non-culturable and coccoid.Campylobacters are Gram negative, but Gram staining may

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be omitted since cell morphology and motility are moresignificant in the identification of these organisms.(Carbol fuchsin [0.5%] is used instead of safranin as acounter stain to improve Gram stain results.) Continueconfirmation of those BFBP cultures that exhibit typical

Campy l obac t er morphology.

b. Inoculate the top 10 mm layer of a tube of semisolid brucella glucose medium with several drops of the aboveBFBP broth culture. Incubate tubes with caps loosened inan anaerobic jar under microaerobic conditions at 42 ±1.0

oC for 1 to 3 days.

i. Glucose fermentation test: Campylobacters arenonfermentative, so the color of the medium willremain red-orange. A positive reaction shows ayellow color (acid with phenol red indicator) in thesemisolid brucella glucose medium.

ii. Catalase test: After reading the results of theglucose fermentation test, add 1 ml of 3% hydrogen peroxide to the semisolid brucella glucose medium culture, let sit for two to three minutes, thengently invert the tube to distribute the reagent.Examine after 1 to 10 minutes for formation of bubbles, indicating a positive reaction. C. j ej un i and C. col i are catalase positive.

c. Add about six drops of the BFBP broth culture to a BFBPagar plate, and spread the inoculum over the surface witha sterile swab or a bent glass rod. Aseptically place a

disc of nalidixic acid (30 µµg) and a disc of cephalothin

(30 µµg) on each plate. Press each disc with sterileforceps to adhere it to the agar surface. Incubate the plates in an anaerobic jar at 42 ± 1.0

oC for 1 to 3 days

in a microaerobic atmosphere.

i. Susceptibility to nalidixic acid and cephalothin:Observe the growth patterns surrounding theantibiotic impregnated discs. C. j ej un i and C. col i are sensitive to nalidixic acid, and a clear zone ofinhibition will exist around the disc. A zone ofany size indicates sensitivity. The organisms are

both resistant to cephalothin, so growth will be present right up to the disc. Lawns ofCampy l obac t er growth may be very light and can bedifficult to see, so it is helpful to tilt the plateat an angle under a light for viewing.

ii. Oxidase test: Place a 2 cm square piece of filter

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paper in an empty petri dish and add 1 to 2 drops ofoxidase reagent to the paper. Heavily smear cellsfrom the above BFBP agar plate onto thereagent-impregnated paper in a spot 3 to 5 mm indiameter using a platinum or plastic loop. The test

is positive if the cell mass turns dark purple within 30 seconds. Alternatively, the Difco

DrySlide™™ oxidase test may be used. Campylobactersare oxidase positive.

d. Optional tests

Other biochemical tests useful for differentiation ofcatalase-positive campylobacters include nitrate and nitrite reduction, H2S production, growth in 1% glycine,growth in 3.5% NaCl, and growth at 25, 30.5, 37, and 42

oC.

C. j ej un i / col i grow well at 42oC and are curved or

S-shaped with darting, corkscrew-like motility.Biochemically, they are catalase positive, oxidase positive, nonfermentative, nalidixic acid sensitive, and cephalothin resistant. Distinguishing between C. j ej un i and C. col i is usually not necessary in a food microbiology laboratory since both are causes of humancampylobacteriosis. The few existing tests to separatethese species are not dependable. Hippurate hydrolysisappears to be the most reliable and useful test for this purpose. A convenient rapid disk method is available(Cacho et al., 1989). C. j ej un i is positive for thistest, while C. col i yields a negative reaction.

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6.5 Multiple Start Days

Analysis should begin on a Monday, Tuesday, Wednesday, or Thursdayto avoid weekend work. Samples received on a Friday should beanalyzed immediately or begun on Saturday; starting either day will

require weekend work. Follow the table below according to the dayanalysis is to begin.

Analysis To Be Done On Days

StartingDate

Enrichment Plating PickColonies

InoculateBiochemicals

Read/Perform Tests

MON MON TUE WED THU FRI

TUE TUE WED THU FRI MON

WED WED THU FRI MON WED

THU THU FRI MON TUE THUFRI FRI SAT MON TUE THU

SAT SAT SUN MON TUE THU

6.6 Storage and Transport of Stock Cultures

Inoculate overnight BFBP broth cultures into tubes of Brucella broth with 0.15% agar. Loosen the screw-caps and incubate for 24 to 48 hat 42 ± 1.0

oC in an atmosphere of 5% O2, 10% CO2, and 85% N2. Store

refrigerated under this atmosphere for up to a month without serial passage. Cultures in this medium can be transported by mail. Seal

tightened caps with adhesive tape to prevent leakage duringshipment.

Cultures grown in enriched semisolid brucella medium may be stored under atmospheric conditions at room temperature with capstightened, for at least three weeks. This medium is also suitablefor transporting cultures by mail.

Cultures may also be preserved frozen. To prepare these stocks,swab 6 drops of a 24 h BFBP broth culture onto a BFBP agar plate and incubate microaerobically at 42 ± 1.0

oC for 24 to 48 h. Then remove

the plate growth with a swab and suspend the cells in 4 ml ofBrucella broth with 15% sterile glycerol. The suspension can bestored frozen at -70oC in 1 ml portions for 6 months or longer.Thawing and refreezing these stocks will usually result in loss ofviability.

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6.7 Media Quality Control

Pay strict attention when preparing all media to assure propersupplement additions. Ingredients, reagents, and media thatare past expiration date should be discarded. It is important

to discard all unused liquid media more than one month old and all plating media more than two weeks old, since absorbed oxygen willgenerate peroxides which can be detrimental to campylobacters.Store all media refrigerated, tightly sealed, and shielded from light.

Inoculated media controls should be incubated with each batch oftests to assure proper media formulation and atmospheric conditions.When enriching, include a Qwik Seal® bag of HEB inoculated with an

actively growing BFBP broth culture of C. j ej un i as a control.Similarly, in each anaerobic jar, include an appropriate agar plateor broth inoculated with a known C. j ej un i strain. Use of positiveand negative controls for all biochemical tests is also recommended.

An uninoculated control of all test media should also be included to allow assessment of sterility and any changes that may occur inthe medium.

Listed below are some recommended controls for the Campy l obac t er biochemical tests:

a. Glucose fermentation test:Inoculate a semisolid brucella glucose tube with anEscher i ch i a co l i strain and incubate aerobically togenerate a positive reaction. Inoculate a C. j ej un i strain and incubate microaerobically to yield anegative reaction.

b. Catalase test:Use a C. j ej un i strain as a positive control and aSt r ept ococ cus spp. as a negative control.

c. Susceptibility to nalidixic acid and cephalothin:Use a C. j ej uni strain to demonstrate the desired sensitive/resistant pattern.

d. Oxidase Test:Use a C. j ej un i strain as a positive control and anE. col i strain as a negative control.

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Blankenship, L. C., S. E. Craven, J. Y. Chiu, and G. W. Krumm.1983. Sampling methods and frozen storage of samples fordetection of Campy l obac t er j ej un i on freshly processed broiler

carcasses. J. Food Prot. 46: 510-513.

Cacho, J. B., P. M. Aguirre, A. Hernanz, and A. C. Velasco.1989. Evaluation of a disk method for detection of hippuratehydrolysis by Campy l obac t er spp. J. Clin. Microbiol. 27:359-360.

Holdeman, L. V., E. P. Cato, and W. E. C. Moore. 1977.Campy l obac t er , p.114-115. I n Anaerobe Laboratory Manual, 4thEdition. Virginia Polytechnic Institute and State University,Blacksburg, Va.

Hunt, J. M. 1992. Campy l obac t er , p. 77-94. I n FDA

Bacteriological Analytical Manual, 7th Edition. Association ofOfficial Analytical Chemists International, Inc., Gaithersburg, MD 20877.

Hutchinson, D. N., and F. J. Bolton. 1984. Improved blood freeselective medium for the isolation of Campy l obac t er j ej un i from faecal specimens. J. Clin. Pathol. 37: 956-957.

Smibert, R. M. 1984. Campy l obac t er , p. 111-118. I n N. R.Krieg and J. G. Holt (ed.), Bergey's Manual of SystematicBacteriology, vol. 1. Williams & Wilkins, Baltimore, MD.

Stern, N. J., C. M. Patton, M. P. Doyle, C. E. Park, and B. A. McCardell. 1992. Campy l obac t er , p. 475-495. I n C. Vanderzantand D. F. Splittstoesser (ed.), Compendium of Methods for the Microbiological Examination of Foods, 3rd Edition. Amer. Publ.Hlth. Assoc., Washington, D.C.

Stern, N. J., and S. U. Kazmi. 1989. Campy l obac t er j ej uni , p.71-110. I n M. P. Doyle (ed.), Foodborne Bacterial Pathogens. Marcel Dekker, Inc., New York.

Stern, N. J., and A. W. Kotula. 1982. Survival ofCampy l obac t er j ej un i inoculated into ground beef. Appl.Environ. Microbiol. 44:1150-1153.

Wang, W. L. L., N. W. Luechtefeld, L. B. Reller, and M. J.Blaser. 1980. Enriched Br uc el l a medium for storage and transport of cultures of Campy l obac t er f et us subsp. j ej uni . J.Clin. Microbiol. 12:479-480.

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CHAPTER 7. ISOLATION AND IDENTIFICATION OF AEROMONAS SPECIESFROM MEAT AND POULTRY PRODUCTS

Bonnie E. Rose and Anita J. G. Okrend

7.1 Introduction

Members of the genus Aer omonas typically are aquatic bacteria and sometime pathogens of fish and cold-blooded vertebrates thatinhabit wet environments. Nevertheless, aeromonads are isolated (often in considerable numbers) from various foods of animalorigin. These include seafood, raw milk, beef, pork, lamb, and poultry. They grow readily at refrigeration temperatures.Production of enterotoxins can be demonstrated using variouslaboratory assays, and indirect epidemiological evidence suggests

that members of the genus Aer omonas have been involved in sporadichuman gastroenteritis outbreaks involving seafood. However, nofully confirmed foodborne outbreak has been described in thescientific literature.

The method presented describes procedures for isolation and identification of species of the Aer omonas hy dr ophi l a group whichconsists of A. hy dr ophi l a , A. sob r i a and A. ca v i ae . A procedurefor detection of hemolysin(s) is also provided. Burke et al.,1983, reported a 97% correlation between hemolysin production and enterotoxin production among Aer omonas species.

7.2 Equipment, Reagents and Media

7.21 Equipment

(isolation/identification)

a. Incubator, static 28 ± 1oCb. Osterizer-type blender with sterilized cutting

assemblies and adapters for use with Mason jars, or

Stomacher™™ (Tekmar) with sterile Stomacher™™ bagsc. Sterile bent glass rods ("hockey sticks")

(hemolysin test)

d. Incubator, static 37oCe. Microtiter plate reader equipped to read at 540 nm f. Centrifuge capable of 12,000 RPM

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7-2

g. Shaker incubator (30oC; 210 RPM)h. Screw-cap Erlenmeyer flasks, 125 mli. Sterile screw-cap centrifuge tubes: 15 ml conical and

50 ml round bottom j. 96-well microtiter plates

k. Membrane filters, 0.2 µm l. Bench top clinical centrifuge

7.22 Reagents


a. Butterfield's phosphate diluent (BPD) b. Mineral oil, sterilec. N,N-dimethyl-p-phenylenediamine monohydrochloride

(1% aqueous solution)

(hemolysin test)

d. Rabbit blood, defibrinated e. Phosphate buffered saline (PBS)f. Distilled water, sterile

7.23 Media


a. Tryptic soy broth plus 10 µg/ml ampicillin (TSBA) b. Starch-ampicillin (SA) agarc. Triple sugar iron (TSI) agard. Nutrient agare. Mannitol fermentation broth with Andrade's indicatorf. Arginine decarboxylase broth (Moeller)g. Ornithine decarboxylase broth (Moeller)h. Decarboxylase broth base (Moeller)i. Glucose fermentation broth with Andrade's indicatorj. Bile esculin agar

(hemolysin test)

k. Brain heart infusion (BHI) broth

7.3 Isolation Procedure

Serial dilutions of meat samples may be surface-spread directly onSA agar. However better recovery of Aer omonas will be achieved by

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using enrichment procedures, particularly when the aeromonads have been freeze-injured or are low in number.

a. Blend 25 g of meat in 225 ml TSBA with a blender or

Stomacher™™ for 2 minutes. Incubate at 28oC for 18 to

24 h.

b. After incubation prepare serial dilutions of theenrichment cultures in BPD. Transfer 0.1 ml of the10-4 to 10-6 dilutions onto the surface of SA plates.Evenly spread the inoculum with sterile bent glass rods.The plates must be free of surface moisture if singlecolonies are to be obtained. Incubate the plates at28oC for 18 to 24 h.

c. Pick three typical colonies per sample from the SA agar

plates to TSI agar and nutrient agar slants. Incubateovernight at 28oC. Aer omonas colonies are typically 3to 5 mm in diameter and appear yellow to honey-colored on SA agar.

7.4 Identification

a. Read the TSI reactions. Aer omonas reactions on TSI areas follows: acid butt, acid or alkaline slant, H2Snegative, positive or negative gas production.

b. Perform the oxidase test on the nutrient agar slants. Add a few drops of a N,N-dimethyl-p-phenylenediamine monohydrochloride solution (prepared fresh daily) to thegrowth on the nutrient agar slant. Oxidase positivecultures develop a pink color which successively becomes maroon, dark red, and black in 10 to 30 min. Allaeromonads are oxidase-positive and fermentative.

c. Transfer all oxidase-positive fermenters from the TSIagar slants to the following media for biochemicalconfirmation: mannitol fermentation broth, argininedecarboxylase broth, ornithine decarboxylase broth,glucose fermentation broth, and bile esculin agar. Afterinoculation, layer the decarboxylase media with sterile mineral oil and incubate at 28oC for 48 h. Incubate theremainder of the confirmation media at 28oC for 24 h.

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d. Record the biochemical characteristics of each isolate. All aeromonads produce acid from mannitol and arearginine positive, ornithine negative. Species of theA. hy dr ophi l a group can be differentiated according tothe biochemical characteristics shown below:

Test (Substrate) A. hy dr o ph i l a A. s ob r i a A. c av i a e

Gas from Glucose + + -

Esculin hydrolysis + - +

NOTE: Esculin hydrolysis imparts a dark brown color to themedium.

e. Transfer isolates of suspected Aer omonas that are to be

tested for hemolysin production from TSI agar tonutrient agar slants and incubate overnight at 28oC.

7.5 Hemolysin Test

The hemolysin test described below is based on that of Burke etal., 1983 and 1984.

7.51 Preparation of Culture Filtrate

a. Transfer growth from the nutrient agar slant to BHI

broth (25 ml broth in a 125 ml Erlenmeyer flask).Incubate overnight at 30oC on a shaker incubator at 210RPM.

b. Centrifuge the broth culture at 11,950 RPM (SS-34Dupont-Sorvall rotor) for 30 minutes. Decant and savethe supernatant liquid; discard the cell pellet.

c. Filter sterilize the supernatant through a steriledisposable membrane filter (0.2 µm).

d. Hold the sterile culture filtrate at 4oC until needed,and test it for hemolysin activity within 24 h of preparation.

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7.52 Preparation of Rabbit Erythrocyte Suspensions

a. Centrifuge 10 ml of defibrinated rabbit blood in a 15-mlconical centrifuge tube at 2400 RPM in a bench topclinical centrifuge for 5 minutes.

b. Remove the supernatant and white blood cell layer bysuction and discard.

c. Add 10 ml of cold PBS to the packed erythrocytes, mixgently, and centrifuge as described above. Discard supernatant.

d. Wash the erythrocytes in PBS two more times, asdescribed above.

e. After the final wash, note the volume of packed erythrocytes in the centrifuge tube. Prepare a 10% and a 1% erythrocyte suspension in PBS. Hold the twosuspensions at 4oC until needed (use within 24 h).

7.53 Preparation of Hemoglobin Standard Curve

a. Transfer 1 ml of the 10% erythrocyte suspension into 8 ml of sterile distilled water. Shake the mixture untilall cells are lysed. Add 1 ml of 10X PBS to obtain a 1%hemoglobin solution.

b. Add 1% hemoglobin solution and 1% erythrocyte suspensionto conical centrifuge tubes in the following volumes:

Volume(ml)

% hemoglobin

0 10 20 30 40 50 60 70 80 90 100

Hemoglo-bin

0 .1 .2 .3 .4 .5 .6 .7 .8 .9 1.0

Erythro-cytes

1.0 .9 .8 .7 .6 .5 .4 .3 .2 .1 0

c. Centrifuge tubes at 2400 RPM for 5 minutes in a clinicalcentrifuge. Transfer 0.5 ml of supernatant from eachtube into wells of a 96-well microtiter plate. Hold the plate for the hemolysin test (Section 7.54).

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7.54 Hemolysin Test

a. Add 1 ml of sterile culture filtrate (Section 7.51) to 1 ml of the 1% erythrocyte suspension (Section 7.52) in aconical centrifuge tube and mix gently.

b. Incubate at 37oC for 1 h, then incubate for anadditional 1 h at 4-5oC.

c. Centrifuge at 2400 RPM for five minutes.

d. Transfer 0.5 ml of supernatant to the 96-well platecontaining the standards (Section 7.53).

e. Read the plate on a microtiter plate reader at 540 nm.

f. A positive hemolysin test is defined as the productionof an O.D. reading > the O.D. of the 20% hemoglobinstandard in the standard curve prepared above in Section7.53.

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Buchanan, R. L., and S. A. Palumbo. 1985. Aer omonas

hy dr ophi l a and Aer omonas sob r i a as potential food poisoningspecies: a review. J. Food Safety 7:15-29.

Burke, V., M. Gracey, J. Robinson, D. Peck, J. Beaman, and C.Bundell. 1983. The microbiology of childhood gastroenteritis: Aer omonas species and other infectiveagents. J. Infect. Dis. 148:68-74.

Burke, V., J. Robinson, M. Cooper, J. Beaman, K. Partridge,D. Peterson, and M. Gracey. 1984. Biotyping and virulencefactors in clinical and environmental isolates of Aer omonas

species. Appl. Environ. Microbiol. 47:1146-1149.

Okrend, A. J. G., B. E. Rose, and B. Bennett. 1987.Incidence and toxigenicity of Aer omonas species in retail poultry, beef, and pork. J. Food Protect. 50(6):509-513.

Palumbo, S. A., F. Maxino, A. C. Williams, R. L. Buchanan,and D. W. Thayer. 1985. Starch-ampicillin agar for thequantitative detection of Aer omonas hy dr oph i l a . Appl.Environ. Microbiol. 50(4):1027-1030.

Palumbo, S. A., D. R. Morgan, and R. L. Buchanan. 1985.Influence of temperature, NaCl, and pH on the growth of

Aer omonas hy dr ophi l a . J. Food Sci. 50:1417-1421.

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_________________________________________________________________________________

_______________________________________________________________________________________________





Notice of Change


Title: Isolation and Identification of Listeria monocytogenes from Red Meat, Poultry,

Egg, and Environmental Samples

Effective Date: 8/3/09 Description and purpose of change(s):

Environmental sponge sample compositing is described in section 8.5.2.b. An additional test kit, described in Section 8.6.2, is described as an option for

performing biochemical confirmation.

Tumbling motility observation was changed to an optional confirmatory test in section

8.6.1.c.

The more accurate term “Primary Enrichment plating” replaced “Direct plating”

throughout the chapter.



acknowledges that equivalent products may be available for laboratory use.



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United States Department of Agriculture Food Safety And Inspection Service, Office of Public Health Science


Title: Isolation and Identification of Listeria monocytogenes from Red Meat, Poultry, Egg and Environmental

Samples


Procedure Outline

8.1 Introduction

8.2 Safety Precautions8.3 Quality Control Procedures

8.4 Equipment, Supplies, Media, Reagents, and Test Systems

8.4.1 Equipment8.4.2 Supplies

8.4.3 Media8.4.4 Reagents and Test Systems8.4.5 Cultures

8.5 Detection and Isolation Procedures

8.5.1 Sample Preparation8.5.2 Primary Enrichment in UVM Broth

8.5.3 Secondary Enrichment in FB or MOPS-BLEB and Primary Enrichment Plating of

UVM8.5.4 Examination of UVM-streaked MOX, Interpretation/Plating of 26 h FB, and Plating

of MOPS-BLEB

8.5.5 Examination of MOX Plates and Interpretation/Plating of 48 h FB

8.5.6 Isolation and Purification Procedures8.6 Confirmation and Identification Procedures

8.6.1 Preliminary Confirmation Tests for Listeria spp.

8.6.2 Biochemical Tests8.6.3 CAMP/CAMP Factor Test

8.6.4 Genetic Identification Tests

8.7 Culture Storage and Shipment8.8 References

8.1 Introduction

Listeria monocytogenes is a gram-positive rod-shaped bacterium associated with a variety of environments including soils, water, sewage, silage, as well as plant and animal food

products. Although reported cases of human foodborne listeriosis are rare, the incidence of serious illness and death in affected individuals is high. Immunocompromised individuals,

pregnant women, neonates and the elderly are particularly vulnerable.

Among all species in the genus Listeria, only L. monocytogenes is typically implicated in

human foodborne illness. The method described below employs well-established media and

Issuing Authority: Director, Laboratory Quality Assurance Division (LQAD)

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tests for the isolation and specific identification of β-hemolytic L. monocytogenes. The

method is broadly applicable to raw or cooked ready-to-eat red meat and poultry products,

including egg products, and environmental sponge samples. This protocol supercedes previous USDA/FSIS protocols for L. monocytogenes.


a. Laboratory personnel must abide by CDC guidelines for manipulating Biosafety

Class II pathogens. A Class II laminar flow biosafety cabinet is recommended for activities with potential for aerosolization of pathogens.

b. Pregnant women and potentially immunocompromised individuals should be prohibited from laboratory rooms or areas where L. monocytogenes isolation or

identification procedures are in progress. Although a properly sanitized

laboratory area should not harbor L. monocytogenes or other pathogens,supervisors should use their own discretion in allowing high-risk individuals into

these areas when not in use for these activities.


The correct performance of all stages of the analysis, including enrichment, screening

tests, plating and all confirmatory tests, must be verified through the use of appropriatecontrols.

a. For enrichment, screening, plating and verification of tumbling motility, controlcultures must be transferred from one medium to the next in the sequence of

analysis used for the samples. For these stages of analysis, the following

requirements apply:i. One L. monocytogenes positive control and one uninoculated media

(negative) control are required for each set of concurrently analyzed

samples.

ii. For the uninoculated control, use an aliquot of UVM broth. For all

subsequent uninoculated control tests, use one unit of the medium at the

volume specified for the test. Investigate the source of any contaminatingorganisms.


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b. Specific control requirements for each confirmatory test are addressed in theappropriate sections of this protocol.

c. Additional control cultures may be employed for individual tests or the entiresequence of analysis at the discretion of the laboratory.

d. L. monocytogenes and L. innocua control culture inocula are prepared and used asdeemed appropriate for the media to be tested. Ideally, an empirically determined

standardized quantity of inoculum should be employed.

8.4 Equipment, Supplies, Media, Reagents, and Test Systems

8.4.1 Equipment

a. Electronic top-loading balance capable of weighing a minimum of 25 ± 0.1 g(500 g capability recommended)

b. Stomacher ™ (model 400 by Tekmar, or comparable model), or blade-type blender, sterilized blender cutting assemblies and jars

c. Incubator, 30 ± 2°C

d. Incubator, 35 ± 2°C

e. Incubator, 20 or 25 ± 2°C

f. Vortex mixer

g. Phase-contrast microscope (40X and 100X objectives)h. Fluorescent desk lamp or natural spectrum light source

8.4.2 Supplies

a. Supplies for all samples:

i. Sterile sample preparation supplies: scalpels, chisels, knives,scissors, spatulas, forceps, disposable or reusable dishes, pans or

trays

ii. Stomacher ®

Strainer 400™

sterile filter bags or non-filtered bags or

equivalent

iii. Non-filter plastic bags for double-bagging sample homogenates(optional)

iv. Pipets (e.g. 1 ml)


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v. Pipettor and sterile disposable tips for dispensing 100 μl

vi. Microscope slides, cover slips and immersion oil

vii. Disposable plastic and/or platinum inoculating needles and loopsviii. Wax or waterproof ink marker

ix. Sterile cotton-tipped applicators (i.e. swabs)

b. Additional supplies necessary for environmental sponge samples:

Non-bactericidal sampling sponges (e.g. Nasco cat # B01299WA, or equivalent)

c. Additional supplies necessary for environmental aqueous chilling solutions:

i. 0.45 μm hydrophobic grid membrane filter system (e.g. SELeCT

56 mm/100-ml 0.45-μm Monitor, cat# BPM9261, BioPath Inc.,

2611 Mercer Ave., W. Palm Beach, FL 33401, 800-645-2302, or equivalent)

ii. Vacuum flask/hose and vacuum source

iii. Sterile disposable scalpels

iv. Sterile forcepsv. Whirl pak filter bag

vi. Glass fiber filter (e.g. Whatman Glass microfibre GF series GradeGF/D: 2.7µm)

vii. 500 ml graduated polypropylene beaker with handle

viii. Sterile 500 - 1000 ml non-polystyrene sample container

8.4.3 Media

Refer to the MLG Media Appendix for formulations and preparation instructions.

a. Media required for enrichment, plating and preliminary confirmation tests

i. Modified University of Vermont broth (UVM, also known as

UVM1)

ii. Morpholinepropanesulfonic acid-buffered Listeria enrichment broth

(MOPS-BLEB) or iii. Fraser broth (FB)

iv. Modified Oxford agar (MOX)


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v. Horse blood overlay agar (HL, also known as HBO)vi. Trypticase soy agar with 5% sheep blood (TS-SBA, also known as

CAMP test agar)

vii. Brain heart infusion broth (BHI broth)

b. Additional media for environmental samples

Dey-Engley (D/E) neutralizing broth (e.g. Nasco cat# B01256WA, or

equivalent)

c. Optional media Trypticase Soy Agar-Yeast Extract (TSA-YE)

8.4.4 Reagents and Test Systems

a. β-lysin CAMP factor discs (Remel #21-120, or equivalent)

b. Biochemical test panel (MICRO-ID®

Listeria, Listeria API®

system,

VITEK ® 2 Compact or equivalent)

c. Ribosomal RNA-based test system (i.e. GenProbe Accuprobe®

L. monocytogenes-specific test, GeneTrak ®

L. monocytogenes-specific test,or equivalent)

8.4.5 Cultures

a. At least one L. monocytogenes positive control strain is required.

Appropriate cultures include ATCC®

19111, NCTC 7973 or other L.

monocytogenes cultures validated to perform in an equivalent manner.

b. At least one L. innocua negative control culture is required. Appropriate

cultures include ATCC® 33090 or other L. innocua validated to perform inan equivalent manner.

c. Other Listeria spp., such as L. seeligeri, L. grayi and L. ivanovii, may benecessary as controls for additional confirmatory testing.


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d. If the β-lysin CAMP factor test is not employed, Staphylococcus

pseudintermedius ATCC® 49444 or Staphylococcus aureus ATCC® 25923,

and Rhodococcus equi ATCC®

6939 are required to perform the traditionalCAMP test.

8.5 Detection and Isolation Procedures

8.5.1 Sample preparation

a. Meat, poultry and egg products: A 25 ± 1.0 g portion is used for raw and processed red meat, poultry and egg product testing. The test portion

should be a composite representative of the entire sample or available

samples common to a specific lot.

i. Intact retail packages must be disinfected at the incision sites

immediately prior to incision for sampling. Appropriate

disinfectants include but are not limited to ca. 3% hydrogen peroxide, ca. 70% ethanol or ca. 70% isopropanol. If the package

does not appear to be clean, scrub gently using soapy water and

rinse thoroughly prior to disinfection. A sterile scalpel may behelpful for cutting the packaging. Aseptically pull the packaging

away to expose the product for sampling.

ii. Using a sterile scalpel, knife, spoon, chisel or other tool cut small pieces from representative sites on the sample and/or a variety of

same-lot samples to prepare a composite sample.

iii. Place 25 ± 1.0 g of the composite sample in a sterile Stomacher ™ bag (i.e. filter bag recommended). If necessary, double-bag with anon-filter bag prior to stomaching (e.g. zip-lock bag).

iv. If analysis of the test portion is not to be initiated within 1 h, store at

≤ -10°C. Do not dilute the sample until ready to initiate analysis.

v. For analysis, proceed to Section 8.5.2.

b. Outbreak/recall samples: Some samples or sample lots, particularly those

implicated in foodborne illness outbreaks, may require analysis of up to


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thirteen 25 g test portions. The test portion should be a compositerepresentative of the entire sample or available samples common to a

specific lot. The need for multiple subsample analyses must be

determined on a case-by-case basis, or by client requirements.

c. Most Probable Numbers (MPN) Determination: Follow MPN instructions

given in the specific program protocol or see MLG Appendix 2, MostProbable Number Procedure and Tables.

8.5.2 Primary Enrichment in UVM broth

a. For all meat, poultry and egg product samples (including outbreak/recall

samples):

To the 25 ± 1.0 g test portion, dispense 225 ± 5 ml (or 225 ± 5 g) of UVM

broth. Stomach or blend for 2 ± 0.2 minutes. If blended, aseptically transfer

the homogenate to an appropriate sterile container. Incubate the homogenate

at 30 ± 2°C for 22 ± 2 h. Proceed to Section 8.5.3.

b. For environmental sponge samples:

Add 225 ± 5 ml (or 225 ± 5 g) of UVM broth to each bagged single sponge

sample or add 100± 2 ml of UVM per sponge to each bagged compositesponge sample that contains up to five sponges. For example, a composite

of 5 sponge samples would require 500± 10 ml of UVM. Stomach for 2 ± 0.2 min. Hand mixing is an acceptable alternative for stomaching. To hand

mix, massage each sponge until no purple fluid is released from the

sponge.

Incubate at 30 ± 2°C for 22 ± 2 h. Proceed to Section 8.5.3.

c. For environmental aqueous chilling solutions and surface rinse solutions:

Aqueous chilling solutions may include water, brine and propylene glycolsolutions.


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i. Pour 500 ± 2 ml of sample solution into a whirl pak filter bag. Filter

the solution by pouring it through a glass fiber filter and a 0.45 μmhydrophobic grid membrane filter in a vacuum filter system. These

filters can be easily clogged by particulates. Therefore, more than

one filter may be necessary to filter the entire test portion. Whenthe sample has been filtered, aseptically remove the glass filter and

transfer it back to the whirl pak filter bag for enrichment.

ii. Aseptically remove the membrane from the membrane filter plastichousing and transfer to the same filter bag as above. A sterilescalpel may be used to cut around the circumference of the

hydrophobic grid membrane to excise it from the filter housing

(e.g. SELeCT®

system). All membranes associated with a givensample are combined in the single enrichment bag.

iii. Add 225 ± 5 ml (or 225 ± 5 g) of UVM broth. Stomach 2 ± 0.2

minutes. Ensure that the filters are submerged. Incubate the

homogenate at 30 ± 2°C for 22 ± 2 h. Proceed to Section 8.5.3.

8.5.3 Secondary Enrichment in FB or MOPS-BLEB and Primary Enrichment

Plating of UVM

a. Transfer 0.1 ± 0.02 ml of the UVM enrichment to 10 ± 0.5 ml of FB or

MOPS-BLEB. As per media preparation instructions, be sure that

appropriate supplements have been added to the FB prior to inoculation.

Incubate inoculated FB tubes at 35 ± 2°C for 26 ± 2 h or inoculated MOPS

BLEB tubes at 35 ± 2°C for 18-24 h.

b. Streak a MOX plate. Streak a loopful or a drop approximating 0.1 ml of

the UVM over the surface of the plate. Alternatively, dip a sterile cotton-tipped applicator or equivalent into the UVM and swab 25-50% of the

surface of a MOX plate. Use a loop to streak for isolation from the

swabbed area onto the remainder of the plate. Incubate the MOX at 35 ± 2°C for 26 ± 2 h.


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8.5.4 Examination of UVM-streaked MOX, Interpretation/Plating of 26-h FB, and

Plating of MOPS-BLEB

a. Examine the UVM-streaked MOX for colonies with morphology typical of Listeria spp. At 26 ± 2 h, suspect colonies are typically small (ca. 1 mm)

and are surrounded by a zone of darkening due to esculin hydrolysis.

i. If suspect colonies are present on MOX, transfer suspect colonies to

HL agar as described in Section 8.5.6.

ii. If no suspect colonies are evident, re-incubate the MOX plate for an

additional 26 ± 2 hour.

iii. Proceed to step b below for FB or step c for MOPS-BLEB.

b. After 26 ± 2 h of incubation, examine the FB for the potential presence of L. monocytogenes, by visual examination of the broth for darkening due to

esculin hydrolysis.

i. If any degree of FB darkening is evident, aseptically dispense a dropapproximating 0.1 ± 0.02 ml of FB onto a MOX plate. Swab or

streak 25-40% of the surface of the MOX plate with the FBinoculum. Use a loop to streak for isolation from the initial

swab/streak quadrant onto the remainder of the plate. Incubate the

MOX plate at 35 ± 2°C for 26 ± 2 h. Proceed to Section 8.5.5.

ii. If no FB darkening is evident, re-incubate the FB at 35 ± 2°C until a

total incubation time of 48 ± 2 h has been achieved. Proceed toSection 8.5.5.b.

c. After 18-24 h, streak a MOX plate using a loopful of the MOPS-BLEB, or by streaking a drop approximating 0.1 ml or aseptically dip a sterile

cotton-tipped applicator or equivalent into the MOPS-BLEB and swab 25

50% of the surface of a MOX plate. Use a loop to streak for isolationfrom the swabbed area onto the remainder of the plate. Incubate the MOX

at 35 ± 2°C for 26 ± 2 h. Proceed to Section 8.5.5.a.


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8.5.5 Examination of MOX Plates and Interpretation/Plating of 48 h FB

a. Examine and select suspect colonies from any MOX agar plate pending

analysis (i.e. MOX plates streaked from 26 ± 2 h FB, 18-24 h MOPS

BLEB, and/or UVM) as described in Section 8.5.4.a.i and ii.

b. Re-examine the FB for evidence of darkening after 48 ± 2 h of total

incubation.

i. If any degree of darkening is evident, swab, streak and incubate a

MOX plate as described in Section 8.5.4.i.

ii. If no darkening of FB is evident and no suspect MOX and/or HL

colonies have been demonstrated, the sample is considered negativefor L. monocytogenes.

8.5.6 Isolation and Purification Procedures

a.

If suspect colonies are present on MOX from any source, use a loop or equivalent sterile device to contact a minimum of 20 (if available) suspect

colonies and collectively streak for isolation on one or more HL agar plates. Alternatively, a swipe of suspect growth representing at least 20

colonies may be used. Incubate the streaked HL at 35 ± 2°C for 22 ± 4 h.

b. After incubation, examine the HL plate(s) against backlight for translucent

colonies surrounded by a small zone of β-hemolysis.

i. If at least one suspect colony is clearly isolated, proceed to

confirmatory testing (Section 8.6 below). Hold all HL plates

containing suspect colonies (room temperature or refrigeration)until confirmatory testing is complete.

ii. If suspect colonies or β-hemolytic growth are present on HL but not

clearly isolated, re-streak representative suspect colonies/growth onto

one or more fresh HL plates and incubate per section 8.5.6.a.


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iii. If no suspect isolates are present on HL, pursue follow-up of MOXand/or HL isolates from other branches of analysis (e.g. FB follow-

up vs. UVM Primary Enrichment streak follow-up). If no branch of

the analysis produces suspect β-hemolytic colonies on HL, the

sample may be reported as negative for L. monocytogenes.

Note: Removal of a few colonies may assist the analyst in observing mediumclearing for weakly hemolytic strains.

8.6 Confirmation and Identification Procedures

Confirmatory identification of L. monocytogenes consists of preliminary confirmation tests(Section 8.6.1) followed by biochemical tests (Section 8.6.2). The CAMP/CAMP factor test

(Section 8.6.3) and genetic tests (Section 8.6.4) may be required in certain circumstances.

All confirmatory identification tests require a pure culture.

8.6.1 Preliminary Confirmation Tests for Listeria spp.

a. Inoculation of preliminary confirmation test media

Use one isolated HL colony only to inoculate, in order, a BHI broth aliquot and

(optionally) a fresh HL plate to confirm purity. In addition, media required for inoculation of biochemical test systems (e.g. HL, BHI agar, TSA-YE, TSA-SBA or

equivalent as described in Section 8.4.3) must be inoculated from the same colony or

growth subcultured from that colony. A minimum of one colony must beconfirmed. If the first selected suspect HL colony does not confirm as L. monocytogenes, confirmation must be attempted for additional suspect HL

colonies, if available, until at least three isolates from the test portion have failedconfirmation.

Incubate BHI broth at 18-25°C for 16-18 h. Proceed to step 8.6.1.c.

Incubate the optional HL purity streak plate at 35 ± 2°C for 22 ± 4 h. Proceed to

Section 8.6.2.


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b. Purity streak test (optional)

Examine the re-streaked HL plate for consistent morphological characteristics. The

colonies on this second HL plate should represent a single clone. If the culture

appears to be mixed, repeat Steps 8.5.6.b.ii. A pure culture is required for allconfirmatory tests.

c.Tumbling motility test (optional)

After 16-18 h of incubation of BHI broth at 18-25°C, prepare a wet-mount. Using

the 100X oil immersion objective (phase contrast microscopy recommended),

examine the wet-mount culture for small rods that exhibit an active end-over-endtumbling/rotating movement characteristic of Listeria spp.

i. If cell morphology and motility is not characteristic of Listeria spp.,

and the culture appears pure, report the sample as negative for L. monocytogenes.

ii.

If a mixture of typical Listeria cells and cells that do not have thecharacteristic Listeria morphology (i.e. small rods) are present, streak

a loopful of the contaminated BHI broth onto fresh HL agar for

further purification (Step 8.5.6.b.ii).

iii. If no growth is evident at 16-18 h, re-incubate at 18-25°C untilgrowth is evident or up to a total of 48 h.

iv. If cell morphology is typical, tumbling motility is evident and the

culture appears pure, proceed with biochemical confirmation(Section 8.6.2 below).

8.6.2 Biochemical Tests

Using a pure culture, perform confirmatory biochemical tests. Commercially

available test systems (MICRO-ID®

Listeria, Listeria API®

test system or

VITEK ®

2 Compact) or validated equivalent systems, including well-establishedschemes involving traditional tube biochemical media (e.g. Compendium of

Methods for the Microbiological Examination of Foods, Bacteriological


http://slidepdf.com/reader/full/8.5.6.b.ii

http://slidepdf.com/reader/full/8.5.6.b.ii

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Analytical Manual), may be employed. However, exercise caution in interpretingthe identification of atypical Listeria spp. isolates when using biochemical

systems. Cultures identified as “ L. monocytogenes/innocua” or any beta-

hemolytic Listeria spp. that is biochemically indeterminate or identified as L.innocua must be further characterized using a ribosomal RNA-based test system

as described in Section 8.6.4.a.

a. MICRO-ID®

Listeria test system

i. Follow the instructions provided by the manufacturer for

inoculation and interpretation of the test panel.

ii. TSA-YE, BHI, TSA-SBA or HL agar inoculated and incubated as

described in Section 8.6.1.a may be used for preparing the

inoculum suspension. All growth on the agar of choice mustrepresent the same clone.

iii. A CAMP/CAMP factor test (Section 8.6.3) must be performed to

augment MICRO-ID®

results.

iv. At minimum, one L. monocytogenes positive control must be

analyzed concurrently with sample isolates.

b. Listeria API®

test system

i. Follow the instructions provided by the manufacturer for inoculation and interpretation of the test panel.

ii. TSA-YE, BHI, TSA-SBA or HL agar may be used for preparingthe inoculum suspension. All growth on the agar of choice must

represent the same clone.

iii. At a minimum, one L. monocytogenes positive control must be

analyzed concurrently with sample isolates.


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c. VITEK ®

2 Compact

i. Follow the instructions provided by the manufacturer for

inoculation and loading the VITEK 2®

Compact test system.

ii. TSA-YE, BHI, TSA-SBA or HL agar may be used for preparing

the inoculum suspension. All growth on the agar of choice must

represent the same clone.

iii. At a minimum, one L. monocytogenes positive control must beanalyzed concurrently with sample isolates.

8.6.3 CAMP/CAMP Factor Test

A CAMP/CAMP factor test is required to augment traditional biochemical or

MICRO-ID®

test results, or to resolve the hemolytic capability of indeterminatestrains. Either of two test options may be employed:

a. β-lysin CAMP factor test

This test system may provide results that are easy to interpret compared to the

traditional CAMP test. Therefore, the β-lysin CAMP factor test is recommended

over the traditional test.

i. Aseptically place a β-lysin disc in the approximate center of a

TS-SBA plate. (A 9 ± 1-ml TS-SBA plate is easier to interpret than

thicker plates and is recommended.)

ii. Individually and aseptically streak four to eight isolates each as

straight lines radiating away from the disc. The inoculation line

should almost but not quite touch the disc. Be sure to include positive and negative control cultures. A non-hemolytic L.

innocua is an appropriate negative control.

iii. Incubate at 35 ± 2 °C for 24 ± 2 h.


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iv. An arrowhead-shaped zone of β-hemolysis surrounding the

inoculum line proximal to the disc indicates a positive CAMP

factor reaction. L. monocytogenes, L. seeligeri and L. ivanovii areCAMP factor-positive by this test. However, L. ivanovii

demonstrates relatively intense β-hemolysis distal to the disk and,therefore, can be distinguished from the other two species. Non

hemolytic Listeria spp. are CAMP factor-negative.

v. If a suspected β-hemolytic Listeria spp. does not produce a CAMP

factor-positive reaction at 24 ± 2 h, continue to incubate the culture

at 35 ± 2 °C until a total incubation time of 48 ± 2 h has been

achieved. Re-examine as before. If a CAMP factor-positive

reaction is still not evident at 48 ± 2 h, ribosomal RNA-based

testing is required to determine the genetic identity of the isolate

(Section 8.6.4.a).

b. Traditional CAMP test

If the CAMP test is necessary and β-lysin discs are not available, performthe following test:

i. For the traditional culture CAMP test, apply single-line streaks of S. pseudintermedius ATCC

®49444 or S. aureus ATCC

®25923

and R. equi (ATCC®

6939) reference cultures on a TS-SBA platein parallel and 3-4 cm apart.

ii. Streak test cultures between and perpendicular to the two referencecultures (i.e. like rungs of a ladder). The test culture streak must

be 2-4 mm from each reference culture streak. Test and reference

cultures must not touch or be cross contaminated in any manner.

iii. Incubate 24 ± 2 h at 35 ± 2°C.

iv. Examine the test culture streaks for enhanced β-hemolysis at both

ends proximal to the reference cultures. The zone of enhanced

β-hemolysis may resemble an arrowhead, circle or rectangle. The

presence of this zone indicates a CAMP-positive reaction.


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Absence of enhanced β-hemolysis indicates a CAMP-negative

reaction. L. monocytogenes and L. seeligeri are CAMP-positive to

the Staphylococcus reference strain and CAMP-negative to R. equi.In contrast, L. ivanovii is CAMP-positive to the R. equi reference

strain and CAMP-negative to the Staphylococcus reference strains.

v. If a suspected β-hemolytic Listeria spp. does not produce a

CAMP-positive reaction with either reference culture at 24 ± 2 h,

continue to incubate the culture at 35 ± 2°C until a total incubation

time of 48 ± 2 h has been achieved. Re-examine as before.

vi. If the culture does not produce a CAMP-positive reaction with the

Staphylococcus culture, but has or does clearly demonstrate β

hemolysis on HL agar, ribosomal RNA-based testing is required to

determine the genetic identity of the isolate (Section 8.6.4.a).

8.6.4 Genetic Identification Tests

a. Ribosomal RNA-based tests

Ribosomal RNA-based tests may be performed as a confirmatory test for all

biochemically identified L. monocytogenes strains. However a ribosomal RNA- based test is required to resolve the identity of any atypical suspected L.

monocytogenes strain. In some circumstances, available phenotypic tests cannot

clearly distinguish strains of L. monocytogenes from L. innocua. In particular,

rhamnose-negative β-hemolytic L. monocytogenes and phospholipase C-negative,

weakly hemolytic L. monocytogenes can be misidentified as L. innocua on

biochemical testing. And β-hemolytic L. innocua strains exist. Several L.

monocytogenes-specific ribosomal RNA-based tests are commercially available.

Follow the test kit instructions provided by the manufacturer for performing andinterpreting these tests.

A L. monocytogenes positive control, a L. innocua negative control and anuninoculated sterility control must be analyzed concurrently with sample isolates.


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A positive ribosomal RNA-based test result from either of the above test systemsindicates that atypical phenotypes are confirmed L. monocytogenes. A negative

result indicates that atypical phenotypes are not L. monocytogenes.

b. Pulsed-field gel electrophoresis (PFGE)

PFGE is a highly discriminative and reliable means of subtyping L.

monocytogenes strains. The pulsotype (i.e. “genetic fingerprint”) derived from

PFGE analysis is used in tandem with epidemiologic evidence to link clinical andfood isolates implicated in foodborne illness. Standardized PFGE methodologyand internet-based exchange of federal and state laboratory pulsotype data (i.e.

PulseNet) facilitate epidemiologic and traceback investigations.

8.7 Culture Storage and Shipment

a. BHI or TSA-YE slants may be used for short-term storage of Listeria spp. Theculture should be stabbed into the agar using an inoculating needle. Tubes should be

sealed with Parafilm®

or equivalent to prevent desiccation and stored at 2-8°C.Under these conditions, Listeria spp. can remain viable for many months.

b. For long-term storage (i.e. for more than one year) or to assure that the genetic

character of the strain does not change over time (e.g. lose plasmids or other unstable genetic elements), cultures should be lyophilized and/or frozen

at -20 to -80° C. Fetal calf serum or commercially available cryobead products areappropriate media for frozen storage of Listeria spp.

8.8 References

Bailey, J. S., M. D. Pratt, D. A. Blank, L. E. Manis, O. A. Soto, and P. A. White. 1992.

Recovery and speciation of Listeria from raw and cooked meat and poultry products. J. Rap.

Meth. Automat. Microbiol. 1:93-100.

Bacto®

Listeria Antigens and Antisera. 1998. pp. 648-651. In: Difco Manual. 11th

Edition.

Difco Laboratories/Becton Dickinson and Co., Sparks, MD.

Curiale, M. S., and C. Lewus. 1994. Detection of Listeria monocytogenes in samplescontaining Listeria innocua. J. Food Prot. 57:1048-51.


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Fraser, J. A. and W. H. Sperber. 1988. Rapid detection of Listeria spp. in food andenvironmental samples by esculin hydrolysis. J. Food Prot. 51:762-5.

Graves, L. M. 1998. Standardized protocol for molecular subtyping of Listeria

monocytogenes by pulsed-field gel electrophoresis (PFGE). The Centers for Disease Control

and Prevention, Atlanta, GA.

Hayes, P. S., L. M. Graves, B. Swaminathan, G. W. Ajello, G. B. Malcomb, R. E. Weaver,

R. Ransom, K. Deaver, B. D. Plikaytis, A. Schuchat, J. D. Wenger, R. W. Pinner, C. V.Broome, and The Listeria Study Group. 1992. Comparison of three selective enrichmentmethods for the isolation of Listeria monocytogenes from naturally contaminated foods. J.

Food Prot. 55(12):952-9.

Hendrickson, D. A., and M. M. Krenz. 1991. Reagents and Stains. pp. 1289-1314. In:

Manual of Clinical Microbiology. 5th

Edition. A. Balows, W. J. Hausler, Jr., K. L.

Herrmann, H. D. Isenberg, and H. J. Shadomy (eds). American Society for Microbiology,Washington, D. C.

Hitchins, A. D. 2003. Chapter 10, “Detection and Enumeration of Listeris monocytogenes in

Foods” in FDA Bacteriological Analytical Manual. Online

Johnson, J. L., and C. P. Lattuada. 1993. Comparison of nucleic acid hybridization assays

and biochemical characterization tests for the confirmation of Listeria monocytogenes. J.Food Prot. 56:834-840.

Kornacki, J. L., D. J. Evanson, W. Reid, K. Rowe, and R. S. Flowers. 1993. Evaluation of the USDA protocol for detection of Listeria monocytogenes. J. Food Prot. 56:441-443.

McClain, D., and W. H. Lee. 1988. Development of USDA-FSIS method for isolation of Listeria monocytogenes from raw meat and poultry. J. Assoc. Off. Anal. Chem. 71:660-4.

McClain, D., and W. H. Lee. 1989. FSIS method for the isolation and identification of Listeria monocytogenes from processed meat and poultry products. LaboratoryCommunication No. 57. USDA, FSIS, Microbiology Division, Beltsville, MD.

Petran, R. L., and K. M. J. Swanson. 1992. Simultaneous growth of Listeria monocytogenes

and Listeria innocua. J. Food Prot. 56(7):616-18.


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Ryser, E. T., and C. W. Donnelly. 2001. “ Listeria”. Chapter 36. pp.343-356 In:Compendium of Methods for the Microbiological Examination of Foods. 4

thEdition. F. P.

Downes and k. Ito (eds). American Public Health Association, Washington, D.C.

Ryser, E. T., and E. H. Marth. 1999. Listeria, Listeriosis, and Food Safety. 2nd

Edition.

Marcel Dekker, Inc., New York, NY.

Sallam, S. S., and C. W. Donnelly. 1991. Destruction, injury, and repair of Listeria species

exposed to sanitizing compounds. J. Food Prot. 55(10):771-6.

Schuchat, A., B. Swaminathan, and C. V. Broome. 1991. Listeria monocytogenes CAMP

reaction. Clin. Microbiol. Rev. 4:396.

Seeliger, H. P. R., and D. Jones. 1986. Genus Listeria, pp. 1235-1245 In: Bergey's Manual

of Systematic Bacteriology, Vol. 2. Edited by P. H. A. Sneath, N. S. Mair, M. E. Sharp, and

J. G. Holt., Williams & Wilkins, Baltimore, MD.

Smola, J. 1989. Possibilities of differentiation of listerial hemolysins by synergistic

hemolytic reactions (CAMP reactions). Int. J. Food Microbiol. 8:265-267.

Vázquez-Boland, J. A., L. Domínguez, J. F. Fernández-Garayzábal, and G. Suárez. 1992. Listeria monocytogenes CAMP reaction. Clin. Microbiol. 5:343.

Warburton, D. W., J. M. Farber, A. Armstrong, R. Caldeira, N. P. Tiwari, T. Babiuk, P.

Lacasse, and S. Read. 1991. A Canadian comparative study of modified versions of the

"FDA" and "USDA" methods for the detection of Listeria monocytogenes. J. Food Prot.54:669-676.

Wiedmann, M., J. L. Bruce, C. Keating, A. E. Johnson, P. L. McDonough, and C. E. Batt.1997. Ribotypes and virulence gene polymorphisms suggest three distinct Listeria

monocytogenes lineages with differences in pathogenic potential. Infect. Immun.65(7):2707-16.


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_________________________________________________________________________________

_______________________________________________________________________________________________




Laboratory GuidebookNotice of Change

Chapter new, revised, or archived: MLG 8A.04

Title: FSIS Procedure for the Use of a Listeria monocytogenes Polymerase Chain

Reaction (PCR) Screening Test


Description and purpose of change(s):The more accurate term “Primary Enrichment plating” replaced “Direct plating” throughout the chapter.



acknowledges that equivalent products may be available for laboratory use.



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Title: FSIS Procedure for the Use of a Listeria monocytogenes Polymerase Chain Reaction (PCR) Screening Test


Procedure Outline

8A.1 Introduction8A.2 Safety Precautions


8A.3.1 Culture Controls8A.3.2 Sterility Control

8A.4 Equipment, Reagents, and Media


8A.6 Secondary Enrichment and Primary Enrichment Plating8A.7 The BAX

®System for Screening L. monocytogenes Test Procedure

8A.8 Cultural Confirmation8A.9 Interpretation of Results

8A.10 Completion of Testing if BAX®

Unavailable8A.11 Selected References

8A.1 Introduction

This method describes the use of a commercial PCR based screening procedure as

described in MLG 8 Section 8.4.5 to screen-test processed meat, poultry, pasteurized

liquid egg products and environmental sponge samples for the presence of Listeriamonocytogenes. All samples identified as presumptively positive for Listeria

monocytogenes by these tests are subject to cultural confirmation.


CDC guidelines for the handling of BioSafety Level 2 organisms should be followed whenever live cultures of Listeria monocytogenes are used. All available Material Safety

Data Sheets (MSDS) must be obtained from the manufacturer for the media, chemicals,

reagents, and microorganisms used in the analysis. The personnel who will handle thematerial should read all MSDS sheets, and all MSDS requirements should be followed.

Pregnant women and potentially immunocompromised individuals should be prohibited from laboratory rooms or areas where L. monocytogenes isolation or identification

procedures are in progress. Although a properly sanitized laboratory area should not

harbor L. monocytogenes or other pathogens, supervisors should use their own discretion

in allowing high-risk individuals into these areas when not in use for these activities.


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8A.3.1 Culture Controls

a. At least one L. monocytogenes positive control strain is required.

Appropriate cultures include ATCC®

19111, NCTC 7973 or other L.

monocytogenes cultures validated to perform in an equivalent manner.

b. At least one L. innocua negative control culture is required. Appropriate

cultures include L. innocua strain ATCC®

33090 or other L. innocua strainsvalidated to perform in an equivalent manner.

8A.3.2 Sterility Control

Prepare one “blank” (incubated but un-inoculated pre-enrichment/ enrichment broth) to provide a sterility control for the process.

8A.4 Equipment, Reagents, and Media

In addition to equipment, reagents, and media used in analysis of samples as described in

MLG 8, the following materials will be needed.

a. PCR tube holder (Qualicon)

b. Cell Lysis Tube Cooling Block (Qualicon) held at 4 ± 2°C

c. Techne DB-2A Heating block set at 55 ± 2°C

d. Techne DB-2A Heating block set at 95 ± 3°C

e. Eppendorf Repeater Plus Pipettor (or equivalent) set at 200:l μl, and tips

f. Corning Lambda 20 Pipettor (or equivalent) set at 5 ± l μl, and tips

g. Corning Lambda 200 Pipettor (or equivalent) set at 150 ± l μl, and tipsh. 12 X 75 mm (Falcon 352063, or equivalent) tubes

i. Cell Lysis Tubes and Caps, Cell Lysis Tube Rack and box (Genemate 8 strip

tubes, ISC Bioexpress, T-3120-5)

j. Pipettor and 5 ml pipettes

k. BAX®

Assay for Screening L. monocytogenes (Qualicon # 17710609) held at

4 ± 2°C

l. Morpholinepropanesulfonic acid-buffered Listeria enrichment broth (MOPS-BLEB)

medium:


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BBL Listeria enrichment broth (BBL #12333, or equivalent) MOPS free acid (Sigma #1254, or equivalent) MOPS sodium salt (Sigma #M9381, or equivalent)


Perform sample preparation and pre-enrichment in as described in MLG 8, Section 8.5.1

and 8.5.2.

8A.6 Secondary Enrichment and Primary Enrichment Plating

a. Transfer 0.1 ± 0.02 ml of the UVM enrichment to 10 ± 0.5 ml of MOPS-BLEB.

Incubate inoculated MOPS-BLEB tubes at 35 ± 2°C for 18-24 h.

b. Streak a MOX plate. Streak a loopful or a drop approximating 0.1 ml of the UVM

over the surface of the plate. Alternatively, dip a sterile cotton-tipped applicator or equivalent into the UVM and swab 25-50% of the surface of a MOX plate.

Use a loop to streak for isolation from the swabbed area onto the remainder of the

plate. Incubate the MOX at 35 ± 2°C for 26 ± 2 h.

8A.7 The BAX®

System for Screening L. monocytogenes Test Procedure

Follow the current BAX® User’s Guide for preparing reagents, performing the test, and

reading the results. The equipment must be set up, and operated, and all records must be

documented, according to laboratory work instructions.

8A.8 Cultural Confirmation

a. Streak a MOX plate using a loopful of the MOPS-BLEB, or by streaking a drop

approximating 0.1 ml or aseptically dip a sterile cotton-tipped applicator or

equivalent into the MOPS-BLEB and swab 25-50% of the surface of a MOX

plate. Use a loop to streak for isolation from the swabbed area onto the remainder

of the plate. Incubate the MOX at 35 ± 2°C for 26 ± 2 h.

b. Proceed with all isolation and purification procedures as per MLG 8, Sections

8.5.4.a.i, 8.5.6, and 8.6.


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8A.9 Interpretation of Results

a. Samples that test BAX®

-negative will be reported as negative if the concurrent

24h Primary Enrichment Plating is also negative. Cultural analysis will continue

on samples that are BAX®

-negative but have typical colonies on the 24 h Primary

Enrichment Plating MOX plates, or have a BAX®

-positive, BAX®

-indeterminateor have an invalid result. Or based on the findings of a cause analysis, the

laboratory may analyze the indeterminate or invalid result samples by:


analysis from the rack loading step or

• preparing new BAX® tubes and repeating the analysis. b. In analytical runs where the positive control tests negative, either the reserve

samples will be retested or the laboratory shall complete the cultural method by

streaking all samples and controls from MOPS-BLEB medium onto MOX plates.Proceed with all isolation and purification procedures as per MLG 8, Sections

8.5.6 and 8.6.

8A.10 Completion of Testing if BAX®

Unavailable

If circumstances (e.g. a power outage or equipment failure) do not allow testing using the

BAX®

system, the laboratory shall complete the cultural method by streaking all samples

and controls from MOPS-BLEB medium onto MOX plates. Proceed with all isolationand purification procedures as per MLG 8, Sections 8.5.6 and 8.6.

8A.11 Selected References

Centers for Disease Control and Prevention and National Institutes of Health (CDC/NIH).

2007. BioSafety in Microbiological and Biomedical Laboratories, 5th

ed. U.S. GovernmentPrinting Office, Washington, D.C. (also found on the internet at:


BAX®

System PCR Automated Detection for Bacterial Screening User Guide, Dupont

Qualicon.




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9-1

CHAPTER 9.ISOLATION & IDENTIFICATION OF PATHOGENIC YERSI NI A

ENTEROCOLI TI CA FROM MEAT AND POULTRY PRODUCTS

Jennifer L. Johnson

9.1 Introduction

Yer s i ni a en t er oc ol i t i ca and other Yer s i ni a species such as

Y. f r eder i k sen i i and Y. k r i s t ensen i i are ubiquitous in the naturalenvironment, and may be recovered from water, soil, animals, and food. There is considerable variation within the species

Y. en t er oc ol i t i ca , and member organisms range from the so-called "Y. en t er oc ol i t i ca -like" organisms and "environmental" strains ofY. en t er oc ol i t i ca to strains capable of causing serious disease inhumans. Hogs have been shown to be a reservoir for certain types

of pathogenic Y. en t er oc ol i t i ca and pork products have beenimplicated in human disease. The presence of pathogenic

Y. en t er oc ol i t i ca on food products is a special concern sincethose organisms are capable of growth at refrigeratortemperatures.

Pathogenic Y. en t er oc ol i t i ca organisms are significant causes ofhuman disease in many parts of the developed world.Epidemiological evidence from Belgium, Norway, Denmark, The Netherlands, Japan, Canada, and elsewhere strongly implicatesconsumption of pork products in human disease. In fact, diseasedue to Y. en t er oc ol i t i ca in the United States may be on the rise,and more information on contamination of meat (especially pork)and poultry is needed.

The term "pathogenic serotype", when used in reference to Y.

en t er oc ol i t i ca , typically refers to one of 11 O-antigen groups inthe Y. en t er oc ol i t i ca serotyping scheme. Some strains belongingto these serotypes have been implicated in human disease and havedemonstrated pathogenicity in animal models or tissue culture cellinvasiveness tests. Until recently, serotypes O:4,32; O:8;O:13a,13b; O:18; O:20; and O:21 have accounted for the majority of pathogenic serotypes recovered in the U.S. Only recently have

serotype O:3 organisms been identified as a common cause ofyersiniosis in the United States of America. In a recent Americansurvey of hospitalized gastroenteritis patients, 92% of theY. en t er oc ol i t i ca isolates were serotype O:3 while 5% wereserotype O:5,27. Serotypes O:3, O:9, and O:5,27 are well-established human pathogens in other areas of the world. The so-called "North American serotypes" of Y. en t er oc ol i t i ca (serotypes

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O:8, O:13, and O:21) represent a genetically distinct lineage from that of the other pathogenic serotypes.

While the term "pathogenic serotype" is in common usage, severalauthors have stated that terms such as "pathogenic phenotype",

"pathogenic bio-serotype", and "pathogenic bio-serogroup" are moredescriptive since they differentiate between pathogenic and nonpathogenic members of a generally pathogenic serotype.Biogrouping, the phenotypic characterization of Y. en t er oc ol i t i ca ,can serve as a useful indication of the likely pathogenicity of agiven strain. Testing for markers of pathogenicity like calcium dependence, crystal violet dye binding, auto-agglutination, and pyrazinamidase activity provide additional information. Markersare not perfectly correlated with pathogenicity but provide usefulinformation under conditions where animal testing is undesirableor impractical.

Virulence in Y. en t er oc ol i t i c a is mediated by both chromosomal and plasmid-borne genes. While chromosomal determinants are stable, plasmids containing virulence genes may be lost during culture and

confirmational procedures. Temperatures above 30°°C are known tocause the loss of virulence plasmids in pathogenic

Y. en t er oc ol i t i ca , but plasmid loss may also occur under other,less well-defined, circumstances.

Numerous enrichment schemes have been described for the recoveryof Yer s i ni a en t er oc ol i t i ca from meat samples. These enrichment procedures include cold enrichment for up to a month, directselective enrichment, or two-step pre-enrichment/selectiveenrichment procedures. It appears that some enrichment proceduresare better suited for the recovery of pathogenic Y. en t er oc ol i t i ca

than others, though recovery may be influenced by the type of meat product. Even when using an enrichment and plating schemereported to give good recovery from a particular meat product,considerable variation in recovery may be observed. Methodsreported to provide good recovery of pathogenic Y. en t er oc ol i t i ca

in one part of the world may not work so well in anothergeographical area, possibly due to differences in levels of Y.

en t er oc ol i t i ca and competing flora.

Recovery of pathogenic Y. en t er oc ol i t i ca is contingent upon anumber of factors including: the level of background flora on the product; the amount of background flora coming through enrichmentand plating; the level of pathogenic Y. en t er oc ol i t i ca present onthe sample; the numbers of non-pathogenic Y. en t er oc ol i t i ca and non-pathogenic Yer s i ni a spp. present on the product; and loss of

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virulence factors during enrichment and plating. Furthermore, arecovery method which gives good recovery of one serotype of pathogenic Y. en t er oc ol i t i ca may not be suited to other serotypes.In order to recover any of the important pathogenic serotypes of

Y. en t er oc ol i t i ca which might be present, multiple enrichment

broths and plating media are usually recommended for the recoveryof the organism from naturally-contaminated foods.

As there is no "universal" enrichment scheme capable of reliablyisolating all important pathogenic serotypes of Y. en t er oc ol i t i ca ,recovering serotypes O:3, O:8, and O:5,27 necessitates the use of parallel procedures. This protocol specifies the use of threeseparate enrichment procedures in combination with twoselective/differential agars. Even with the use of multiplecultural enrichment schemes, however, shortcomings of conventionalcultural procedures for the recovery of pathogenic

Y. en t er oc ol i t i ca undoubtedly result in an under-estimation of the prevalence of this organism in foods and in clinical specimens. A study reported that while 18% of raw pork products were found tocontain Y. en t er oc ol i t i ca serotype O:3 by two cultural procedures,use of a genetic probe on plated enrichments gave a detection rateof 60%. One of the main difficulties encountered duringconventional cultural isolation of pathogenic Y. en t er oc ol i t i ca

appeared to be overgrowth of small numbers of pathogenic Y.

en t er oc ol i t i ca by nonpathogenic yersiniae and other microorganisms. The use of conventional cultural procedures forthe detection and recovery of pathogenic Y. en t er oc ol i t i ca by FSISsets the stage for a move towards use of genetically-based detection methods.

A great deal of effort must be expended in the recovery and characterization of presumptively-pathogenic Y. en t er oc ol i t i ca .Sequential levels of characterization tests include:identification of presumptive Yer s i ni a , speciation toY. en t er oc ol i t i ca , biogrouping the Y. en t er oc ol i t i ca , followed bytesting for pathogenicity markers. Y. en t er oc ol i t i ca is more

active biochemically at 25°°C than at 35-37°°C, meaning thatdisparate results for a given test may be obtained depending onincubation temperature. This characteristic, coupled with the

known temperature-sensitivity of the Y. en t er oc ol i t i ca virulence plasmid, makes strict adherence to temperature and timerequirements a necessity. A word to the reader: although theextensive characterization protocol appears intimidating, the vast majority of non-Y. en t er oc ol i t i ca are effectively eliminated with minimal work by the first tier of testing.

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The enrichment and characterization procedures described in this protocol are well-documented in the literature. The inclusion ofthese procedures in the latest edition of the "Compendium of Methods for the Microbiological examination of Foods" is furtherevidence of their acceptance by the scientific community.


9.21 Equipment

a. Sterile scissors, forceps, knives, pipettes, hockeysticks, and other supplies

b. Balance (sensitivity of ± 0.1 g)c. Inoculating needles and loopsd. Vortex mixer

e. Stomacher™™ and sterile stomacher bags

f. Freezer (-70°°C)g. Stereomicroscope and oblique lighting (optional)h. Incubators capable of holding temperatures at

4 ± 1°°C, 25 ± 1°°C, 28 ± 1°°C, 30 ± 1°°C, 32 ± 1°°C, 35 ± 1°°C

and 37 ± 1°°C.

9.22 Reagents

a. 0.25% KOH in 0.5% NaCl aqueous solution b. Crystal violet (85 µg/ml aqueous solution)c. Sterile mineral oil

d. 1% Ferrous ammonium sulfate (prepare fresh on day ofuse)

e. Kovacs' reagentf. Voges-Proskauer (VP) test reagentsg. Oxidase reagent or reagent-impregnated disc/striph. Glycerol (sterile)i. 1 N HCl solution

9.23 Media

a. Irgasan-Ticarcillin-Cholate (ITC) broth

b. Trypticase Soy Broth (TSB)c. Bile-Oxalate-Sorbose (BOS) brothd. 0.01 M Phosphate Buffered Saline (PBS, pH 7.6)e. Cefsulodin-irgasan-novobiocin (CIN) agar (MUST BE MADE

ACCORDING TO FORMULATION IN APPENDIX)f. Salmonella Shigella Deoxycholate Calcium (SSDC) agarg. Kligler's Iron agar (KIA) slants

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h. Simmon's Citrate agar slantsi. Christensen's urea agar slantsj. Lysine decarboxylase medium (0.5% lysine)k. Ornithine decarboxylase medium (0.5% ornithine)l. CR-MOX (Congo Red Magnesium Oxalate) agar

m. Methyl Red-Voges Proskauer (MR-VP) brothn. ββ-D-Glucosidase test medium o. Purple broth with 1% filter-sterilized salicin p. Purple broth with 1% filter-sterilized xyloseq. Purple broth with 1% filter-sterilized sucroser. Purple broth with 1% filter-sterilized trehaloses. Purple broth with 1% filter-sterilized rhamnoset. Esculin agar slantsu. Sterile Saline (0.85% NaCl)v. Tween 80 agar (lipase test agar) w. DNase test agar

x. Tryptophan broth (indole test medium)y. Pyrazinamide agar slantsz. Veal infusion brothaa. Trypticase Soy agar or Brain Heart Infusion agar plates

NOTE: Formulations for all the very specialized media and reagents used for the isolation and identification of

Yer s i ni a are presented at the end of this chapter.

9.3 Isolation Procedures

9.31 Preparation of Sample Homogenate

a. For meat samples other than surface samples: Add 25 gof sample to 100 ml of 0.01 M Phosphate Buffered Saline(PBS: pH 7.6). Homogenize for 2 minutes in a

Stomacher™™. Allow homogenate to stand undisturbed atroom temperature for 10 minutes to allow settling oflarge meat particles.

b. For carcass surface samples: Add PBS to surface sampleso as to prepare a 2:1 ratio of volume to surface area(e.g. add 100 ml PBS to a 50 cm 2 sample). Homogenize

for 2 minutes in a Stomacher™™. Allow homogenate tostand undisturbed at room temperature for 10 minutes.

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9.32 Enrichment & Plating Procedures

In order to improve the chances of recovering pathogenicY. en t er oc ol i t i ca , three enrichment procedures (ITC, TSB/BOS, and PBS) should be used. Although this will increase a laboratory's

work-load, it is the best way to insure that any serotype of pathogenic Y. en t er oc ol i t i ca present in the product will berecovered. ITC broth provides good recovery of serotype O:3 and probably serotype O:9 Y. en t er oc ol i t i ca . TSB/BOS permits recoveryof serotype O:8. PBS-cold enrichment has been shown to recoverserotype O:5,27. KOH treatment of Y. en t er oc ol i t i ca enrichmentcultures decreases background flora. Two selective plating media,SSDC and CIN agars, are recommended for the isolation of pathogenic Y. en t er oc ol i t i ca . Figure 1 illustrates the enrichment procedures which are included in this protocol.

a. ITC broth: Transfer 2 ml of sample homogenatesupernatant into 100 ml ITC broth contained in an

Erlenmeyer flask. Incubate at 25°°C for 2 days. Spread- plate 0.1 ml onto SSDC agar and incubate the plates at

30°°C for 24 h. Spread-plate 0.1 ml onto CIN agar, and

incubate the plates at 32°°C for 18 h. Also, remove 0.5 ml of the ITC enrichment, treat it with KOH, then streak

onto CIN. Reincubate the ITC enrichment at 25°°C foranother 24 h. After the plate incubation is complete,examine the plates as described below. If colonieshaving typical Y. en t er oc ol i t i ca morphology are not

visible on the plates, the ITC culture should be plated out as before.

b. TSB/BOS: Transfer 20 ml of sample homogenate

supernatant into 80 ml TSB. Incubate at 25°°C for 24 h.Transfer 0.1 ml of the TSB culture into 10 ml BOS.

Incubate at 25°°C for 3 days. Spread-plate 0.1 ml onto

SSDC agar and incubate the plates at 30°°C for 24 h.Spread-plate 0.1 ml onto CIN agar, and incubate the

plates at 32°°C for 18 h. Also, remove 0.5 ml of theBOS enrichment, treat it with KOH, then streak onto CIN.

Reincubate the BOS enrichment culture at 25°°C for 2additional days, then plate as before.

c. PBS: Refrigerate the remainder of the PBS homogenate at

4°°C for 14 days. Spread-plate 0.1 ml onto CIN agar, and

incubate the plates at 32°°C for 18 h. Also, remove 0.5

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ml of the PBS enrichment, treat it with KOH, then streakonto CIN. Also, use KOH treatment with plating ontoCIN.

d. KOH treatment: Add 0.5 ml of enrichment culture to 4.5

ml KOH/NaCl. Vortex briefly (3-4 sec) and IMMEDIATELYstreak a loop-full of the KOH-treated broth onto CINagar (Do NOT use KOH treatment in combination with SSDCagar).

9.33 Selection of Colonies from Plating Media

Due to the fact that SSDC and CIN agars are not completelyinhibitory to non-yersiniae, a variety of non-Yer s i ni a organisms may be recovered from these agars. Some of these organisms (e.g.strains of Ci t r obac t er and Ent er obac t er ) have a colonial

morphology similar to that of Y. en t er oc ol i t i ca . Care must beexercised in the selection of suspect colonies from SSDC and CINagars in order to minimize picking non-yersiniae. It may behelpful for the analyst to compare colonies growing on sample plates to colonies on the positive control plates. Colonyappearance can change over time so strict adherence totime/temperature recommendations is necessary.

a. SSDC: On SSDC, Y. en t er oc ol i t i ca colonies are typicallyround, about 1 mm in diameter and opaque or colorless. When observing plates through a stereomicroscope withoblique transillumination, look for irregular colonyedges with a finely granular colony center (neveriridescent). Non-yersiniae present either an entireedge or a coarser pattern or both.

b. CIN: On CIN, typical Y. en t er oc ol i t i ca colonies have ared bulls-eye which is usually very dark and sharplydelineated. The bulls-eye is surrounded by atransparent zone with varying radii, with the edge ofthe colony either entire or irregular; colony diameteris ca. 1-2 mm (larger colonies are usually not

Yer s i ni a ). Again, the use of a stereomicroscope and

oblique transillumination may facilitate examination of plates.

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9.4 Identification and Confirmation Procedures

9.41 Identification of Yer s i ni a

Select a colony on CIN or SSDC having morphology typical of

Y. en t er oc ol i t i ca and emulsify colony in about 1 ml of sterilesaline (0.85%). Use this to first inoculate a slant of Simmon'sCitrate Agar, then inoculate Kligler's Iron Agar, and a tube ofurea agar. Repeat until 5 colonies having morphology typical of

Y. en t er oc ol i t i ca have been selected from each plate of selectiveagar. Table 1 presents the testing scheme to which isolatesrecovered from SSDC and CIN will be submitted.

a. Simmon's Citrate: Only Streak-inoculate the slant of atube of Simmon's Citrate agar; do NOT stab the butt.

Incubate at 28°°C for 24 h. Presumptive

Y. en t er oc ol i t i ca are citrate negative (-) and thecitrate slant will remain the original green color (a positive (+) reaction is characterized by the agarturning a vivid blue color).

b. Kligler's Iron Agar: Stab-inoculate the butt and streak

the slant. Incubate at 28°°C for 18-24 h. PresumptiveY. en t er oc ol i t i ca should present an alkaline (red) slantand acid (yellow) butt, without gas or H2S on KIA.

c. Christensen's urea agar: Streak the slant with a heavy

inoculum load; do NOT stab the butt. Incubate at 28°°Cfor 24-72 h. Presumptive Y. en t er oc ol i t i ca are (+) forurease and will turn the agar to an intense red-pinkcolor.

9.42 Confirmation and Biogrouping of Yer s i ni a en t er oc ol i t i ca

Any organism which is citrate negative (-), urease positive (+),and gives an alkaline slant/acid butt without gas or H2S on KIA should be submitted to further testing. Inoculum for furthertesting may be obtained from the KIA slant; the KIA slant should

then be refrigerated pending the test results. THE TESTS LISTEDBELOW ARE ALL NECESSARY TO CONFIRM AND BIOGROUP POTENTIALLY-PATHOGENIC Y. en t er oc ol i t i ca . Do NOT attempt to biogroup anyisolate until the results are available from ALL tests!Similarly, do NOT discard any culture until ALL tests have beencompleted. See Holt et al., 1994, for additional information onspeciating Yer s i ni a .

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a. Oxidase test: Test colony growth from the KIA slant ofany presumptive Y. en t er oc ol i t i ca isolates using oxidasereagent or commercially-available, reagent-impregnated test strips/discs. Yer s i ni a are oxidase negative (-).

b. Lysine and ornithine decarboxylase: Inoculate one tubeeach of lysine decarboxylase medium and ornithinedecarboxylase medium; overlay each inoculated tube withsterile mineral oil (4-5 mm deep layer). Incubate at

28°°C for 4 days. Y. en t er oc ol i t i ca are LYS negative (-)and ORN positive (+).

c. Rhamnose, sucrose, xylose, and trehalose utilization:Inoculate one tube of each of these carbohydrate broths,

and incubate at 25°°C for 10 days, reading after 1,2,3,7,and 10 days. Y. en t er oc ol i t i ca are rhamnose negative (-)

and sucrose positive (+). Xylose and trehalosereactions vary between biogroups.

d. Salicin utilization: Inoculate a tube of salicin broth,

and incubate at 35°°C, reading after 1,2,3, and 4 days.Salicin reactions vary between biogroups.

e. Esculin hydrolysis: Inoculate a tube of esculin agar.

Incubate at 25°°C for 10 days, reading after 1,2,3,7 and 10 days. Blackening indicates esculin hydrolysis.Esculin reactions vary between biogroups of Y.

en t er oc ol i t i ca .

f. Indole test: Inoculate a tube of Tryptophan broth(indole test medium). Incubate (with loosened caps) at

28°°C for 48 h. Add 0.5 ml of Kovacs' reagent, mixgently, then allow tubes to stand about 10 minutes. A dark red color developing below the solvent layer isevidence of a positive (+) test while the color willremain unchanged in a negative (-) test. Indole testresults vary with biogroup of Y. en t er oc ol i t i ca .

g. VP test: Inoculate a tube of MR-VP broth, and incubateat 25°°C for 24 h. After incubation, add 0.6 ml αα-naphthol to the tube, and shake well. Add 0.2 ml 40%KOH solution with 0.3% creatine and shake. Read resultsafter 15 minutes and 1 hour. Development of a pink toruby red color is a positive test. Results vary with biogroup.

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h. ββ-D-Glucosidase test: Emulsify culture in saline to McFarland 3 turbidity. Add 0.75 ml of culture

suspension to 0.25 ml of ββ-D-glucosidase test medium.

Incubate at 30°°C overnight (16-20 h). A distinct yellowcolor indicates a positive reaction. Results vary with biogroup.

i. Lipase test: Inoculate Y. en t er oc ol i t i ca isolate onto a plate of Tween 80 agar (more than one isolate may be

tested per plate). Incubate at 28°°C, and examine after2 and 5 days. Lipase activity is evidenced by an opaquehalo surrounding the streak, and varies with biogroup.

j. Deoxyribonuclease (DNase) test: Inoculate

Y. en t er oc ol i t i ca strain onto a plate of DNase test agar by streaking the medium in a band (about 3/4 inch length

streak). Four or more strains may be tested per plate.

Incubate plates at 28°°C for 18-24 h. Followingincubation, examine plates as follows. For DNase testagar, flood plate with 1 N HCl. A zone of clearingaround a colony indicates a positive test. Observe forclear zones surrounding the streak (no clearing or auniformly opaque agar indicates a negative reaction).DNase test agars containing toluidine blue or methylgreen may also be used; follow manufacturer'sinstructions for interpreting results.

k. Pyrazinamidase test: Inoculate strains over entireslant of pyrazinamide agar and incubate at 25°°C for 48h. Flood slant surface with 1 ml of freshly prepared 1%(w/v) aqueous solution of Fe+2 ammonium sulfate. Read after 15 minutes; a pink to brown color indicates PYR positive (+); (presence of pyrazinoic acid) while nocolor development is observed with PYR negative (-)strains. Pathogenic strains are PYR negative (-).

9.43 Testing for Pathogenicity Markers

Presumptive pathogenic Y. en t er oc ol i t i ca are LYS negative (-),ORN positive (+), sucrose positive (+), salicin negative (-) and esculin negative (-). Once the results from all the biogroupingtests are available, Table 2 should be consulted for informationon biogroup designation. Y. en t er oc ol i t i ca isolates belonging toBiogroups 1B, 2, 3, 4, or 5 should be subjected to further testingfor pathogenicity markers.

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a. Auto-agglutination in MR-VP broth: Inoculate 2 tubes of

MR-VP broth; incubate one at 25°°C for 24 h, and the

other at 35°°C for 24 h. After incubation, the tubeincubated at the lower temperature should exhibitturbidity from cell growth. The tube which had been

incubated at 35°°C should show agglutination (clumping)of bacteria along the walls and/or bottom of tube and clear supernatant fluid. Test is plasmid-dependent.

b. Congo red binding/crystal violet binding: Grow isolates

in TSB at 25°°C for 16-18 h, then dilute in saline toobtain about 104 cfu/ml and dilute to 10-5. Spread-plate10 µl of diluted suspension on CR-MOX plates. Incubate

plates at 37°°C for 24 h. A predominance of tiny red colonies is indicative of a positive response for bothcongo red binding and calcium dependency (some largecolorless colonies [CR-MOX negative] may be present dueto loss of the virulence plasmid). Perform crystalviolet binding on the same agar by flooding each plate with about 8 ml of crystal violet (85 µg/ml), allowingthis to stand for 2 minutes, then decanting off the dye.If desired, plates may be observed with a stereodissecting microscope at 40X magnification. Examinecolonies as soon as possible as color tends to fade withtime; positive isolates display small, intensely purplecolonies. CR-MOX permits demonstration of calcium dependency, Congo red binding, and crystal violet dye

binding. Test is plasmid-dependent.

9.5 Method Quality Control Procedures

Due to the variety of bio-serogroups of Y. en t er oc ol i t i ca whichcan be found on meat and poultry, a cocktail of control cultures(including serotypes O:3 and O:8) should be used as a positivecontrol. In addition, an uninoculated media control should beutilized for each of the different enrichment media.

Inoculate control strains into separate tubes of TSB. Incubate at

25°°C for 18-24 h. In order to provide ca. 30-300 cfu/ml, make a10-7 dilution of each culture in sterile saline. Add 1 ml of the10-7 dilution of each culture to a single bottle containing 50 mlPBS. Mix well. From this point forward, treat thePBS/Y. en t er oc ol i t i ca positive-control cocktail as a sample,following the instructions given above in Section 9.32. Confirm

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at least one isolate (of each morphological type present on eachof the agars) recovered from the positive-control sample.

9.6 Storage of Isolates

9.61 Maintenance of Y. en t er oc ol i t i ca Control Strains

Because of the possibility of plasmid loss in virulentY. en t er oc ol i t i ca , it is recommended that control strains of

Y. en t er oc ol i t i ca be immediately subcultured upon receipt

(incubating at temperatures below 30°°C), then preserved in afrozen state.

Inoculate a tube of veal infusion broth with each control strain.

Incubate for 48 h at 25°°C. Add sterile glycerol to a finalconcentration of 10% (e.g. 0.3 ml in 3 ml veal infusion broth),

dispense into several sterile vials, and freeze immediately at-70°°C. Preparation of a batch of vials for each strain isrecommended so that one vial can be held in reserve to serve as asource of inoculum for preparation of a new batch of frozenstocks.

When a fresh culture of a control strain is needed, a small portion of frozen suspension may be removed aseptically and

transferred to a tube of TSB. Incubation should be at 25°°C for 24h, followed by streaking onto a non-selective agar such as TSA or

BHI agar with incubation at 25°°C for 24 h.

Strains may be kept on TSA or BHI slants at 4°°C for short periodsof time, but it is not recommended that such strains betransferred due to the possibility of plasmid loss.

Periodically, control cultures should be tested for pathogenicity markers as described above. Cultures which have lost thevirulence plasmid should be destroyed, and replaced by a freshsubculture from the frozen stock preparation.

9.62 Maintenance of Isolates During Confirmation

Due to the possibility of plasmid loss during extensive

subculturing (even at temperatures below 30°°C), it is recommended that presumptive Y. en t er oc ol i t i ca isolates be frozen following Y.

en t er oc ol i t i ca confirmation testing.

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From the KIA slant of a presumptive Y. en t er oc ol i t i ca isolate,inoculate a tube of veal infusion broth.

Incubate for 48 h at 25°°C. Add sterile glycerol to a final

concentration of 10%, and freeze immediately at -70°°C.

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Anonymous. 1993. Yer s i ni a en t er oc ol i t i ca enrichment and plating media. Int. J. Food Microbiol. 17:257-263.

Aulisio, C. C. G., I. J. Mehlman, and A. C. Sanders. 1980. Alkali method for rapid recovery of Yer s i ni a en t er oc ol i t i ca

and Yer s i ni a ps eudot ube r cul osi s from foods. Appl. Environ. Microbiol. 39:135-140.

Bhaduri, S., Conway, L. K., and R. V. Lachica. 1987. Assayof crystal violet for rapid identification of virulent plasmid-bearing clones of Yer s i ni a en t er oc ol i t i ca . J. Clin. Microbiol. 25:1039-1042.

Boer, E. de. 1992. Isolation of Yer s i ni a en t er oc ol i t i ca

from foods. Int. J. Food Microbiol. 17:75-84.

Bottone, E. J., J. M. Janda, C. Chiesa, J. W. Wallen, L.Traub, and D. H. Calhoun. 1985. Assessment of plasmid profile, exoenzyme activity, and virulence in recent humanisolates of Yer s i ni a en t er oc ol i t i ca . J. Clin. Microbiol.22:449-451.

Caugant, D. A., S. Aleksic, H. H. Mollaret, R. K. Selander,and G. Kapperud. 1989. Clonal diversity and relationshipsamong strains of Yer s i ni a en t er oc ol i t i ca . J. Clin. Microbiol. 27:2678-2683.

Chiesa, C. L. Pacifico, and G. Ravagnan. 1993.Identification of pathogenic serotypes of Yer s i ni a

en t er oc ol i t i ca . J. Clin. Microbiol. 31:2248.

Farmer, J. J. III., G. P. Carter, V. L. Miller, S. Falkow,and I. K. Wachsmuth. 1992. Pyrazinamidase, CR-MOX agar,salicin fermentation-esculin hydrolysis, and D-xylosefermentation for identifying pathogenic serotypes of Yer s i ni a

en t er oc ol i t i ca . J. Clin. Microbiol. 30:2589-2594.

Farmer, J. J. III, G. P. Carter, I. K. Wachsmuth, V. L. Miller, and S. Falkow. 1993. Identification of pathogenicserotypes of Yer s i ni a en t er oc ol i t i ca . J. Clin. Microbiol.31:2248-2249.

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Holt, J. G., N. R. Krieg, P. H. A. Sneath, J. T. Staley, and S. T. Williams. 1994. Genus Yer s i ni a , p. 189, 220, and 249-252. I n Bergey's Manual of Determinative Bacteriology, 9thEdition. Williams & Wilkins. Baltimore, MD.

Kandolo, K., and G. Wauters. 1985. Pyrazinamidase activityin Yer s i ni a en t er oc ol i t i ca and related organisms. J. Clin. Microbiol. 21:980-982.

Kotula, A. W., and A. K. Sharar. 1993. Presence of Yer s i ni a

en t er oc ol i t i ca serotype O:5,27 in slaughter pigs. J. Food Prot. 56:215-218.

Kwaga, J. K. P., and J. O. Iversen. 1992. Laboratoryinvestigation of virulence among strains of Yer s i ni a

en t er oc ol i t i ca and related species isolated from pigs and

pork products. Can. J. Microbiol. 38:92-97.

Kwaga, J., J. O. Iversen, and J. R. Saunders. 1990.Comparison of two enrichment protocols for the detection of

Yer s i ni a in slaughtered pigs and pork products. J. Food Prot. 53:1047-1049.

Laack, R. L. J. M. van, J. L. Johnson, C. J. N. M. van derPalen, F. J. M. Smulders, and J. M. A. Snijders. 1993.Survival of pathogenic bacteria on pork loins as influenced by hot processing and packaging. J. Food Prot. 56:847-851,873.

Lee, L. A., A. R. Gerber, D. R. Lonsway, J. D. Smith, G. P.Carter, N. D. Puhr, C. M. Parrish, R. K. Sikes, R. J. Finton,and R. V. Tauxe. 1990. Yer s i ni a en t er oc ol i t i ca O:3infections in infants and children associated with thehousehold preparation of chitterlings. N. Engl. J. Med.322(14):984-987.

Lee, L. A., J. Taylor, G. P. Carter, B. Quinn, J. J. FarmerIII, R. V. Tauxe, and the Yer s i ni a en t er oc ol i t i ca

Collaborative Study Group. 1991. Yer s i ni a en t er oc ol i t i ca

O:3: an emerging cause of pediatric gastroenteritis in theUnited States. J. Infect. Dis. 163:660-663.

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Nesbakken, T., G. Kapperud, K. Dommarsnes, M. Skurnik, and E.Hornes. 1991. Comparative study of a DNA hybridization method and two isolation procedures for detection of Yer s i ni a

en t er oc ol i t i ca O:3 in naturally contaminated pork products. Appl. Environ. Microbiol. 57:389-394.

Portnoy, D. A., S. L. Moseley, and S. Falkow. 1981.Characterization of plasmids and plasmid-associated determinants of Yer s i ni a en t er oc ol i t i ca pathogenesis.Infect. Immun. 31:775-782.

Riley, G., and S. Toma. 1989. Detection of pathogenic

Yer s i ni a en t er oc ol i t i ca by using Congo red-magnesium oxalateagar medium. J. Clin. Microbiol. 27:213-214.

Schiemann, D. A. 1979. Synthesis of a selective agar medium

for Yer s i ni a en t er oc ol i t i c a . Can. J. Microbiol. 25:1298-1304.

Schiemann, D. A. 1982. Development of a two-step enrichment procedure for recovery of Yer s i ni a en t er oc ol i t i ca . Appl.Environ. Microbiol. 43:14-27.

Schiemann, D. A. 1983. Comparison of enrichment and plating media for recovery of virulent strains of Yer s i ni a

en t er oc ol i t i ca from inoculated beef stew. J. Food Prot.46:957-964.

Schiemann, D. A., and G. Wauters. 1992. Yer s i ni a , p. 433-450. I n C. Vanderzant and D. F. Splittstoesser (ed.),Compendium of Methods for the Microbiological Examination ofFoods, 3rd Edition. Amer. Publ. Hlth. Assoc., WashingtonD.C. 20005.

Tauxe, R. V., G. Wauters, V. Goossens, R. van Noyen, J. Vandepitte, S. M. Martin, P. de Mol, and G. Thiers. 1987.Yer s i ni a en t er oc ol i t i ca infections and pork: the missinglink. Lancet 1:1129-1132.

Toma, S., and V. R. Deidrick. 1975. Isolation of Yer s i ni a en t er oc ol i t i ca from swine. J. Clin. Microbiol. 2:478-481.

Wauters, G. 1973. Improved methods for the isolation and recognition of Yer s i ni a en t er oc ol i t i ca . Contrib. Microbiol.Immunol. 2:68-70.

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Wauters, G., K. Kandolo, and M. Janssens. 1987. Revised biogrouping scheme of Yer s i ni a en t er oc ol i t i ca . Contrib. Microbiol. Immunol. 9:14-21.

Wauters, G., V. Goossens, M. Janssens, and J. Vandepitte.

1988. New enrichment method for isolation of pathogenicYer s i ni a en t er oc ol i t i ca serogroup O:3 from pork. Appl.Environ. Microbiol. 54:851-854.

Weagant, S. D., P. Feng, and J. T. Stanfield. 1992. Yer s i ni a

en t er oc ol i t i ca and Yer s i ni a ps eudo t ube r cul os i s , p. 95-109.I n FDA Bacteriological Analytical Manual, 7th Edition. AOACInternational Inc., Gaithersburg, MD. 20877.

Zink, D. L., J. C. Feeley, J. G. Wells, C. Vanderzant, J. C. Vickery, W. D. Rood, and G. A. O'Donovan. 1980. Plasmid-

mediated tissue invasiveness in Yer s i ni a en t er oc ol i t i ca . Nature 283:224-226.

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Figure 1. Enrichment schemes used for the recovery of pathogenic Y. en t er oc ol i t i ca from meat or poultry samples.

Homogenize Sample in PBS

2 ml into 100 ml ITC broth 20 ml into 80 ml TSB remainder

of homogenate

2 days 1 day 14 days

25°°C 25°°C 4°°C

--Onto SSDC --0.1 ml TSB culture + 10 ml BOS --Onto CIN

24 h 25°°C

30°°C 3 days --KOHOnto CIN

--Onto CIN --Onto SSDC18 h

32°°C --Onto CIN

--KOH treatment -KOH treatmentOnto CIN Onto CIN

After 1 additional daya After 2 additional daysof broth incubation of broth incubation

--Onto SSDC --Onto SSDC

--Onto CIN --Onto CIN

--KOH treatment --KOH treatment

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Onto CIN Onto CIN

a Plating should only be done if colonies having typical Y. en t er oc ol i t i c a morphologyare not present on plates inoculated on previous day.

Table 1. Sequence of Confirmation, Biogrouping, and Pathogenicity-marker Tests used forY. en t er oc ol i t i ca

Yer s i ni a Simmons' Citrate Kligler's Iron Agar Christensen'sConfirmation slant slant & butt urea agarTests

28°°C, 24-72 h 28°°C, 18-24 h 28°°C, 18-72 h

Citrate (-) Alk/Acid Urea (+)(green) no H2S (pink)little/no gas

Y. Oxidaseen t er oc ol i t i ca Lysine decarboxylaseConfirmation Ornithine decarboxylase

Tests Rhamnose utilizationSucrose utilization

Y . Lipaseen t er oc ol i t i ca DNaseBiogrouping IndoleTests Xylose

VP

ββ-D-Glucosidase

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PyrazinamidaseSalicin; EsculinTrehalose; Nitrate Reduction

Pathogenicity- Autoagglutination in MR-VP broth Marker Congo Red BindingTests Crystal Violet Binding

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Table 2. Biogrouping Scheme for Yer s i ni a en t er oc ol i t i ca a

Biogroups b

1A 1Bc 2c 3c 4c 5c

Lipase (Tween-esterase) + + - - - -Esculin/salicin 24 hd +,- - - - - -

Indole + + (+)

e

- - -Xylose + + + + - V f

Trehalose/NO3g + + + + + -

Pyrazinamidase + - - - - -

ββ-D-Glucosidase + - - - - - Voges-Proskauer + + + +h + (+)DNase - - - - + +

a Modified from Wauters et al., 1987.

b Reactions from tests incubated at 25-28°°C, with the exception of ββ-D-Glucosidase which

was incubated at 30°°C and salicin which was incubated at 35°°C. Incubation at othertemperatures may result in different results and biogroupings.

c Biogroup contains pathogenic strains.

d Esculin and salicin reactions for a given strain of Y. en t er oc ol i t i ca are nearlyalways identical so they are listed together in this table.

e Indicates a delayed positive reaction.

f Indicates variable reactions.

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g Trehalose and nitrate reduction reactions for a given strain of Y. en t er oc ol i t i ca arenearly always identical so they are listed together in this table.

h Rarely, a serotype O:3 strain may be negative for VP.

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ADDENDUM

Formulations for Media and Reagents for Yer s i ni a en t er oc ol i t i ca

Isolation and Identification

ββ-D-Glucosidase test

Add 0.1 g 4-nitrophenyl-ββ-D-glucopyranoside to 100 ml 0.666 M NaH2PO4 (pH 6.0), dissolve, then filter-sterilize.

BOS broth

Na2HPO4*7H2O 17.25 g Na oxalate 5.0 gBile salts No. 3 (Difco) 2.0 g NaCl 1.0 g

0.1% solution of MgSO4*7H2O 10.0 mlDistilled deionized H2O 639.0 ml

Combine ingredients and mix until dissolved, adjust pH to 7.6

with 5 N HCl, then autoclave at 121°°C for 15 minutes.

Add the following filter-sterilized solutions:

100 ml of 10% sorbose100 ml of 1.0% asparagine100 ml of 1.0% methionine

10 ml of 2.5 mg/ml metanil yellow10 ml of 2.5 mg/ml yeast extract10 ml of 0.5% Na pyruvate1 ml of 0.4% solution of Irgasan DP300 (2,4,4'-trichloro-2'-hydrox

Adjust pH to 7.6 with either 5 N NaOH or HCl as required.

Store at 4°°C for up to 7 days.

On day of use, add 10 ml of 1.0 mg/ml Na furadantin (from

stock solution stored at -70°°C) to the above complete base. Aseptically dispense 10 ml portions into sterile tubes.

CIN agar

MUST CONTAIN Cefsulodin at 4 mg/L:

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This formulation is commercially available from Difco; premixes available from other manufacturers contain differentlevels of cefsulodin.

Oxoid special peptone 20.0 g

Yeast extract 2.0 g Mannitol 20.0 g Na pyruvate 2.0 g NaCl 1.0 g0.1% aqueous stock solution of MgSO4*7H2O 10.0 ml Na deoxycholate 0.5 gOxoid No. 4 (L11) agar 12.0 gDistilled deionized H2O 748.0 ml

Bring to a boil in order to dissolve agar completely (do NOT

autoclave). Cool to around 80-85°°C.

Add 10 ml of Irgasan DP300 (2,4,4'-trichloro-2'-hydroxydiphenyl ether, Ciba Geigy) solution (0.04% in 95%ETOH). Shake vigorously to disperse ethanol. Cool in a

water bath to ca. 50-55°°C.

Add 1 ml of 5 N NaOH, then 10 ml of each of the followingaqueous, filter sterilized (0.22 µm pore size) stocksolutions:

neutral red (3 mg/ml)

crystal violet (0.1 mg/ml)cefsulodin (0.4 mg/ml)novobiocin (0.25 mg/ml).

[Stock antibiotic solutions are stored at -70°°C and thawed atroom temperature just before use]

Adjust final pH to 7.4 with 5 N NaOH. Store prepared plates

at around 20-25°°C for up to 9 days.

CR-MOX agar

Tryptic soy agar 40.0 gDistilled deionized H2O 825.0 ml

Mix and autoclave at 121°°C for 15 minutes. Cool basal medium

to 55°°C.

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Add the following solutions:a) 80 ml of 0.25 M sodium oxalate (Sigma) solution

(sterilized by autoclaving at 121°°C for 15 minutes) b) 80 ml of 0.25 M magnesium chloride solution (sterilized

by autoclaving at 121°°C for 15 minutes)

c) 10 ml of 20% D-galactose solution (sterilized byautoclaving at 115°°C for 10 minutes)

d) 5 ml of 1% Congo red solution (sterilized by autoclaving

at 121°°C for 15 minutes)

Mix well and dispense into 15 X 100 mm petri dishes. Store

prepared media in plastic bags at 4°°C for up to 3 months.

DNase test Agar

Tryptose 20.0 gDeoxyribonucleic acid 2.0 gSodium chloride 5.0 g Agar 15.0 gDistilled water 1.0 L

Suspend all ingredients and heat to boiling to dissolvecompletely. Sterilize in the autoclave at 121oC for 15 minutes, final pH = 7.3. Dispense into sterile Petri dishes.

Esculin agar

Polypeptone (Oxoid) 10.0 gEsculin 1.0 gFerric ammonium citrate 1.0 g Agar 5.0 gDistilled deionized H2O 1.0 L

Mix well. Dispense into tubes, and autoclave at 121°°C for 15 minutes.

Indole test medium

Prepare a 1% solution of Bacto Peptone (Difco) OR 1% Trypticase peptone (BBL) OR use Tryptone Water (Oxoid). Dispense 5 ml

quantities into tubes. Sterilize by autoclaving at 121°°C for 15 minutes.

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ITC broth

Tryptone 10.0 gYeast extract 1.0 g MgCl2*6H2O 60.0 g

NaCl 5.0 g0.2% (w/v) malachite green solution (aqueous) 5.0 mlKClO3 1.0 gDistilled deionized H2O 1.0 L

Mix above ingredients, autoclave at 121°°C for 15 minutes,cool. Then add,

a) 1 ml of Ticarcillin solution (1 mg/ml in H2O; filter-sterilized) (Ticarcillin available from Sigma)

b) 1 ml of Irgasan DP300 (1 mg/ml in 95% ethanol); AKA

2,4,4'-trichloro-2'-hydroxydiphenyl ether (CIBA-Geigy,Basel)

c) Mix well. Dispense 100 ml into sterile 100 mlErlenmeyer flasks (it is important to minimize the

surface area:volume ratio). Store at 4°°C for up to 1 month.

Kligler's iron agar (KIA) slants

Polypeptone peptone 20.0 gLactose 20.0 g

Dextrose 1.0 g NaCl 5.0 gFerric ammonium citrate 0.5 gSodium thiosulfate 0.5 g Agar 15.0 gPhenol red 0.025 gDistilled water 1.0 L

Heat with agitation to dissolve completely. Dispense into13 X 100 mm screw-cap tubes and autoclave for 15 minutes at121oC. Cool and slant to form deep butts. Final pH = 7.4.

KOH solution

NaCl 5.0 gKOH 2.5 gDistilled deionized H2O 1.0 L

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Dispense 4.5 ml amounts in small screw-cap tubes, and

sterilize at 121°°C for 15 minutes. Tighten caps when cool. Make only a small number of tubes at a time since pH

decreases with storage time; store at 4°°C for no more than 7days.

Pyrazinamide agar

Tryptic soy agar (Difco) 30.0 gPyrazine-carboxamide (Merck) 1.0 g0.2 M Tris-maleate buffer (pH 6) 1.0 L

Mix well, dispense 5 ml amounts in tubes (160 X 16 mm).

Autoclave at 121°°C for 15 minutes. Slant for cooling.

SSDC agar

SS agar (quantity per liter as stated by a particular Manufacturer)

Yeast extract 5.0 g Na deoxycholate 10.0 gCaCl2 1.0 gDistilled deionized H20 1.0 L

Adjust pH to 7.2 to 7.3 Bring agar almost to a boil on a hot

plate (Do NOT autoclave). Temper agar to 55-60°°C, mix and

pour while still warm, making thick plates. Store prepared plates for 7 days at 20-25°°C in the dark. Do NOT store at

4°°C.

Tween 80 agar (Lipase test agar)

Peptone 10.0 g NaCl 5.0 gCaCl2*H2O 0.1 g Agar 15.0 gDistilled deionized H2O 1.0 L

Sterilize agar base by autoclaving at 121°°C for 15 minutes.

Temper to 45-50°°C.

Sterilize Tween 80 by autoclaving at 121°°C for 20 minutes.

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Add sterile Tween 80 to tempered agar base to give a finalconcentration of 1% (v/v). Mix well. Dispense into Petridishes, and allow to solidify.

Veal infusion broth

Veal, infusion from 500.0 gProteose peptone # 3 10.0 g NaCl 5.0 gDistilled water 1.0 L

Heat with agitation to dissolve all ingredients. Dispense7 ml portions into 16 X 150 mm tubes and autoclave at 121oCfor 15 minutes. Final pH = 7.4.

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CHAPTER 10. EXAMINATION OF HEAT PROCESSED, HERMETICALLY SEALED(CANNED) MEAT AND POULTRY PRODUCTS

George W. Krumm, Charles P. Lattuada,Ralph W. Johnston, James G. Eye, and John Green

10.1 Introduction

Thermally processed meat and poultry products in hermeticallysealed containers include both shelf stable products as well asthose that must be kept refrigerated (i.e. perishable product).There are a wide variety of packages designed to totally excludeair. These include traditional rigid containers, such as metalcans and glass jars; semi-rigid containers such as plastic cans, bowls and trays; and flexible containers such as retortable

pouches and bags. The microbiological examination of these food products requires knowledge and a thorough understanding of food microbiology, food science, and packaging technology and engineering. Many books and scientific articles are available onthe processing and the laboratory testing of these products.Individuals who perform these analyses should be familiar with thecurrent procedures and methods. Some of these references arelisted in section 10.6.

10.2 Important Terms and Concepts

a. Shelf Stability (commercial sterility):

The term "shelf stability" traditionally has been used by the Agency and is synonymous with the terms"commercial sterility" or commercially sterile". Shelfstability is defined in CFR title 9, part 318, SubpartG, 318.300 (u) of the Food Safety and Inspection Service(meat and poultry) USDA regulations. Shelf stability(commercial sterility) means "the condition achieved byapplication of heat, sufficient, alone or in combination with other ingredients and/or treatments, to render the product free of microorganisms capable of growing in the

product at non-refrigerated conditions (over 50°°F, 10°°C)at which the product is intended to be held duringdistribution and storage". Such a product may containviable thermophilic spores, but no mesophilic spores orvegetative cells. These products usually are stable for

years unless stored at temperatures of 115-130°°F (46-

55°°C) which may allow swelling or flat sour spoilage to

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occur because of germination and growth of thethermophilic spores. Many low acid canned meat/poultry products contain low numbers of thermophilic spores.For this reason, samples of canned foods are not

routinely incubated at 55°°C because the results usually

will be confusing and provide no sound information.Canned food lots that are to be held in hot vending machines or are destined for tropical countries areexceptions to this rule.

b. Hermetically Sealed Container:

A container that is totally sealed to prevent the entryor escape of air and therefore secure the productagainst the entry of microorganisms.

c. Adventitious contamination:

Adventitious contamination may be defined as theaccidental addition of environmental microorganisms tothe contents of a container during analysis. This canoccur if the microbiologist has not sterilized the puncture site on the container surface or the openingdevice adequately, or is careless in manipulatingequipment or cultures. Strict attention to proper procedures is required to avoid this type ofcontamination.

d. Cured Meat/Poultry Products:

Many canned meat/poultry products contain curing saltssuch as mixtures of sodium chloride and sodium nitrite. When included in a canned meat/poultry productformulation, sodium chloride and sodium nitrite inhibitthe outgrowth of bacterial spores, particularlyclostridial spores. Lowering the pH and increasing thesodium chloride concentration enhance the inhibitoryaction of sodium nitrite. Thus, most canned, cured meat/poultry products are minimally heat processed and are rendered shelf stable by the interrelationship ofheat, pH, sodium chloride, sodium nitrite and a lowlevel of indigenous spores. Spoilage in canned cured meat/poultry products attributed to underprocessing israre. When it occurs, it is usually the result ofimproper curing rather than inadequate heating. Theheat processes used for canned, cured, shelf stable meat/poultry products are unique in that they usually

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are not designed to destroy mesophilic bacterial spores but merely to inhibit their outgrowth.

e. Uncured Meat/Poultry Products:

Canned uncured meat/poultry products are given a much more severe heat treatment than canned cured products.The treatment given to canned uncured meat/poultry products is commonly referred to as a "full retortcook".

10.21 Classification of Containers

a. Metal and plastic cans with metal double sealed end(s):

Cans must be at room temperature for classification.

Cans are classified as NORMAL if both ends are flat orslightly concave; FLIPPER when one end of a normal-appearing can is struck sharply on a flat surface, theopposite end "flips out" (bulges) but returns to itsoriginal appearance with mild thumb pressure; SPRINGER if one end is slightly convex and when pressed in willcause the opposite end to become slightly convex; SOFTSWELL if both ends are slightly convex but can be pressed inward with moderate thumb pressure only toreturn to the convex state when thumb pressure isreleased; HARD SWELL if both ends are convex, rigid and do not respond to medium hard thumb pressure. A can witha hard swell will usually "buckle" before it bursts.Hard swollen cans must be handled carefully because theycan explode. They should be chilled before openingexcept when aerobic thermophiles are suspected. Neverflame a can with a hard swell, use only chemicalsanitization.

b. Glass jars:

Classify glass jars by the condition of the lid (closure) only. Do not strike a glass jar against a

surface as you would a can. Instead shake the jarabruptly to cause the contents to exert force againstthe lid; doing so occasionally reveals a flipper.Scrutinize the contents through the glass prior toopening. Compare the contents of theabnormal/questionable jar with the contents of a normaljar (e.g., color, turbidity, and presence of gas bubbles), and record observations.

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c. Flexible containers (pouches):

Pouches usually are fabricated from laminates consistingof two or more layers (plies) of material. Retortable pouches are the most common type of flexible container

used for canned, shelf-stable products. Most pouchesare 3-ply: an outer ply of polyester film, a middle plyof aluminum foil, and an inner ply of polypropylene.The polyester functions as the heat resistant, tough protective layer; the aluminum foil as a moisture, gasand light barrier; and the polypropylene functions asthe food contact surface and the film for heat sealing.The polypropylene also provides added strength, and protects the aluminum film against corrosion by the food product. Not all retortable pouches contain an aluminum foil ply. Pouches and paperboard containers used for

non-retorted, shelf stable products (e.g. pH-controlled and hot-filled product) or aseptically filled containers may be quite different from retortable pouches inconstruction. Pouches and other flexible containers areeither factory-formed and supplied ready for filling, orare formed by the processor from roll stock.

10.22 Container Abnormalities

To determine the cause of product abnormalities, both normal and abnormal containers from the same production lot should beexamined. All observed microbiological results should becorrelated with any existing product abnormalities (Section 10.46a) such as atypical pH, odor, color, gross appearance, direct microscopic examination, etc. as well as the container evaluationfindings (Section 10.46, b,c). Non-microbial swells (such ashydrogen swells) are usually diagnosed by considering all productattributes because culture results are negative or insignificant.

a. Metal cans, plastic containers and glass jars:

Conditions such as "swells" are defined in Section 10.21(a). The defects and abnormalities associated with

these containers have been extensively detailed byothers. Rather than include extensive descriptions foreach of them in this section, the analyst is referred toseveral excellent references presented in Section 10.6.These references provide detailed information on thenumerous defects and abnormalities that can occur withthese containers. The analyst should be familiar withthese conditions before beginning any analysis of a

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defective or abnormal container. The effect of processing failures, such as overfilling, closure at lowtemperature or high altitude; container damage; and storage temperature changes, must be taken intoconsideration as the analyst evaluates possible causes

for the defect or abnormality. For quick reference, aGlossary of Terms is provided in Appendices I and II.

b. Pouches:

A Glossary of Terms for these containers can be found in Appendix III. It is imperative to follow uniform procedures (Section 10.46,c) when examining defective orabnormal pouches. The APHA, 1966 reference (Section10.6) provides detailed information on the analysis of pouch defects.

10.3 Analysis of Containers

The number of containers available for analysis will vary.However, it is important that the number be large enough to provide valid results. Unless the cause of spoilage is clear cut,at least 12 containers should be examined. With a clear cutcause, one half this number may be adequate. If abnormalcontainers have been reported, but are not available for analysis,incubation of like-coded containers may reproduce the abnormality.

The "normal" cans should be incubated at 35°°C for 10 days prior to

examination. Incubation temperatures in excess of 35°°C should not be used unless thermophilic spoilage is suspected. This

incubation may reproduce the abnormality, and thereby document progressive microbiological changes in the product. Examine theincubated cans daily. Remove any swells from the incubator asthey develop and culture them along with a normal control. Afterthe 10 day incubation period, cool the cans to room temperatureand reclassify. Swollen, buckled and blown containers should NOT be incubated but analyzed immediately along with a normal control. All steps in the analysis should be conducted in sequenceaccording to protocol.

10.31 Physical Examination of Metal and Plastic Containers

a. Before opening, visually examine the double end seam(s)and side seam (if present) for structural defects, flawsand physical damage; record pertinent observations.

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b. Run thumb and forefinger around the inside and outsideof the double seams for evidence of roughness,unevenness, or sharpness.

c. Using a felt marker, make three slash marks at irregular

intervals across the label and the code-end seam.Remove the label and copy any label code-numbers to theside of the container along with a mark indicating thecode end of the can. Correlate any stains on the label with suspicious areas on the side panel (can body) byreturning the label to its exact position relative tothe slash marks.

d. Examine all non-seam areas of the can and ends for anyevidence of physical damage. If the code is embossed,carefully examine it for any evidence of puncturing.

Circle any suspect and/or defective areas with anindelible pen and record this information on the worksheet. For an illustration of these defects see the APHA, 1966 reference (Section 10.6).

10.32 Physical Examination of Glass Jars

a. Before opening, remove the label and, using a good lightsource such as a microscope light, examine the containerfor apparent or suspected defects. Microorganisms mayenter jars through small cracks in the glass. Make noteof any residue observed on the outer surface and thelocation.

b. Test the closure gently to determine its tightness. After sampling has been completed, examine the lid (closure) and the glass rim (sealing surface) of thejar. Look for flaws in the sealing ring or compound inside the closure; for food particles lodged betweenthe glass and the lid; and for chips or uneven areas inthe glass rim.

10.33 Physical Examination of Pouches

a. Pouches should be examined using an illuminated 5X magnifier.

b. Hold the pouch in one hand, examine it forabnormalities, such as swelling, leakage, overfilling,and defects such as delamination and severe distortion.Record any pertinent observations.

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c. Hold the pouch at both ends and examine both sides fornoticeable cuts, cracks, scratches, food residues, punctures, missing labels, foreign materials or otherabnormalities.

d. Carefully examine all seal areas for incomplete fusion.Pay attention to such defects as entrapped product, wrinkles, moisture and foreign material in the seal.Particular attention should be given to the final orclosing seal.

e. All actual and suspected defects should be circled withan indelible marking pen for more detailed examinationafter all sampling is complete.

10.4 Analysis of the Contents

Processing errors occur infrequently with canned products, but mayresult in the improper processing of large quantities of product.Swollen cans, for instance, may signal a microbial spoilage problem. Each abnormality in a "canned" product must beinvestigated thoroughly and correctly. The following proceduresshould be followed carefully.

10.41 Equipment and Material

a. Incubators 20°°, 35°° & 55 ± 1°°C

b. Vertical laminar flow hood c. Microscope, microscope slides & cover slipsd. pH meter equipped with a flat electrodee. Felt-tip indelible markerf. Illuminated 5X magnifierg. Sterile Bacti-disc cutter or other suitable opening

deviceh. Large, sterile plastic or metal funneli. Large autoclavable holding pansj. Sterile towelsk. Clean laboratory coat and hair covering(s)l. Sterile wide bore pipettes or 8 mm glass tubing with

cotton plugs m. Sterile serological pipettes with cotton plugsn. Safety aspiration device for pipetting (e.g. pro-

pipette)o. Sterile petri dishes, beakers, and large test tubes p. Sterile triers, cork borers, scissors, knives and 8"

forceps. Triers can be made from the tail piece of

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chrome finish sink drain pipe, 1 1/2" in diameter,flanged on one end and sharpened on the other end.

q. Sterile cotton swabs with wooden handles in glass testtubes, one per tube, or commercially sterilized swabs in paper sleeves

r. Sterile glovess. Small wire basket to hold pouches in an upright positiont. Seam analysis tools (micrometer, calipers, saw,

countersink meter, metal plate scissors, nippers).u. Vacuum gaugev. Light source such as a microscope light w. Sonic cleaning apparatusx. Transparent acrylic plate with a hole and tubing to a

vacuum sourcey. Bituminous compound in strips (tar type strips usually

available in hardware stores) stored in the 35°°C

incubatorz. Seamtest Type U (Concentrate), Winston Products Co., IncBox 3332, Charlotte, N.C., Dilute 1:300 with distilled water for use.

aa. Wooden dowels, 1/2" diameter bb. Gas cylinder clampcc. Abrasive chlorinated cleaner or a scouring pad

10.42 Media and Reagents

a. Modified Cooked Meat Medium (MCMM) STEAM JUST BEFORE USE

b. Brom Cresol Purple Broth (BCPB) or Dextrose TryptoneBrothc. Plate Count Agard. APT Agare. KF Brothf. Strong's Sporulation Medium g. Gram stain reagentsh. Spore staini. Dishwashing detergentj. Chlorine solution, (Commercial Bleach with approximately

5% available chlorine diluted 1:100 with 0.5 M phosphate buffer, pH 6.2)

10.43 Preparation

a. The Analyst

i. The analyst must wear a clean full lengthlaboratory coat.

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ii. Hair must be completely covered with a clean,disposable operating room type hair cover. A surgical face mask should be worn; if the analysthas facial hair such as beards and sideburns, the mask must completely cover it.

iii. Hands, forearms and face should be washed withgermicidal soap and water.

iv. The analyst should wear safety glasses or goggles, preferably in combination with some type of faceshield when opening swollen cans or cans suspected of being contaminated with Cl os t r i di um s pp .

b. Preparing the Environment

i. If possible, the analysis should be done in avertical laminar flow hood. If a hood is notavailable, the area used must be clean and draft-free.

ii. Flat cans should be opened in the laminar flowhood.

iii. Swells may explode or spew, therefore they should be opened outside the hood and the containertransferred to the hood only after it is opened and all gas released.

iv. Disinfect the work surface before beginning any work.

c. Preparing Metal Cans Prior to Opening

i. Scrub the non-coded end of the metal can withabrasive cleaner or a scouring pad. This removes bacteria-laden oil and protein residues. Rinse well with tap water. Cans with an "easy open" end usually are coded on the bottom. Record the code

exactly and prepare the code end as described above.

ii. Sanitize the cleaned end with chlorine solution(Section 10.42 j) either by placing clean tissuesover the end and saturating it with chlorinesolution or by immersing the end in a shallow pancontaining the solution. Allow a 15-minute contact

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time; wipe dry with sterile towels or tissue. (Analternative sanitization procedure which can beused on Normal-appearing cans ONLY is to heat theentire can surface using a laboratory burner or a propane torch until the metal becomes slightly

discolored from the heat.) Proceed as outlined inSection 10.44.

d. Preparing Jars Prior to Opening

i. Scrub the surface of the jar closure with abrasivecleaner or scouring pads. Rinse well with tap water.

ii. Sanitize the jar closure with chlorine (Section10.42 j) either by placing clean tissues over the

closure and saturating it with chlorine solution orimmersing the closure in a shallow pan containingthe solution. Allow a 15-minute contact time; wipedry with sterile towels or tissue.

e. Preparing Plastic Containers Prior to Opening

i. Scrub the bottom surface of the container withabrasive cleaner or scouring pads. Rinse well withtap water.

ii. Sanitize the bottom with chlorine solution (Section10.42 j) by placing clean tissues over the bottom and saturating it with chlorine or immersing the bottom of the container in a shallow pan containingthe solution. Allow a 15-minute contact time; then wipe dry with sterile towels or tissue.

f. Preparing Normal and Abnormal-Appearing FlexibleRetortable Pouches Prior to Opening

i. Clean the outside of the pouch with a sanitizer and rinse well.

ii. Sanitize the entire pouch in a suitably sized pan with chlorine solution (Section 10.42 j). Allow a15-minute contact time; then wipe dry with steriletowels or tissue.

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g. Preparing Swollen Cans Prior to Opening

i. Scrub the non-coded end of the chilled metal can with an abrasive cleaner or a scouring pad. Thisremoves bacteria-laden oil and protein residues.

Rinse well with tap water.

ii. Sanitize the cleaned end with chlorine solution(Section 10.42 j) either by placing clean tissuesover the end and saturating it with chlorinesolution or immersing the end in a shallow pancontaining the solution. Allow a 15-minute contacttime; then wipe dry with sterile towels or tissue.

h. Opening Devices

i. The preferred type of opening device is theadjustable Bacti-disc cutter (available from the Wilkens-Anderson Company, 4525 W. Division Street,Chicago, IL.; a similar device is available from the American National Can Co., 1301 Dugdale Rd., Waukegan, IL. Order Number WT2437). The openershould be pre-sterilized or heated in a flame toredness. If this type of device is not available,individually packaged and heat sterilized regular,all metal, kitchen-type can openers may be used.The advantage of the Bacti-disc type opener is thatit causes no damage to the double seam (simplifyinglater examination) and the size of the opening can be adjusted.

ii. Sometimes a large can (e.g. a #10 size can) may bedifficult to open. The analyst could be exposed to pathogens or their toxins if the can is not properly secured. The container can be held tightly with a gas cylinder clamp secured in aninverted position in a shallow metal drawer or traylined with a large disposable poly bag or anautoclavable tray to contain any overflow. Place

the #10 container against the clamp and secure thestrap. Rotate the can and continue cutting untilthe opening is completed. The metal tray and liner may be removed for cleaning and the clamp isautoclavable.

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10.44 Sampling

a. Normal-Appearing Metal Cans and Jars with Metal Closures

i. Prepare the area and can or jar closure as

described in section 10.43.

ii. Shake the container to distribute the contents.

iii. Use a sterilized opening device to cut the desired size entry hole. Transfer samples immediately tothe selected media with a sterile pipette or swaband proceed as outlined in Section 10.45.

iv. Aseptically transfer a representative amount of the product to a sterile test tube or other sterile

container as a working reserve. Use a pipet orsterile spoon to accomplish this.

v. Caution: The contents from overfilled cans mayflow out of the hole onto the surrounding lid surface at the time of opening. This material canthen drain back into the can when the openingdevice is removed. Should this occur, terminatethe analysis.

b. Normal and Abnormal-Appearing Plastic Containers

i. Immediately after removing the container from thechlorine solution and wiping the excess liquid, usea very hot, sterilized opening device to cut thedesired size entry hole. Transfer samplesimmediately to the selected media with a sterile pipette or swab and proceed as outlined in Section10.45.

ii. Aseptically transfer a representative amount of the product to a sterile test tube or other sterilecontainer as a working reserve. Use a pipet or

sterile spoon to accomplish this.

c. Normal and Abnormal Appearing Flexible RetortablePouches

i. Place the disinfected pouch upright in a sterile beaker and cut a two inch strip about one quarter

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of an inch under the seam edge using a sterilescissors. If possible, use a pipette to removesome of the pouch contents, otherwise use a swab.Transfer the samples immediately to the selected media with a sterile pipet or swab, proceed as in

section 10.45.

ii. Aseptically transfer a representative amount of the product to a sterile test tube or other sterilecontainer as a working reserve. Fold the edge ofthe opened pouch over against itself several timesand secure with tape until the microbiologicalanalysis is complete.

d. Swollen Cans

i. Cans displaying a hard swell should be chilled before opening. Most foods spoiled by Bac i l l us

s t ear ot her mophi l us will not produce gas (flat sourspoilage). However, if nitrate or nitrite is present in the meat/poultry product, gas may be produced by this microorganism. Cold usually willkill B. s t ear ot her mophi l us resulting in no growthin Bromcresol Purple Broth. If possible, save oneor two cans and store without refrigeration.

ii. NEVER FLAME A SWOLLEN CONTAINER - IT MAY BURST.Place the container to be opened in a large,shallow, autoclavable pan. The side seam, if present, should be facing away from the analyst. A container with a hard swell may forcefully sprayout some its contents, posing a possible hazard tothe analyst if the contents are toxic. Therefore,these cans should be considered a biohazard and precautions must be taken to protect the analyst.Protective gloves should be worn and the lab coatshould be tucked inside the cuffs of the gloves orat least secured around the wrist. Some type offacial shield is also recommended.

iii. Place the sanitized container into a biohazard bagand cover with a sterile towel or invert a sterilefunnel with a cotton filter in the stem over thecan. Place the point of the sterile opening devicein the middle of the container closure. Make asmall hole in the center of the sterilized end/closure. Try to maintain pressure over the

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hole. Release the instrument slowly to allow gasto escape into the towel or funnel.

iv. After the gas pressure has been released, enlargethe opening to the desired size to permit sampling

and aseptically remove some of the containercontents. Sample as outlined in (a) above.

10.45 Culturing

a. Inoculation of Culture Media

i. The sampling and transfer processes must beconducted aseptically; care must be taken to prevent contamination during the various manipulations.

ii. Transfer the sample at once to the selected media,inoculating each tube at the bottom. Whenever possible, use a pipet and pro-pipette to remove 1-2 ml of product for inoculating each tube of medium. When the nature of the meat/poultry product makesit impossible to use a pipet, use a sampling swab(holding it by the very end of the shaft) totransfer 1-2 g of the product to each tube. Thisis accomplished by plunging the swab into the product, then inserting the swab as far as possibleinto the appropriate tube of medium and breakingoff the portion of the shaft that was handled. Useone swab for each tube of medium. When inoculating MCMM, force the broken swab to the bottom of thetube by using the tip of another sterile swab.

iii. For each sample, inoculate 2 tubes of MCMM which were steamed (or boiled) for 10 minutes and cooled just before use and 2 tubes of Bromcresol PurpleBroth. If a tube of KF medium is inoculated at thesame time, the presence of enterococci can bedetermined rapidly.

iv. As a process control, place uninoculated swabs intoeach of two tubes of MCMM and BCP and one swab intoKF broth (if used). Additionally, label twouninoculated tubes of each medium to serve ascontrols. If multiple samples are cultured at thesame time, only one set of control tubes are needed for each medium and each temperature.

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v. After all tubes have been inoculated with a sample,aseptically transfer approximately 30 ml or a 30 g portion of the container contents to a steriletube, Whirl-Pak® or jar for retention as a workingreserve sample. Appropriately label the container

and store it in a refrigerator at approximately4°°C.

vi. Finally, transfer a portion of the containercontents to a sterile Petri plate, clean jar or beaker for pH, microscopic, organoleptic and otherrelevant analyses (10.46).

vii. Cover the hole made in the container with severallayers of sterile aluminum foil, secure the foil with tape and then store the container in a

refrigerator at approximately 4°°C. This serves asthe primary reserve. Re-enter it only as a lastresort. If the sample is a regulatory sample,chain of custody records must be maintained on it.

b. Incubation of Culture Media

i. Incubate one tube each of MCMM and BCP at 35°°C and

one tube each at 55°°C. If used, incubate the tube

of KF medium at 35°°C. For the MCMM and BCP

controls, incubate one tube at 35°° and one at 55°°C.

ii. Observe all tubes at 24 and 48 h. Tubes incubated

at 35°°C that show no growth should be incubated for

5 days before discarding. Tubes incubated at 55°°Cshould be incubated for 3 days before discarding.Subculture any questionable tubes, especially ifthe product under examination contributesturbidity.

c. Identification of Organisms

i. Use conventional bacteriological procedures tocharacterize the type(s) of microbial flora found in the contents of the container.

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ii. Use descriptive terms such as: mixed culture or pure culture, anaerobic or aerobic growth, sporeformer or non-sporeformer, mesophile orthermophile, cocci or rods.

iii. Cultures should be examined using a Gram stain.Gram stains should be done only on 18-24 hcultures. Record the morphological types observed and their Gram reaction. If the container contentsare examined microscopically using a methylene bluestain, record those observations as well. Ifendospores are present, the spore stain can be used for better definition of spore type and placement.

iv. Record all biochemical test results in addition toany characteristic growth patterns on differential

and/or selective media.

v. MCMM tubes showing a bright yellow color withvisible gas bubbles, and containing gram positiveor gram variable rods should be suspected ofcontaining gas-forming anaerobes. If Cl os t r i di um

bot ul i num is suspected, sub-cultures should be madeand incubated for 4-5 days. The original tubeshould be reincubated to check for spores. After 4- 5 days incubation, test the cultures for toxin bythe mouse bioassay (see Chapter 14).

10.46 Supportive Determinations

a. Examination of Container Contents

i. Determine the pH of the sample (10.45, a, vii)using a flat electrode. Disinfect the electrodeafter taking this measurement.

ii. If applicable, determine the water activity of thesample (Section 2.4).

iii. Examine the sample microscopically by making asimple methylene blue or crystal violet stain. A Gram stain is of no value since the age of thecells is not known and Gram-stain reactions may not be dependable in the case of old cells. Prepare aspore stain if the contents of a swollen containershow signs of digestion and few bacterial cells.

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iv. Note abnormalities observed in the containercontents such as off-odors, off-color, changes inconsistency and texture when compared with normal product. DO NOT TASTE!

b. Examination of Metal and Plastic Cans

NOTE: Whenever possible a "normal" companion can should be examined along with the abnormal one.

i. After a reserve sample has been taken and allexaminations are complete, discard any remaining product into an autoclavable bag and terminallysterilize.

ii. Disinfect the inside of the container with a

phenolic disinfectant and carefully clean it with astiff brush or use an ultra sonic bath. Do notautoclave the container since this may destroy anydefects.

iii. Examine the interior lining of metal containers for blackening, detinning and pitting.

iv. The container code should have been recorded priorto analysis; if it was not, do so now. Sometimesembossed codes are poorly impressed and can berevealed by rubbing a pencil on a paper held overthe code. If this does not work, place a thinsmooth piece of paper over the code, hold securelyand rub the paper with a clean finger in order toimpress the paper. Rerub the paper with a fingercoated with graphite. This is superior to using a pencil to rub the code. If that fails, rub thecode with carbon paper. Place transparent adhesivetape over the code and rub the tape with the backof a fingernail. Lift the tape and transfer it toany document requiring the can code. The lattertwo techniques allow a record to be kept of any

partial numbers or symbols. It is also possible to wait until the can is emptied, then view thereverse of the code from the inside. If needed,the code can be viewed in a mirror.

v. When leakage from double seams or side seams issuspected, remove excess metal from the opened end,leaving a 0.5 - 1 cm flange. Dry thoroughly,

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preferably overnight, in the 55°°C incubator. Add leak detection liquid (10.41z) to the can to adepth of 2-4 cm. Place a microleak detector on theopen end of the container. The leak detectorconsists of a transparent acrylic plate with a

vacuum gauge and connector for a vacuum source.Place a gasket (cut pieces of an automobile tireinner tube will do) between the apparatus and thecan. If the fit is not tight (e.g., end seam is bent), use modeling clay to fill in the gaps.Large cans without beading or thin metal canshaving a wider diameter than height may collapse when vacuum is applied. To prevent this from happening, use 1/2" wooden dowels cut to theappropriate length to support the can sides.Bituminous compound on the dowel ends will hold

them in place. Generally, 4 dowels are sufficientfor a #10 can. Apply the gasket and any bituminouscompound, to the open can end and fit the leakdetector plate in place. Connect the vacuum and apply 10 inches vacuum to the can. Swirl theliquid to dissipate bubbles formed by gasesdissolved in the liquid. Examine seams by coveringthem with the diluted Seamtest. Leaks areidentified by a steady stream of bubbles or asteadily increasing bubble size. After carefullyexamining all seams for leaks, increase the vacuum

to 20 inches vacuum and re-examine the seams.Leave the can under vacuum until a leak appears orfor a maximum of 2 h, and examine at half-hourintervals. Mark the location of leaks on the can'sexterior using a marking pen. When reporting, note which seam, and the distance from the side seam orsome other appropriate reference point. If noleaks were found, note test conditions (time and amount of vacuum drawn).

vi. Perform a tear-down examination of the doubleseams. The following references in Section 10.6 will guide you through this process: APHA, 1966;Food Processors Institute, 1988; Double Seam Manual; Evaluating a Double Seam, FDA Bacteriological Analytical Manual, 1992.

vii. The tightness of double seams formed by plasticcans and metal can ends may be evaluated bycomparing the actual seam thickness to the

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calculated thickness of the plastic flange, neck,or metal end. This would include three thicknessesof plastic and two of metal. Also, assesstightness by inspecting the pressure ridge, sinceit reflects the compression of the plastic body

wall. The pressure ridge should be visible and continuous. Each packer may have differentspecifications for the finished seams; ifnecessary, the analyst must call the in-plantinspector and ask for specifications for thecontainer of interest.

c. Examination of Pouches

i. The best way to determine if a pouch has leaked is by the type of microorganisms recovered.

ii. The pouch should be examined microscopicallylooking for points of light coming through thefilm. These are potential leakage sites.

10.47 Interpretation of Results

Use Tables 2, 3 and 4 to arrive at possible causes of spoilage based on all laboratory results. Caution: The tables are based on a single cause of spoilage. If there are multiple causes, thetables may not help.

10.5 Examination of Canned, Perishable Meat/Poultry Products

Perishable meat and poultry products, such as hams, luncheon meats, and loaves are packaged in hermetically-sealed containersand then heat-processed to internal temperatures of not less than

150°°F (65.5oC) and usually not greater than 160°°F (71oC)."Perishable, Keep Refrigerated" must appear on the label of these products. Although they are not shelf stable, good commercial processing usually will destroy vegetative bacterial cells. Thecombined effects of sodium nitrite, salt, refrigeration, and lowoxygen tension retard the outgrowth of the few vegetative cellsand/or spores that may survive the process. Such products canretain their acceptable quality for 1 to 3 years when properly processed and refrigerated.

10.51 Analysis of Containers

See Sections 10.3 - 10.33

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10.52 Analysis of the Contents

a. Equipment and Material

See Section 10.41

b. Media and Reagents

See Section 10.42

c. Preparation

See Section 10.43

d. Sampling

i. Using procedures already described (Section 10.44)remove approximately 50 g of sample with asterilized trier, large cork borers, scissors,knife or forceps.

ii. Place the sample into a sterile blender jar orStomacher bag, add 450 ml of sterile Butterfield'sPhosphate Diluent and homogenize for 2 minutes.This is a 1:10 dilution; make additional dilutionsthrough at least 10-4. Proceed with the culturingsteps given in Section 10.52 (e, f & g).

iii. After sampling, cover the container opening withsterile aluminum foil several layers thick and secure with tape. Place the opened sample unit inthe freezer until the analysis is complete.

e. Aerobic Plate Counts

i. Pipet 1 ml of each dilution prepared in 10.52 (d)into each of two sets of duplicate pour platesaccording to the instructions given in Section 3.4.

ii. Prepare one dilution set with Plate Count Agar.Incubate this set at 35°°C for 48 h.

iii. Substitute APT agar for the Plate Count Agar in the

other set of plates. Incubate this set at 20°°C for96 h.

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iv. Count and record the results from both sets asdescribed in Section 3.4.

f. Gas-Forming Anaerobes (GFAs)

i. Steam tubes of MCMM for 10 minutes and cool just prior to use.

ii. Inoculate each tube with l ml of each dilution prepared in 10.52 (d). Begin with the 1:10dilution and continue with subsequent dilutions.Use a separate pipet for each dilution. Dilutions must be sufficiently high to yield a negativeendpoint. Be sure that the inoculum is deposited near the bottom of the tube.

iii. Incubate these tubes for 48 h at 35°°C, but read daily.

iv. Consider any MCMM tubes showing a bright yellowcolor, containing visible gas bubbles, and containing gram positive or gram variable rods as positive for GFAs.

v. Based upon the highest dilution showing theseorganisms, report the approximate number ofgas-forming anaerobes per gram, calculated as the

reciprocal of the highest positive dilution. Ifskips occur, disregard the final actual dilutionand calculate the end point at the dilution wherethe skip occurred. This is only an approximationof the gas forming anaerobe count. A minimum ofthree tubes per dilution and an MPN table must beused for a more accurate determination.

vi. If Cl os t r i di um bot ul i num is suspected,representative tubes that have not been opened should be reincubated for a total of 4 - 5 days and then tested for botulinum toxin using the mouse bioassay (Chapter 14).

g. Enterococci

i. Transfer 1 ml of each dilution prepared in 10.52(d)to individual tubes of KF broth. Use a separate pipette for each dilution. Begin with the 1:10dilution and continue with each subsequent

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dilution. Dilutions must be sufficiently high toyield a negative end point.

ii. Incubate these tubes at 35°°C for 48 h. Tubesshowing a yellow color, turbidity and buttoning of

growth are presumptive positives.

iii. Confirm all presumptive positives microscopically.Either wet mounts examined under low light or gram stained preparations are suitable for these microscopic determinations. Microscopicdeterminations yielding cells with ovoid streptococcal morphology shall be considered confirmed positive.

iv. Report the approximate number of enterococci per

gram, calculated as the reciprocal of the highest positive confirmed dilution. If skips occur,disregard the final actual dilution and calculatethe end point at the dilution where the skipoccurred. This is only an approximation of thenumber of enterococci. A minimum of three tubes per dilution and an MPN table must be used for a more accurate determination of organisms asdescribed in 10.43-10.45 and Tables 2, 3 and 4.

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APHA 1966. Recommended Methods for the MicrobiologicalExamination of Foods. 2nd Edition. American Public Health Association, Inc., New York, New York.

Bee, G. R. and Denny, C. B., 1972, First Revision.Construction and Use of a Vacuum Micro-Leak Detector for Metal and Glass Containers. National Canners Association,(now NFPA), Washington, D.C.

Crown Cork & Seal. Top Double Seaming Manual. Crown Corkand Seal Co., Inc., 9300 Ashton Road, Philadelphia, PA 19136

Cunniff, P. (ed.). 1995. Official Methods of Analysis of AOAC International, 16th Edition. Sections 17.6 - 17.8. AOAC

International, Inc., Gaithersburg, MD 20877.

Denny, C., Collaborative Study of a Method for theDetermination of Commercial Sterility of Low-Acid Canned Foods, Journal of the Association of Official AnalyticalChemists 55 (3):613 (1972).

Double Seam Manual. Carnaud Metalbox Engineering, 79Rockland Road, Norwalk, Connecticut 06854

Evaluating a Double Seam. W. R. Grace and Company, GraceContainer Products, 55 Hayden Ave., Cambridge, Massachusetts02173

Food and Drug Administration, Bacteriological Analytical Manual, Division of Microbiology, Center for Food Safety and Applied Nutrition, 7th ed., 1992. Association of Official Analytical Chemists, 1111 North 19th Street, Suite 210, Arlington, VA 22209.

Food Processors Institute 1988. Canned Foods: Principles ofThermal Process Control, Acidification and Container ClosureEvaluation. The Food Processors Institute, Washington, D.C.

20005.

Hersom, A. C. and Hulland, E. D., 1964. Canned Foods, AnIntroduction to Their Microbiology. Chemical PublishingCompany, Inc. New York, New York.

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National Food Processors Association, 1979. Guidelines forEvaluation and Disposition of Damaged Canned Food ContainersBulletin 38-L, 2nd Edition. National Food Processors Assoc., Washinton, D.C.

National Food Processors Association, 1989. Flexible PackageIntegrity Bulletin by the Flexible Package IntegrityCommittee of NFPA. Bulletin 41-L. NFPA, Washington, D.C.

Schmitt, H. P. 1966. Commercial Sterility in Canned Foods,Its Meaning and Determination. Assoc. Food and DrugOfficials of the U.S. 30:141.

Townsend, C. T., 1964. The Safe Processing of Canned Foods. Assoc. Food and Drug Officials of the U.S. 28:206.

Townsend, C. T., 1966. Spoilage in Canned Foods. J. MilkFood Tech. 20 (1):91-94.

United States Department of Agriculture, Food SafetyInspection Service. Code of Federal Regulations, Title 9, part 318.300, Subpart G (u).

Vanderzant, C., and D. F. Splittstoesser (ed.). 1992.Compendium of Methods for the Microbiological Examination ofFoods, 3rd Edition. Amer. Publ. Hlth. Assoc., Washington,D.C. 20005.

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Appendix I

Glossary of Metal/Plastic Can Seam Terminologyfor Container Components and Defects

The same terms that are used to describe an all-metal seam applyequally well to the metal end/plastic body seam.

Base Plate: Part of a closing machine which supports cansduring seaming operation.

Beaded Can: A can which is re-enforced by having ringindentations around the body. The bead tends to keep the cancylindrical and helps to eliminate paneling of the can body.

Body: Principal part of a container - usually the largest part in one piece containing the sides (thus sidewall or body wall).

Body Hook: Can body portion of double seam. Prior toseaming, this portion was the flange of the can.

Bottom Seam: Factory end seam. The double seam of the canend put on by the can manufacturer.

Buckling: A distortion in a can end.

Can Size: Two systems are commonly used to denote can size:

i. An Arbitrary system (1, 2, etc.) with no relationto finished dimension.

ii. A system indicating the nominal finished dimensionsof a can; e.g. "307 x 512." In this example, thefirst group of digits ("307") refers to the can'sdiameter and the second set ("512"), the can'sheight. The first digit in each set representsinches, and the next two digits represent

sixteenths of an inch. Hence, the example can hasa diameter of 3-7/16 and a height of 5-12/16 (or 5-3/4) inches.

Chuck: Part of a closing machine which fits inside thecountersink and in the chuck wall of the end during seaming.

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Closing Machine: Also known as a double seamer. Machine which double seams the lid onto the can bodies.

Compound: Rubber or other material applied inside the end curl to aid in forming a hermetic seal when the end is double

seamed on the can body.

Contamination in Weld Area: Any visible burn at one or more points along the side seam of a welded can. This is a majordefect.

Countersink: On a seamed end, the perpendicular distancefrom the outermost end panel to the top seam.

Cover: Can end placed on can by packer. Also known as top,lid, packer's end, canner's end.

Cover Hook: That part of double seam formed from the curl ofthe can end.

Cross Over: The portion of a double seam at the lap.

Cross Section: Referring to a double seam, a section throughthe double seam.

Curl: The semi-circular edge of a finished end prior todouble seaming. The curl forms the cover hook of the doubleseam.

Cut Code: A break in the metal of a can due to improperembossing-marker equipment.

Cut-Over: During certain abnormal double seaming conditions,the seaming panel becomes flattened and metal is forced overthe seaming chuck forming a sharp lip at the chuck wall. Inextreme cases the metal may split in a cut-over.

Dead-Head: An incompletely rolled finished seam. Also knownas a skip, skid or spinner.

Double Seam: The joint between the end and the can bodyformed by rolling the curl under the flange (1st operation)and then pressing the metal together (2nd operation).

Droop: A smooth projection of double seam below the bottom ofa normal seam. While droops may occur at any point of theseam, they usually are evident at the side seam lap. A

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slight droop at the lap may be considered normal because ofadditional plate thickness incorporated into the seam structure.

Excessive Slivers: One or more slivers which are 1/32" or

longer. This is a minor defect of welded cans.

Factory End: Bottom or can manufacturer's end.

False Seam: A seam fault where the end and body hook are notover-lapped (engaged), although they give the appearance of a properly formed seam. Also see Knockdown Flange.

Feather: Beginnings of a cut-over. See Sharp Edge.

First Operation: The first operation in double seaming. In

this operation, the curl of the end is tucked under theflange of the can body which is bent down to form cover and body hook, respectively.

Flange: The flared portion of the can body which facilitatesdouble seaming.

Flange Crack: Any crack at the flange or immediately adjacentto the weld of welded cans. This is a major defect.

Headspace: The free space above the contents of a can and thecan lid.

Heavy Lap: A lap containing excess solder. Also called athick lap.

Hook: (i). The bent over edges of a body blank, which form the side seam lock (ii). The body and cover hooks in adouble seam.

Internal Enamel: A coating applied to the inside of the canto protect the can from chemical action by the contents or to prevent discoloration. A lacquer is usually clear; an enamel

is pigmented and opaque.

Jumped Seam: A double seam which is not rolled tight enoughadjacent to the crossover caused by jumping of the seamingrolls at the lap.

Knockdown Flange: A seam defect in which the flange is bentagainst the body of the can. The cover hook is not tucked

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inside the body hook, but lies outside of it. False seams,knockdown flanges and soft crabs are degrees of the sameeffect. In order to distinguish the degree of the defect,the following terminology is suggested:

False Seam: The cover hook and body hook are not tucked for a distance of less than an inch. Thus it may not be possible to detect a false seam until the can is torndown.

Knockdown Flange: As above, but more than an inch inlength. Body hook and cover hook in contact, but nottucked.

Soft Crab: A defect in which the body of the can is broken down and does not contact the double seam. Thus,

there is a wide open hole in the can below the doubleseam where the body was not incorporated into the seam.

Lap: The soldered but not locked portions of a side seam atthe ends of the can body before seaming and removing the canfrom the chuck at completion of the operation.

Lid: See Cover.

Lip, Spurs or Vees: Irregularities in the double seam due toinsufficient or sometimes absent overlap of the cover hook with the body hook, usually in small areas of the seam. Thecover hook metal protrudes below the seam at the bottom ofthe cover hook in one or more "V" shapes.

Loss of Overlap: Any observable loss of overlap along theside seam of a welded can. This is a critical defect.

Loose Tin: A metal can which does not appear swollen, butslight pressure reveals a looseness.

Mislock: A poor or partial side seam lock, due to improperforming of the side seam hooks.

Neck: The thickness of the top of the sidewall (body wall) ofa plastic tub, one tenth of an inch below the junction of theflange and the sidewall.

Notch: A small cut-away portion at the corners of the bodyblank. This reduces droop when double seaming.

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Oozier: An imperfect can which allows the escape of thecontents through the seam.

Open Lap: A lap failed due to various strains set up during manufacturing operations. Also caused by improper cooling of

the solder (See Weak Lap). A lap which is not properlysoldered so the two halves are not properly joined.

Over Lap: The distance the cover hook laps over the bodyhook.

Paneling: A flattening of the can side. Also used to defineconcentric (expansion) rings in can ends.

Peaking: Permanent deformation of the expansion rings on thecan ends due to rapid reduction of steam pressure at the

conclusion of processing. Such cans have no positiveinternal pressure and the ends can be forced back more orless to their normal position.

Perforation: Holes in the metal of a can resulting from theaction of acid in food on metal. Perforation may come from inside due to product in the can or from outside due to material spilled on the cans.

Pleat: A fold in the cover hook which extends from the edgedownward toward the bottom of the cover hook and sometimesresults in a sharp droop, vee or spur.

Pressure Ridge: A ridge formed on the inside of the can bodydirectly opposite the double seam, as a result of the pressure applied by the seaming rolls during seam formation.

Pucker: A condition which is intermediate between a wrinkleand a pleat in which the cover hook is locally distorted downward without actual folding. Puckers may be graded thesame way as wrinkles.

Sanitary Can: Can with one end attached, the other end put on

by the packer after the can is filled. Also known as packer's can or open top can.

Sawtooth: Partial separation of the side seam overlap at oneor more points along the side seam after performing the pulltest on a welded side seam. This is a critical defect.

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Seam Arrowing: A readily visible narrowing of the weld ateither end of the can body. This is a major defect.

Seam Width: The maximum dimensions of a seam measured parallel to folds of the seam. Also referred to as the seam

length or height.

Seam Thickness: The maximum dimension measured across or perpendicular to the layers of the seam.

Second Operation: The finishing operation in double seaming.The hooks formed in the first operation are rolled tightagainst each other in the second operation.

Sharp Edge: A sharp edge at the top of the inside portion ofthe double seam due to the end metal being forced over the

seaming chuck.

Side Seam: The seam joining the two edges of a blank to form a body.

Skipper / Spinner: See Deadhead.

Uneven Hook: A body or cover hook which is not uniform inlength.

Vee: See Lip.

Weak Lap: The lap is soldered and both parts are together.However, strain on this lap (e.g. by twisting with thefingers) will cause the solderbond to break.

Weld Crack: Any observable crack in a welded side seam. Thisis a critical defect.

Worm Holes: Voids in solder usually at the end of the sideseam. May extend completely through the width of the sideseam.

Wrinkle: The small ripples in the cover hook of a can. A measure of tightness of a seam.

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Appendix II

Glossary of Glass Container Parts

From a manufacturing standpoint, there are three basic parts to aglass container based on the three parts of glass container moldsin which they are made. These are the finish, the body and the bottom.

Finish: The finish is that part of the jar that holds the capor closure. It is the glass surrounding the opening in thecontainer. In the manufacturing process, it is made in theneck ring or the finish ring. It is so named since, in earlyhand glass manufacturing, it was the last part of the glasscontainer to be fabricated, hence "the finish". The finish

of glass containers has several specific areas as follows:

Continuous Thread: A continuous spiral projecting glass ridgeon the finish of a container intended to mesh with the thread of a screw-type closure.

Glass lug: One of several horizontal tapering protrudingridges of glass around the periphery of the finish that permit specially designed edges or lugs on the closure toslide between these protrusions and fasten the number oflugs on the closure and their precise configuration isestablished by the closure manufacture.

Neck Ring Parting Line: A horizontal mark on the glasssurface at the bottom of the neck ring or finish ringresulting from the matching of the neck ring parts with the body mold parts.

Sealing Surface: That portion of the finish which makescontact with the sealing gasket or liner. The sealingsurface may be on the top of the finish, or may be acombination of both top and side seal.

Vertical Neck Ring Seam: A mark on the glass finish resultingfrom the joint of matching the two parts of the neck ring. NOTE: Some finishes are made in a one-piece ring and do nothave this seam.

Body: The body of the container is that portion which is madein the "body-mold" in manufacturing. It is the largest partof the container and lies between the finish and the bottom.

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The characteristic parts of the body of a glass containerare:

Heel: The heel is the curved portion between the bottom and the beginning of the straight side wall.

Mold Seam: A vertical mark on the glass surface in the bodyarea resulting from matching the two parts of the body mold.

Shoulder: That portion of a glass container in which the maximum cross-section or body area decreases to join the neckor finish area. Most glass containers for processed foodshave very little neck. The neck would be a straight area between the shoulder and the bottom of the bead or, with beadless finishes, the neck ring parting line.

Side Wall: The remainder of the body area between theshoulder and the heel.

Bottom: The bottom of the container is made in the "bottom plate" part of the glass container mold. The designated partsof the bottom normally are:

Bearing Surface: That portion of the container on which itrests. The bearing surface may have a special configurationknown as the "stacking feature" which is designed to providesome interlocking of the bottom of the jar with the closureof another jar on which it might be stacked for display purposes.

Bottom Plate Parting Line: A horizontal mark on the glasssurface resulting from the matching of the body mold parts with the bottom plate.

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Appendix III

Glossary of terms - Flexible Retortable Pouches.

Adhesive: A substance applied to ply surfaces to cement thelayers together in a laminated film: (a). Polyurethaneadhesive for the outer layer (b). Maleic anhydride adduct of polypropylene for the inner layer.

Blisters: Bubbles/gaseous inclusions/particulate material, may be present between layers of laminate, usually are found in the seal area.

Bottom of Closing Seal: Portion of closing (packer) sealadjustment to the pouch contents.

Bottom Seal: A seal applied by heat and pressure to the bottom of a flexible pouch.

Cosmetic Seal: Area above the primary seal designed to closethe edges of the pouch thus preventing the accumulation ofextraneous material.

Cuts, Punctures, Scratches: Mechanical defects that penetrateone or more layers of the pouch.

Delamination: Any separation of plies through adhesivefailure. This may result in questionable integrity of the package and safety of the product.

Dirty: Smeared with product or product trapped in top edges(where there are no cosmetic seals).

Disintegrated Container: Evidence of delamination ordegradation after retorting.

Final Seal: A seal formed by heat and pressure by the packerafter pouch filling and prior to retorting.

Foil Flex Cracks/Foil Roll Holes: Visible cracks in thealuminum foil layer caused by flexing of the pouch or pinholes (roll holes) in the foil caused through manufacture ofthe aluminum ply.

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Foreign Materials: Any material (solid food, condensate,grease, voids, blemishes) that may be entrapped between the plies but usually found in the seal area.

Fusion Seal: A seal formed by joining two opposing surfaces

by the application of heat and pressure.

Hard Swell or Blown: Distention or rupture due to internalgas formation.

Inner Ply: Polypropylene coating bonded to the food surfaceside of the aluminum foil.

Laminate: Two or more layers of material held together byadhesive(s).

Leaker: Product leaking through any area of the pouch.

Outer Ply: The polyester film bonded to the exterior surfaceof the aluminum foil.

Over Carton: A separate container (usually cardboard) in which the flexible pouch is packaged for additional protection.

Package Dimensions: The measurements of retortable flexible pouches stated as length, the longest dimension (LGT), widththe second longest dimension (W), and thickness, the shortestdimension (HGT). All are given as internal measurements.

Pin Holes, Roll Holes: Holes in the aluminum foil layer only,originating during manufacturing; usually do not leak.

Preformed Seals: Seals formed by heat and pressure, by the manufacturer of the pouches, along the sides and at the bottom of the pouches.

Primary Seal: A fusion seal formed by the food processor byapplying heat and pressure immediately after filling.

Seal: A continuous joint of two surfaces made by fusion ofthe laminated materials.

Seal Width: The maximum dimension of the seal measured from the leading outside edge perpendicular to the inside edge ofthe same seal.

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Severely Damaged: Punctures, cuts or ruptures which penetrateall layers of the pouch and expose the product tocontamination.

Side Seals: Seals formed by applying heat and pressure to the

sides of the pouch's laminates to form the "preformed pouch".

Tear Nicks or Notch: Notches near the final seal to aid theconsumer in opening the pouch.

Wrinkle: A crease or pucker in the seal (Packer or Factory)areas.

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Appendix IV

Table 1. Normal pH Values for a Few Representative Canned Meat/Poultry Products.

__________________________________________________________________

Kinds of Food pH

Beans with Wieners 5.7Beef Chili 5.6Beef Paté 5.7Beef Stew 5.4 - 5.9Beef Taco Filling 5.8Beef and Gravy 5.9 - 6.1

Chicken Noodle Soup 5.8 - 6.5Chicken Soup with Rice 6.7 - 7.1Chicken Broth 6.8 - 7.0Chicken and Dumplings 6.4Chicken Vegetable Soup 5.6Chicken Stew 5.6Chicken Vienna Sausage 6.1 - 7.0Chorizos 5.2Corned Beef 6.2Corned Beef Hash 5.0 - 5.7Egg Noodles & Chicken 6.5

Ham 6.0 - 6.5Lamb, Strained Baby Food 6.4 - 6.5Pork Cocktail Franks 6.2Pork with Natural Juices 6.2 - 6.4Pork Sausage 6.1 - 6.2Roast Beef 5.9 - 6.0Spaghetti and Meatballs 5.0Spaghetti Sauce with Beef 4.2Stuffed Cabbage 5.9Sloppy Joe 4.4Turkey, Boned in Bouillon 6.1 - 6.2Turkey with Gravy 6.0 - 6.3 Vienna Sausage 6.2 - 6.5 Wieners, Franks 6.2 __________________________________________________________________

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Appendix V

Table 2. KEY TO PROBABLE CAUSE OF SPOILAGE IN CANNED FOODS

Group 1.- Low-Acid Foods pH Range 5.0 to 8.0

Condition of Characteristics of Material in Cans

cans

Odor Appearance Gas(CO2 &H2)

pH Smear Cultures Diagnosis

Swells Normal to"metallic"

Normal to frothy(Cans usually etched or corroded)

More than20% H2

Normal Negative tooccasionalorganisms

Negative Hydrogen swells

Sour Frothy; possibly ropy brine

MostlyCO2

Below Normal

Pure or mixed cultures ofrods, cocci,yeasts or molds

Growth, aerobicallyand/or anaerobically

at 35°°C., and

possibly at 55°°C.

Leakage

Sour Frothy; possibly ropy brine, food particlesfirm with uncooked appearance

MostlyCO2

Below Normal

Pure or mixed cultures ofrods, coccoids,cocci and yeasts


at 35°°C., and

possibly at 55°°C.(If product received high exhaust, onlyspore formers may berecovered)

No process given

Normal tosour-cheesy

Frothy H2 and CO2

Slightly todefinitely

belownormal

Rods, med.Short to med.long, usuallygranular;spores seldom seen

Gas, anaerobically

at 55°°C., and possibly slowly at

35°°C.

Post-processingtemperature abuseThermophilicanaerobes

Cheesy to putrid

Usually frothy withdisintegration ofsolid particles

MostlyCO2;

possiblysome H2


belownormal

Rods; usuallyspores present

Gas anaerobically at

35°°C.

Underprocessing - mesophilic anaerobes(possibility of Cl.

botulinum)

Slightlyoff –

possiblyammoniacal

Normal to frothy Slightly todefinitely

belownormal

Rods; sporesoccasionallyseen


with gas at 35°°C and

possibly at 55°°C.Pellicle in aerobic

broth tubes. Sporesformed on agar and in pellicle.

Underprocessing - B.subtilis type

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Novacuum and/orCans

buckled

Normal Normal No H2 Normal toslightly

belownormal

Negative to moderate numberof organisms

Negative Insufficient vacuum,caused by: 1)Incipient spoilage,2) Insufficientexhaust,3) Insufficient

blanch,4) Improper retortcooling procedures,5) Over fill

Flat

cans(0 tonormalvacuum)

Normal tosour

Normal to cloudy brine


belownormal

Rods, generallygranular in

appearance;spores seldom seen

Growth without gas

at 55°°C. Spore

formation onnutrient agar

Post-Processingtemperature abuse

Thermophilic flatsours.

Normal tosour

Normal to cloudy brine; possibly moldy


belownormal

Pure or mixed cultures ofrods, coccoids,cocci or mold


at 35°°C., and

possibly at 55°°C.

Leakage

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Appendix VI

Table 3. KEY TO PROBABLE CAUSE OF SPOILAGE IN CANNED FOODS

Group 3. Semi-Acid Foods pH Range 4.6 to 5.0

Condition of Characteristics of Material in Canscans

Odor Appearance Gas(CO2 &H2)

pH Smear Cultures Diagnosis

Swells Normal to"metallic"

Normal to frothy(Cans usually etched or corroded)

Morethan20% H2

Normal Negative tooccasionalorganisms

Negative Hydrogen swells

Sour Frothy; possiblyropy brine

MostlyCO2

Below Normal Pure or mixed cultures ofrods, coccoids,cocci, yeastsor molds

Growth,aerobicallyand/oranaerobically at

35°°C., and possibly at

55°°C.

Leakage

Note:Cans areSometimesflat

Sour Frothy; possiblyropy brine, food

particles firm withuncooked appearance

MostlyCO2

Below Normal Pure or mixed cultures ofrods, coccoids,cocci and yeasts

Growth,aerobicallyand/oranaerobically at

35°°C., and possibly at

55°°C. (If product received high exhaust,

only sporeformers may berecovered)

No process given

Normal tosour-cheesy

Frothy H2 and CO2


below normal

Rods - med.Short to med.long, usuallygranular;spores seldom seen

Gas,anaerobically at

55°°C., and possibly slowly

at 35°°C.

Post-processingtemperature abuseThermophilicanaerobes

Normal tocheesy to

putrid

Normal to frothy with disintegrationof solid particles

MostlyCO2;

poss-iblysome H2

Normal toslightly belownormal

Rods; possiblyspores present

Gasanaerobically at

35°°C. Putrid odor

Underprocessing – mesophilicanaerobes(possibility ofCl . Bo t u l i n um)

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Slightly off- possiblyammoniacal

Normal to frothy Slightly todefinitely

below normal

Rods;occasionallyspores observed

Growth,aerobicallyand/oranaerobically

with gas at 35°°Cand possibly at

55°°C. Pelliclein aerobic brothtubes. Sporesformed on agarand in pellicle.

Under- processing - B.

s ubt i l i s type

Butyric acid Frothy, large volumegas

H2 and CO2

Definitely below normal

Rods - bipolarstaining;

possibly spores

Gasanaerobically at

35°°C. Butyricacid odor

Under processing -

butyric acid anaerobe

No vacuum and/or Cans

buckled

Normal Normal No H2 Normal toslightly belownormal

Negative to moderate numberof organisms

Negative Insufficientvacuum, caused by:1) Incipientspoilage,2) Insufficientexhaust,3) Insufficient

blanch,4) Improper retortcooling

procedures, 5)Over fill

Flat cans(0 to normalvacuum)

Sour to"medicinal"

Normal to cloudy brine


below normal

Rods, possiblygranular inappearance

Growth without

gas at 55°°C. and possibly at

35°°C. Growth onthermoaciduransagar

Underprocessing B.

coagu lans

Normal tosour

Normal to cloudy brine; possibly moldy


below normal

Pure or mixed cultures orrods, coccoid,

cocci or mold

Growth,aerobicallyand/or

anaerobically at35°°C., and

possibly at

55°°C.

Leakage

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Appendix VII

Table 4. Characteristics of Normal and Abnormal Perishable Canned Meat/Poultry Products

Condition ofCans

Odor Appearance pH Smear Cultures ProbableCause

Flat Cans (0 to Normal Vacuum)

Normal Normal Normal Negative tooccasionalorganisms

0 to low # APC, APT agar count

Normal product

0 to degrees of

swelling

Sour to off

odor

Normal to mushy,

possible gelliquification

Slightly to

definitely below normal

Mixed culture

of rods &enterococci

Low # mesophiles,

high # psychrophilic non-spore formers(enterococci,lactobacilli

1. Prolonged stor

at low temperatur2. Abnormal highlevels in raw

materials 3.Substandard proce

Swell Sour or offodor, possibly

putrid

Normal to mushy, possible gelliquification


below normal

Mixed cultureof rods, cocci

High # mesophilicspore formers and non-sporeformers

Product held withrefrigeration

Swell Normal to sour Normal Below normal Cocci, rods or both

Enterococci, rodsor both

Leakage if shellhigher than core.Underprocessing icore higher thanshell

Swell Off odor Normal to offcolor

Below normal Rods Psychrotrophicclostridia (rarelyoccurs in U.S.).

Low brine levels

Swell Normal to putrid,depending onlength ofstorage.

Ranges from uncooked appearance todigested

Normal to low,depending onlength ofstorage.

Vary Vary Missed processingcycle.

Most of these aredetected soon aftdistribution.

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CHAPTER 11. TESTS FOR ENZYMES IN MEAT AND MEAT PRODUCTS

Charles P. Lattuada, James G. Eye, John M. Damareand B. P. Dey

11.1 Catalase Test

11.11 Introduction

Tests for catalase in meat are limited to products that have beengiven a heat treatment since the enzyme normally is present in allraw meat. It is particularly useful for roast beef. This procedure will detect under-processing when the product is

scheduled to be heated to 145°°F (62.8oC) or higher internaltemperature. Tests for catalase in cooked beef are indicative of

the presence of somatic catalase. Somatic catalase is destroyed at approximately 145oF and the test indicates whether or nottemperatures higher than 145oF were reached.

Detection of catalase in a canned meat product could be indicativeof flat sour spoilage. At canning temperatures all somaticcatalase should be destroyed, and the presence of the enzyme in afreshly opened can is indicative of bacterial catalase produced bygrowth.

11.12 Equipment and Supplies

a. Clean plastic teaspoon b. Clean paper towelsc. Felt-tip marking pend. Adhesive tape or paper labelse. Whirl-Pak® clear plastic bags (3" x 4")f. Clear plastic Zip-Loc® bags (12" x 12")g. Clean and sanitized slicing knifeh. Clean and sanitized large spoon or spatulai. 3% Hydrogen Peroxide - 1 pintj. Baby Shampook. Active dry baker's yeast

11.13 Procedure

a. Preparation of the Peroxide Reagent

i. Remove the caps from both the peroxide and theshampoo bottles.

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ii. Add one teaspoonful of the shampoo to the pint ofhydrogen peroxide (peroxide reagent).

iii. Replace the caps securely on each bottle.

iv. Slowly invert the peroxide reagent bottle 3-4 timesto mix the contents.

v. Label the reagent bottle "Prepared Reagent"followed by the date of preparation.

vi. Store the peroxide reagent in a refrigerator, theunused shampoo can be stored on a shelf with thechemicals.

b. Testing the Peroxide Reagent

i. Label a 3" x 4" Whirl-Pak® bag "Reagent Test".

ii. Carefully open the Whirl-Pak® bag and pourapproximately 10 granules of the baker's yeast intothe bag.

iii. Hold the Whirl-Pak® bag upright and pourapproximately ½ inch of the peroxide reagent intothe bag.

iv. Securely hold the top of the bag with the fingersof one hand and securely hold the bottom of the bag with the fingers of the other hand. Position the bag so that the fluid/foam level in the bag isaligned along the edge of the work surface. Keepthe bag pressed against the edge of the worksurface. Carefully pull the bag downward toward the open end to expel all excess air from the bag.Fold the top over several times and secure it withthe built-in clips.

v. Securely replace the cap on the peroxide reagent

bottle and then use it to support the upright"Reagent Test" bag.

vi. Record the time and then add 5 minutes to it forthe "Read Time".

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vii. At the read time note whether the bag has abundantfoam and is somewhat inflated (Positive Test) ornon-foamy and flat (Negative Test). Record thisinformation in the appropriate Quality Control Log.If the peroxide reagent gives a positive test,

proceed to the product test, if otherwise, preparea fresh aliquot of the peroxide reagent first.

c. Roast Beef Cooking Temperature Test

i. Prepare the product for sampling and secure a clean

sanitized (145°°F + hot water) slicing knife. Drythe knife with a clean, preferably sterilized, paper towel.

ii. Wipe the knife and slicing surface with a 5%

hypochlorite solution.

iii. Make a slice through the roast beef at the thickest part of the sample (maximum circumference).Examine the two halves to see if there are areasthat appear to be more rare than others.

iv. Label a Whirl-Pak® bag with the sampleidentification number and then carefully open it.

v. Cut a ¼ inch thick slice from one of the surfaces,lay it down on a sterile surface and carve out a 1"square section from what appears to be the leastcooked area of the slice. Using the knife blade,transfer this 1" square to the Whirl-Pak® bag.

vi. Shake the bag to transfer the piece to the bottom of the bag. Cover the piece with Peroxide Reagentand proceed according to steps b. iv through vi, with the exception that the reaction time betweenthe reagent and the sample is extended to 15 minutes.

vii. Record the results on the form that accompanied thesample and proceed as you would with any other positive or negative official sample.

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d. Canned Product

i. Label a 12" x 12" zip-lock® bag with theappropriate sample identification number. Do thesame for a 3" x 4" Whirl-Pak® bag.

ii. Aseptically open the suspect can and transfer thecontents to the large zip-lock® bag. It may benecessary to use a clean and sanitized large spoonor spatula to facilitate this transfer.

iii. Carefully close the zipper, expelling all air inthe process.

iv. Carefully manipulate the contents of the zip-loc® bag in a manner to thoroughly mix the contents.

v. Carefully open the zip-loc® bag, and using a clean,sanitized teaspoon, remove a level spoonful of test material from the bag and transfer it to the Whirl-Pak® bag. Reseal the zip-lock bag and set it and the empty container to one side for possible futureuse.

vi. Add peroxide reagent to the Whirl-Pak® bag with thesub-sample to completely cover the sample and the peroxide reagent fills the bottom third of the bag.Use the teaspoon to evenly disperse the sub-samplethroughout the reagent.

vii. Quickly fold the top of the bag four times the width of the tab tape and secure with the sidetabs. Proceed according to steps b. iv through vi, with the exception that the reaction time betweenthe reagent and the sample is one minute.

viii. Allow the sample test bag to stand undisturbed foran additional 15 minute period and then make afinal reading.

ix. Record the results on the form that accompanied thesample and proceed as you would with any other positive or negative official sample.

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Glenister, P. R., and M. Burger. 1960. A method for thedetection of chill-proofer protease in beer. Proc. Amer.Soc. Brewing Chem.:117.

Moreau, J. R., and E. C. Jankus. 1963. An assay for measuring papain in meat tissue. Food Technol. 94:1048.

Performing the Catalase Enzyme Test: A Self InstructionalGuide 1983. United States Dept. of Agriculture, Food Safetyand Inspection Service, Program Training Division, CollegeStation, TX 77845

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CHAPTER 12. EXAMINATION OF MEAT AND POULTRY PRODUCTS FOR BACI LLUS CEREUS

Charles P. Lattuada and Dennis McClain

12.1 Introduction

Bac i l l us c er eus is one of the few sporeforming, aerobic bacteriarecognized as a bacterial pathogen. It is widespread in soil, milk, the surfaces of meat and poultry, cereals, starches, herbsand spices. Its' role as a food-borne pathogen is relativelyrecent and somewhat uncommon in the United States. Two distincttypes of illness have been attributed to the consumption of food contaminated with B. c er e us . The more common manifestation is adiarrheal illness with an incubation time of 8-16 h characterized

by abdominal pain and diarrhea. The other is an emetic illness with an incubation time of 1-5 h and characterized by nausea and vomiting. While the emetic type is usually associated with cerealtype products such as rice, the diarrheal type is more widelyassociated with many foods.

B. c er e us typically is a very large, aerobic, Gram positive,sporeforming rod with peritrichous flagella. It grows over a wide

temperature range (10 to 48°°C) with an optimum range of 28 to 35°°C.It will grow over a wide pH range (pH 4.9 - 9.3) and in sodium chloride concentrations approximating 7.5%. Microscopically it

may be seen in chains. Macroscopically the colonies have a dullor frosted appearance on a nutrient agar plate. Its association with disease is usually related to counts >105 cfu/g in thesuspect food. Since B. c e r e us does not ferment mannitol, does produce lecithinase and is resistant to polymyxin, a selective medium consisting of mannitol-yolk-polymyxin (MYP) is commonlyused for its isolation. Colonies typically are pink in color and surrounded by a zone of precipitate. An ELISA test is availableto detect the diarrheal toxin.

12.2 Equipment, Reagents, Media

12.21 Equipment

a. Balance capable of weighing to 0.1 g

b. Stomacher™™ (model 400 by Tekmar, or comparable model),sterile plastic bags (with twist ties or self-sealing)

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OR blade-type blender, sterile cutting assemblies and blender jars

c. Sterile supplies, spoons or spatulas, pipettes (1 ml), bent glass rods "hockey sticks", aluminum pie pans (orequivalent)

d. Incubator, 30 ± 1°°Ce. Incubator, 35 ± 1°°Cf. Light or Darkfield Microscopeg. Platinum inoculating loops, 3 mm diameterh. Microscope slides and cover slipsi. Meeker/Bunsen burner with tripod, or hot platej. Pyrex beaker, 250-300 ml size

12.22 Reagents

a. Butterfield's Phosphate Diluent (BPD) for sample

extraction b. BPD dilution blanks, 9 ml volumec. Basic fuchsin staining solution, 0.5% aqueous

12.23 Media

a. Plates of Mannitol Yolk Polymyxin (MYP) Agar b. Nutrient Agar Slantsc. BC Motility Medium d. Nutrient Agar Platese. Blood Agar Plates, 5% Sheep RBC

12.3 Sampling and Dilution Procedure

a. Aseptically composite a 25 g or 25 ml sample in sterile bag or blender jar.

b. Add 225 ml Butterfield's Phosphate Diluent (BPD) to eachsample taken.

c. Stomach or blend for 2 minutes and then prepare serialdilutions of 10-2 to 10-6 in 9 ml BPD dilution blanks.

12.31 Plating and Examination of Colonies

a. Pipette 0.1 ml of the homogenate (10-1) and spread itover the entire surface of duplicate, predried MYP plates with a "hockey stick". Repeat the procedure foreach of the other dilutions through 10-6. Use a

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separate, sterile "hockey stick" for each dilution. Allow the inoculum to dry before incubating the plates.

b. Incubate all plates in an upright position for 20 to

24 h at 30°°C.

c. After incubation, examine all plates for colonies thatare surrounded by a zone of precipitate (lecithinase production) against an eosin pink to lavender agar background (non-fermentation of mannitol). If the areasof lecithinase production coalesce between colonies,look for plates with 10-100 colonies. Count all typicalcolonies and determine the presumptive count per gram.Remember that the count will be tenfold higher than thedilution, because only 0.1 ml was placed on a plate.

12.32 Confirmatory and Differential Procedures/Tests

a. Select 4-6 typical colonies for confirmation. Each ofthese colonies is subcultured on a predried Nutrient

Agar Plate and incubated at 30°°C for 24 - 48 h. Notethe presence or absence of rhizoid growth on thenutrient agar plate.

b. At the same time inoculate a tryptic soy sheep blood agar plate that has been divided into 4 - 6 segments. A 2 mm loop should be used to deposit the inoculum in the

center of the segment. Note the size of the hemolyticzone (and whether it is partial or complete).

c. Motility test - use BC motility medium method by makinga center line stab inoculation with a 3 mm loop and

incubating the tube at 30°°C for 18-24 h. Observe fordiffuse growth into the medium away from the stab as anindication of a motile organism.

Alternatively a microscopic motility test may be used.The slide motility test is done by adding 0.2 ml of

sterile water to a nutrient agar slant and theninoculating the aqueous phase with a 3 mm loopful of a

24 h slant culture. Incubate for 6-8 h at 30°°C. Placea loopful of the liquid culture on a glass slide and overlay with a cover slip. B. c er e us and B.

t hur i ngi ens i s are actively motile while B. ant hr ac i s and the rhizoid strains of B. c er e us are non-motile.

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d. Rhizoid growth - to test for rhizoid growth, inoculateseveral well isolated areas of a predried Nutrient AgarPlate. Use a 3 mm inoculating loop to make a point ofcontact inoculation. Incubate the plate in an upright

position at 30°°C for 24-48 h. If hair-like projections

(rhizoids) develop outward from these colonies, theisolate is B. c er e us var. myc oi des and not considered to be a human pathogen.

e. Protein toxin crystal stain - Make a smear on a microscope slide with sterile water from a 2-3 day old nutrient agar plate or slant. Allow the slide to airdry and then gently heat fix it. After cooling, flood the slide with methanol, wait 30 seconds and pour itoff. Then flood the slide with 0.5% aqueous solution of basic fuchsin. Gently heat the slide until steam is

observed, remove the heat, wait 1-2 minutes and repeatthe procedure. Let the slide cool and rinse well with water. Examine under oil immersion for free spores and darkly stained, diamond shaped, toxin crystals. Toxincrystals should be present if the cells have lysed and free spores are observed. The presence of toxincrystals is strongly indicative that the organism is B.

t hur i ngi ens i s .

f. Other Tests - If further biochemical testing is warranted, consult either Bergey's Manual of Systematic

Bacteriology or the Compendium of Methods for the Microbiological Examination of Foods.

12.33 Interpretation of Test Results

a. B. ce r eus usually is: lecithinase positive, stronglyhemolytic on sheep blood agar, actively motile, does not produce rhizoid colonies and does not produce proteintoxin crystals (diamond shaped).

b. Other lecithinase positive or weakly positive cultures may be B. c er e us var. myc oi des , B. t hur i ngi ens i s , or B.

ant hr ac i s . Caution: non-motile, non-hemolytic coloniescould be B. ant hr ac i s and should be handled with specialcare.

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12.4 Method Quality Control Procedures

A minimum of three method control cultures is recommended for use whenever a new batch of medium is made or acquired as well as eachtime that an analysis is performed. These controls should consist

of at least one strain each of B. c er e us , B. c er e us var. myco i des ,and B. t hur i ngi ens i s . This also will assist the analyst in becoming more familiar with the morphological and culturaldifferences of these B. c e r e us variants.

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Claus, D., and R. C. W. Berkeley. 1986. Genus Bac i l l us , p. 1105-1139. I n Bergey's Manual of Systematic Bacteriology, Volume 2. Williams & Wilkens, Baltimore, MD.

Harmon, S. M. 1982. New method for differentiating membersof the Bac i l l us c er eus group: collaborative study. J. Assoc.Off. Anal. Chem. 65:1134-1139.

Harmon, S. M., J. M. Goepfert, and R. W. Bennett. 1992.Bac i l l us c er eus , p. 593-604. I n C. Vanderzant and D.F.Splittstoesser (ed.), Compendium of Methods for the Microbiological Examination of Foods, 3rd Edition. Amer.Publ. Hlth. Assoc., Washington, D.C. 20005.

Johnson, E. A. 1990. Bac i l l us c er eus food poisoning, p. 127-135. I n Foodborne Diseases. Academic Press, New York, N.Y.

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CHAPTER 13. EXAMINATION OF MEAT AND POULTRY PRODUCTS FOR CLOSTRI DI UM PERFRI NGENS

Ann Marie McNamara and Charles P. Lattuada

13.1 Introduction

Cl os t r i di um per f r i ngens is a spore-forming, anaerobic bacterium that is widespread in soil, water, foods, spices, and theintestinal tract of humans and animals. Viable, sporulatingstrains that produce typical foodborne illness belong to Type A and produce an enterotoxin that causes typical symptoms of acuteabdominal pain and diarrhea. Symptoms of nausea, vomiting and fever are rare. Symptoms usually appear 8-12 (range 6-24) hoursafter ingestion of a contaminated food, usually cooked meat or

poultry. The infectious dose for humans is high, generallyconsidered to be 106 - 107 cells/g. In foodborne diseaseoutbreaks, findings of hundreds of thousands or more organisms pergram of food supports a diagnosis of C. per f r i ngens foodborneillness when appropriate clinical and epidemiological evidenceexists. There are four other types of C. per f r i ngens : types B, C,D and E. Some strains of type C produce an enterotoxin thatcauses a rare form of necrotic enteritis that is often fatal and rarely seen outside of New Guinea.

This method for isolating and identifying C. per f r i ngens in foodsis a modification of the C. per f r i ngens method found in theCompendium of Methods for the Microbiological Examination ofFoods, 3rd Edition (Labbe & Harmon, 1992).

For use in the FSIS Nationwide Microbiological Baseline DataCollection Programs and product surveys, the following"presumptive" isolation and enumeration method will suffice. This method is considered to be a "presumptive" method because otherspecies of Cl os t r i di a besides per f r i ngens can reduce sulfite and produce black colonies which are egg-yolk positive in TSC and EY-free TSC agar (Labbe and Harmon, 1992). Additionally, somestrains of C. per f r i ngens may not produce a halo surrounding their

black colonies, so all black colonies should be counted whether ahalo is present or not (Labbe and Harmon, 1992). For outbreakinvestigations or investigation of epidemiologically-linked cases,the more lengthy and time-consuming confirmation method should beused.

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All samples should be shipped as refrigerated samples (0 - 10°°C);this is particularly important with outbreak samples. Samplesshould be analyzed promptly upon laboratory receipt (Labbe and Harmon, 1992). C. per f r i ngens in foods stored for prolonged periods of time or frozen many lose viability. If frozen samples

must be shipped, food samples should be treated with buffered glycerol salt solution to give a 10% final concentration ofglycerol. Samples should be shipped on dry ice and be stored frozen at -55oC to -60oC until the samples are analyzed.


13.21 Equipment

a. Incubator at 35 ± 1°°C b. Anaerobic containers

c. Anaerobic gas mixture consisting of 90% N2 + 10% CO2d. Colony counter with a piece of white tissue paper over

the counting background area to facilitate counting black colonies

e. Stomacher™™ 400 and sterile stomacher bags or Blenderand sterile blender jars

f. Vortex mixer

g. Water bath 46 ± 1°°Ch. Sterile, bent, glass rods ("hockey sticks")

13.22 Reagents

a. Nitrate reduction reagents (Method 1) b. 0.1% peptone water diluentc. Phosphate-buffered saline (PBS)d. Physiological saline (0.85% sodium chloride)e. Butterfield's Phosphate Diluentf. Buffered Glycerol Salt Solution (for frozen samples)

13.23 Media

a. Tryptose Sulfite Cycloserine (TSC) agar

b. EY-free TSC agarc. Trypticase Peptone Glucose Yeast Extract Broth

(buffered)d. Fluid Thioglycollate Medium e. Motility-Nitrate Medium (buffered)f. Lactose Gelatin Medium

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g. Spray's Fermentation Medium (1% salicin, or 1%raffinose)

13.3 Presumptive Test

13.31 Sample Preparation

a. Meat Samples:

i. Label a sterile stomacher bag so that itcorresponds to the label on the sample bag.

ii. Aseptically remove portions of the sample at random to obtain 25 grams. Place these portions in thesterile stomacher bag.

iii. Add 225 ml Butterfield’s Phosphate Diluent (BPD) tothe stomacher bag of each sample taken.

iv. Stomach for 2 minutes. Prepare serial dilutions of10-2 to 10-6.

b. Poultry Samples:

i. Prepare serial dilutions of 10-1 to 10-3 of the whole bird rinse.

13.32 Enrichment and Plating

a. Make duplicate spread plates on thin (6-7 ml) TSC withegg yolk agar base, using 0.1 ml/plate of undiluted sample rinse/extract as well as each dilution.

b. Equally distribute the inoculum using sterile "hockeysticks". Use a new sterile "hockey stick" for eachdilution.

c. After the inoculum has dried slightly, overlay thesurface with approximately 10 ml or more of egg yolk

free TSC agar. Allow the plates to solidify before placing them, lid side up, in an anaerobic jar. Flushjar 3 or 4 times with 90% N2 + 10% CO2 leaving thisatmosphere in after the last flush, or alternatively usea system which catalytically removes oxygen.

d. Incubate all plates for 24 h at 35°°C.

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13.33 Examination of Plates

a. After incubation, count the number of presumptiveC. per f r i ngens colonies. These colonies will be blackand usually surrounded by a 2-4 mm opaque zone (halo).

b. Multiply the number of colonies counted by 10 (sinceonly 0.1 ml used) and then multiply by the appropriatedilution factor to obtain your total count.

13.4 Confirmatory Procedure (for epidemiologically linked cases)

13.41 Colony Selection

a. Select 10 representative black colonies from each TSCagar plate counted and inoculate each into a freshly

boiled (deaerated) and cooled tube of fluid thioglycollate broth.

b. Incubate for 4 h in a water bath at 46°°C or overnight at

35°°C. After incubation prepare a Gram stain from eachtube and examine microscopically. C. per f r i ngens

organisms are short, fat Gram positive rods. Endosporesare rarely produced in fluid thioglycollate medium.

c. If contaminants are observed, re-streak the contaminated culture onto the surface of a TSC (with egg yolk) agar

plate (do not overlay) and incubate anaerobically before proceeding with any confirmatory tests. Surfacecolonies will appear as yellowish-grey colonies measuring approximately 2 mm in diameter. If re-streaking was done, it is necessary to repeat a. and b.of Section 13.41 (above).

13.42 Confirmatory Tests

a. Motility - nitrate reduction test

i. Stab inoculate each tube of motility-nitrate medium with two, 2 mm loopfuls of the fluid thioglycollate medium culture.

ii. The medium contains 0.5% each of glycerol and galactose to improve the consistency of the nitrate

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reduction reaction with different strains of theorganism.

iii. Incubate the inoculated medium at 35°°C for 24 h and check motility. Since C. per f r i ngens is non-

motile, growth should occur only along the line ofinoculum and not diffuse from the stab line.

iv. Test for reduction of nitrate to nitrite. A red ororange color indicates reduction of nitrate tonitrite. If no color develops, test fluid thioglycollate for residual nitrate by addition of powdered zinc.

b. Lactose gelatin medium

i. Stab inoculate each tube of lactose gelatin medium with two, 2 mm loopfuls of the fluid thioglycollate medium culture.

ii. Incubate at 35°°C for 24 to 48 h. Lactosefermentation is indicated by gas bubbles and achange in color of the medium from red to yellow.Gelatin usually is liquefied by C. per f r i ngens

within 24 to 48 h.

c. Carbohydrate fermentation

i. Inoculate 0.15 ml of the fluid thioglycollate brothculture into 1 tube of freshly deaerated Spray'sfermentation medium containing 1% salicin, 1 tubecontaining 1% raffinose, and 1 tube of medium without carbohydrate for each isolate.

ii. Incubate these three media at 35°°C for 24 h and then check for production of acid. To test foracid, transfer 1 ml of culture to a test tube orspot plate and add 2 drops of 0.04% bromthymol

blue. A yellow color indicates that acid has been produced.

iii. Reincubate negative raffinose tubes for anadditional 48 h and retest for the production ofacid.

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iv. Salicin is rapidly fermented with the production ofacid by culturally similar species such as

C. par aper f r i ngens , C. bar at i i , C. s ar di ni ens e ,C. absonum , and C. ce l a t um, but usually not byC. per f r i ngens .

v. Acid is produced from raffinose within 3 days by

C. per f r i ngens but is not produced by culturallysimilar species.

13.43 Quantitation of C. per f r i ngens Populations Based onConfirmed Anaerobic Plate Counts

a. Cultures obtained from presumptive C. per f r i ngens blackcolonies on selective, differential TSC or EY-free TSC medium are confirmed as C. per f r i ngens if they are:

i. nonmotileii. reduce nitrateiii. ferment lactoseiv. liquefy gelatin within 48 hv. produce acid from raffinose.

b. Calculate the number of confirmed C. per f r i ngens pergram of food sample as follows:

i. Average the paired plates counted, then adjust theaverage presumptive plate count to 1.0 ml by multiplying by 10.

ii. Multiply the adjusted presumptive plate count bythe reciprocal of the dilution plated to arrive atthe total of presumptive C. per f r i ngens colonies.

iii. The confirmed colony count is then determined byusing the ratio of the colonies confirmed asC. per f r i ngens to the total colonies tested.

13.5 Quality Control

a. The following authentic, reference cultures can be used as control organisms in the above procedures:

C. per f r i ngens ATCC 13124C. absonum ATCC 27555

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b. The expected reactions produced by these controlorganisms are as shown in the following table:

Organism Motility H2S Gelatin

liq.

Nitrate

reduct.

Lactose

ferm.

Salicin

ferm.

Raffinose

ferm.

C. p er f r i n ge ns

ATCC 13124- + + ± + - d

C. absonum ATCC

27555±* + d + + +w -

* usually + in young cultures; d = delayed; w = weak

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Granum, E. 1990. Cl os t r i di um per f r i ngens toxins involved infood poisoning. Intl. J. Food Micro. 10:101-112.

Jay, J. M. 1996. Food poisoning caused by Gram-positivesporeforming bacteria, p. 451-458. I n Modern Food Microbiology, 5th Edition. Chapman and Hall, New York, NY10003

Labbe, R. G., and S. M. Harmon. 1992. Cl os t r i di um

per f r i ngens , p. 623-635. I n C. Vanderzant and D. F.Splittstoesser (ed.), Compendium of Methods for the Microbiological Examination of Foods, 3rd Edition. Amer.Publ. Hlth. Assoc., Washington, D.C. 20005.

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CHAPTER 14. METHODS FOR THE DETECTION OF CLOSTRI DI UM BOTULI NUM

TOXINS IN MEAT AND POULTRY PRODUCTS

L. Victor Cook, Wei Hwa Lee, Charles P. Lattuadaand Gerri M. Ransom

14.1 Introduction

Botulinum toxin (botox) types A-G are produced by heterogeneousstrains of Cl os t r i di um bot ul i num . In recent years, other strainsof Cl os t r i di um s pp . have been shown to produce botulinum toxin(e.g. F-toxin C. bar at i ). Botox types A, B, E and F have caused serious, and sometimes fatal, cases of foodborne illness inhumans. The vast majority of botulism outbreaks in red meat and poultry products have involved either toxin A or B.

Botulinum toxin exists naturally as a stable complex of 2 protein molecules called "progenitor" or "bimolecular" toxin. Thecomponent proteins are the neurotoxin and non-toxic proteins,typically hemagglutinins.

The current botulinum toxin test method used by FSIS (Section14.4) is the mouse bioassay procedure, published as Official Method 977.26 in Official Methods of Analysis of AOACInternational, 16th Edition, 1995.

14.2 Supplies and Equipment

a. Microcentrifuge b. Refrigerated centrifugec. Microfuge tubes, 1.5 ml, PGC Scientific 505-120 or

equivalentd. Sterile 50 ml stainless steel centrifuge tubes, Sorvall

00579 or equivalente. Polycarbonate screw cap centrifuge tubes, 30 ml size

f. Sterile Stomacher™™ bagsg. White laboratory mice, 15-18 gh. Hypodermic syringes, 1 or 3 ml size, fitted with 25 gage

5/8 inch needles.

14.21 Media and Reagents

a. Botulinum toxin typing antisera, monovalent types A through F, or polyvalent A-F (CDC, Atlanta, GA,

C. bot ul i num laboratory).

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b. Citric-Phosphate Buffer, pH 5.5; combine the following:

0.1 M Citric acid 21.6 ml0.2 M Na2HPO4 28.4 ml

c. Sterile Glycerol

Glycerol is sterilized at 121°°C for 30 min. It is used in the first 1:2 dilution (50% v/v) of neat (1X)antisera or toxic botulinum culture supernatant fluids

prior to storage at -20°°C.

d. Botox Stabilizing Diluent:

This diluent is used to stabilize botox culturesupernatants. Combine the following:

pH 5.5 citric-phosphate buffer 25 mlSterile fetal bovine serum 25 mlSterile glycerol 50 ml

Store at -20°°C

e. Modified Cooked Meat Medium (MCMM)

f. Gelatin Phosphate Buffer

Gelatin 2 g

Na2HPO4 4 gDistilled Water 1 l

Dissolve the ingredients by heating gently in 800 ml ofdistilled water. Adjust the pH to 6.2 with HCl and bring the volume to 1 liter. Autoclave for 20 minutesat 1210C.

g. Trypsin 1:250 (Difco or equivalent), 10% in saline

h. Physiological saline, 0.85% NaCl in distilled water

14.3 Preparation of Culture Fluids for Toxin Analysis

a. Select well isolated, typical, C. bot ul i num coloniesfrom the resulting growth on appropriate solid plating medium incubated under anaerobic conditions for 48 h at350C and inoculate steamed, cooled tubes of Modified

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Cooked Meat Medium (MCMM). Incubate MCMM tubes at 350Cfor 5 days.

b. Remove 10 ml of the MCMM culture fluid and place it insterile 50 ml stainless steel or 30 ml polycarbonate

screw cap centrifuge tubes placed inside stainless steelcentrifuge cups. Clarify by centrifugation at 15,000 XG for 15 minutes at 50C

c. Remove clarified culture fluid from centrifuge tube and place it in another sterile tube. Make a 1:10 dilutionof this culture fluid in Gelatin Phosphate Buffer.

d. Proceed to test the culture fluid at undilute and 1:10for the presence of toxins by the mouse bioassay procedure described below (Section 14.41).

14.31 Preparation of Solid Food Samples for Direct Toxin Analysis

a. Following the procedures given in Chapter 10.4, theentire contents, if possible, of a canned product (e.g.corned beef or canned chicken) should be transferred to

a sterile stomacher™™ bag.

b. Place the sample bag inside a second stomacher™™ bag to minimize the possibility of uncontained leakage. Hand mix by gently squeezing the outside of the bags (knead)

for 2 minutes to distribute any pockets of C. bot u l i num toxins prior to removing the food sample for testing.Remove at least 50 g for analysis.

c. The aliquot of the food sample removed should be placed in a sterile metal blender jar with a screw cap and blended with an equal volume of Gelatin PhosphateBuffer, pH 6.2., until thoroughly homogenized.

d. Ten ml of the food extract should be clarified by

centrifugation at 15,000 X G for 15 minutes at 5°°C in

sterile 50 ml stainless steel or 30 ml polycarbonatescrew cap centrifuge tubes placed inside stainless steelcentrifuge cups.

e. An aliquot should be cultured following Chapter 10.45

a., the remainder held in reserve at 4°°C.

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f. Clarified food supernatants should be tested using the mouse bioassay procedure (Section 14.41) undiluted (1:2of food) and at 1:5 (1:10 of food) dilution. Dilutionis made in Gelatin Phosphate Buffer.

14.32 Sample Reserves

a. Samples reserves may be held at 4°°C for 1-2 weeks.

b. For long term storage, culture supernatants should be

stabilized with 50% glycerol and stored at -20°°C.

14.4 Mouse Bioassay Screening Test for C. bot u l i num Toxins inFoods and Cultures: Introduction

The current FSIS botulinum toxin testing procedure is the mouse

bioassay published in Official Methods of Analysis of AOACInternational as Official Method 977.26 G.

In years of testing, the vast majority of gas-forming anaerobecultures isolated by FSIS from meat and poultry product samples were botulinum toxin negative. Therefore FSIS found it feasibleto slightly abbreviate the AOAC official mouse bioassay test for botulinum toxins as follows:

14.41 Bioassay Procedure

14.411 Non-treated Preparation

a. Remove approximately 2 ml of each food sample extract orculture fluid (at specified dilutions) being tested and place it in an appropriately labeled sterile containersuitable for syringe and needle aspiration.

14.412 Trypsinization

Trypsin treatment potentiates nonproteolytic strains of types Band E that otherwise could escape detection.

a. Remove 3.6 ml of each food sample extract or culturefluid (at specified dilutions) being tested and adjustthe pH to 6.0 - 6.2 with HCl.

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b. Add 0.4 ml of the 10% Trypsin to 3.6 ml of the above

aliquot. Incubate 1 h at 35-37°°C with occasional gentleagitation.

c. Place the trypsinized material in an appropriately

labeled sterile container suitable for syringe and needle aspiration.

14.413 Boiled Control

Botulinum toxin is destroyed by heating to 80°°C for 10 minutes.

a. Transfer 2 ml of each food sample extract or culturefluid (at specified dilutions) being tested to anappropriately labeled sterile container suitable forsyringe and needle aspiration.

b. Heat in boiling water for 10 minutes. Allow to cool prior to injection.

14.414 Test

a. Using a 1 or 3 ml syringe fitted with a 25 gage needle,inject two mice intraperitoneally (IP) with 0.5 ml eachof the original, non-treated supernatant (treatment14.411).

b. Inject two mice IP with 0.5 ml of the trypsinized supernatant (treatment 14.412).

c. Inject two mice IP with 0.5 ml of the boiled extract(treatment 14.413), these are the negative controls.

d. Observe all mice periodically for 48 h for typicalneurological symptoms. Hold mice and observe for onsetof neurological symptoms for an additional 2 days.Record symptoms and time of death of mice.

e. The presence of toxin is presumptively indicated bydevelopment of typical symptoms and death from treatment14.411 and/or 14.412 but not treatment 14.413. Typical botulism symptoms in mice sequentially consist ofruffled fur, labored but not rapid breathing, weaknessof the limbs progressing to total paralysis, gasping for breath (opening of lower jaw), followed by death due to

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respiratory failure. Be aware that tetanus toxin may produce similar symptoms in mice. Death in the absenceof neurological symptoms is not an acceptable indicationof mouse botulism; death may be non-specifically caused by other microorganisms, chemicals present in test

fluids or injection trauma.

14.415 Confirmation and Toxin Typing by Protection Tests

Any food extracts or culture fluids (non-treated and/or trypsintreated preparations) which produce death in mice with typical botulinum toxin symptoms should be subjected to confirmation and typing of the botulinum toxin present by specific mouse protectiontests by the current AOAC Official Method 977.26 H published inOfficial Methods of Analysis of AOAC International, 16th Edition,1995.

a. Obtain the same toxic preparations demonstrated to havecaused death of mice in procedure 14.414.

b. Either the untreated or trypsin treated toxic preparations may be used. If the trypsin treated preparation is to be used, a freshly trypsinized portionof the untreated fluid should be prepared, as thecontinued action of trypsin in the original preparation may destroy the toxin.

c. Prepare 1:10, 1:100 and 1:1000 dilutions of the toxic preparations in Gelatin Phosphate Buffer.

d. Rehydrate the lyophilized vials of specific botulinum antitoxin obtained from CDC according to their specificinstructions.

e. Dilute the monovalent antitoxin types A, B, C, D, E and F respectively in physiological saline to contain 1international Unit per 0.5 ml. Prepare a sufficientquantity of each diluted antitoxin to inject 0.5 ml intoeach of two mice for each dilution of each toxic

preparation to be tested.

f. Protect six separate groups of mice by injecting each mouse in a group IP with 0.5 ml (1 international Unit)of one of the above antitoxins (types A, B, C, D, E and F). Each protected group corresponds specifically totypes A, B, C, etc.

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g. Within 30 minutes to 1 h of antitoxin administration,inject protected mice groups IP with 0.5 ml of eachtoxic preparation at undiluted, 1:10, 1:100 and 1:1000dilutions (2 mice per dilution) and also injectsimilarly pairs of unprotected mice with these same

toxic preparations.

h. Observe mice periodically over 48 h for typical symptomsof botulism; record all symptoms and time of deaths. Note specifically the comparative protection afforded toeach pair of specific antitoxin protected mice (A, B, C,D, E and F) relative to the unprotected control mice foreach respective toxic preparation tested.

i. Protection of mice from death and botulism by one of the monovalent botulinum antitoxins, while unprotected mice

die, confirms the presence of botulinum toxin and determines the homologous serological type of toxin inthe tested preparation.

j. If all antitoxin protected mice should die, in additionto the unprotected mice, this may be an indication oftoo high a quantity of toxin in the original test preparation, the presence of more than one toxinserotype or the presence of some other non-botulinum toxic substance.

k. To determine which of the above factors may beresponsible in such a case, retest the toxic preparations at higher dilutions of the test fluidsand/or use mixtures of antitoxin (or polyvalentantiserum) in place of the monovalent antisera for passive protection of mice groups.

14.5 Method Quality Control

a. The quality control procedures are built in to the above procedure. Because of the nature of this test noadditional methods control are recommended. However it

is very important to adhere to the following safety procedures and waste disposal.

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14.51 Safety Procedures and Waste Disposal

Botulinum toxins are among the most potent toxins known. In a1989 outbreak, consumption of hazelnut yogurt (125 g) contaminated with only about 200 pg/ml of toxin B caused 28 cases of botulism

and one death in England. Therefore, the following precautions must be strictly observed at all times:

a. MOUTH PIPETTING SHOULD NEVER BE USED IN THIS ASSAY.

b. At the completion of an assay, all of the equipment and reagents that have been in contact with the culture orfood extract must be autoclaved. Laboratory benchsurfaces and non-autoclavable materials and equipment must be treated with 1% sodium hypochlorite, pH 11.0,(1:4 dilution of household bleach in water) to destroy

any toxin. Accidental spills can be detoxified usingthe chlorine bleach solution. A supply of fresh bleachsolution must ALWAYS BE AT HAND for emergencysituations.

c. The work area should be sequestered from the work areaused for other analyses if at all possible and should be prominently posted with biohazard signs.

d. A Class II or better Biological Containment Hood should be used whenever possible. Botulinum toxin analyses areconsidered Biosafety Level II analyses.

e. When centrifuging toxic materials, use a tightly closed centrifuge equipped with safety cups.

f. Botox analysts should be immunized to a protective level with a toxoid vaccine (CDC) before working with this microorganism or its' toxins in order to minimize the possibility of accidental intoxication.

g. Emergency phone numbers should be posted prominently inthe lab.

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Cunniff, P. (ed.). 1995. Official Methods of Analysis of AOAC International, 16th Edition. Chapter 17, p. 46-48. AOACInternational, Inc., Gaithersburg, MD 20877

Kautter, D. A., H. M. Solomon, D. E. Lake, D. T. Bernard, and D. C. Mills. 1992. Cl os t r i di um bot ul i num and its toxins, p. 605-621. I n C. Vanderzant and D. F. Splittstoesser (ed.),Compendium of Methods for the Microbiological Examination ofFoods, 3rd Edition. Amer. Publ. Hlth. Assoc., Washington,D.C. 20005.

Smith, D. S. L. 1977. Botulism: The Organism, its Toxins,the Disease. Charles C. Thomas Publisher, Springfield, IL.

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CHAPTER 16. AGAROSE THIN-LAYER ISOELECTRIC FOCUSING (TLIEF)FOR SPECIES DETERMINATION OF RAW MUSCLE TISSUES

Richard P. Mageau

16.1 Introduction

Improvements in the developed biochemical technique of isoelectricfocusing have allowed the application of this technique to be used for species determination of raw muscle tissue. This method provides for the relatively rapid species determination of a largenumber of samples in a definitive, less subjective manner, in asingle analytical run without the use of anti-species sera. The principle of this technique involves the separation and focusing of proteins under an electrical field in a stable pH gradient

dependent upon differences in the isoelectric points of the various proteins present. Since various species tissues contain multiple proteins of different isoelectric points, an aqueous extract of a particular species tissue when subjected to TLIEF will produce astained protein band pattern unique and distinct for that species.By using the method described below, a total of 24 samples (48 ifsample filter papers are cut in half along their long axis) may beanalyzed in a single determination in one day as to their correctspecies. The use of this established method is intended to aid inthe rapid species analysis of a large influx of raw tissue samplesresulting from particular meat species problems which may beencountered in the Agency's inspection system.

16.2 Materials and Equipment

a. Multiphor for high Performance Analytical Electrofocusingin Agarose; to include 2117-301 Multiphor Basic Unit,2117-107 Analytical Electrofocusing Lid, 2117-701Capillary Gel Casting Kit, and 1850-100 Agarose-EF Accessory Kit. (LKB Instruments.)

b. 2197-001 D.C. Power Supply for Electrofocusing and Electrophoresis. (LKB Instruments.)

c. 185-101 Multiphor Gelbond film, 124 x 258 mm. (LKB

Instruments.)d. 2030-710 Bayonet female plastic tubing connector and

2030-702 Bayonet male plastic tubing connector. (LKBInstruments.)

e. 2117-109 Multiphor Staining Kit. (LKB Instruments.)f. 1403 Coomassie Brilliant Blue R-250 dye (Fisher).g. S-460 D-sorbitol powder, reagent grade (Fisher).

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h. A-322 Trichloroacetic acid, reagent grade (Fisher).i. A-297 5-sulfosalicylic acid, crystal, reagent grade

(Fisher).j. 14-198-5A High pressure hose clamps, 1/4" to 5/8" size

(Fisher).

k. K-10 Kerosene (Fisher).l. 17-0468-01 Agarose IEF (Pharmacia Fine Chemicals). m. 17-0453-01 Pharmalyte Carrier Ampholyte, pH 5-8 range

(Pharmacia).n. Schleicher and Schuell #470 filter paper, 12.5 x 26 cm

size and Schleicher and Schuell #577 filter paper, 12.5 x26 cm size (PGC Scientific Corp.).

o. W 3237-10 Lauda Brinkman, Model K-4/RD Circulating water bath. (American Scientific Products.)

p. B-1206-2 Whirl-Pak® bags, 3" x 5". (American ScientificProducts.)

q. R5316-8 Tygon tubing, formula S-50-HL, 5/16" x 1/16".(American Scientific Products.)r. Hair dryer (hot and cold).s. Rubber print roller, 6" wide.t. Silicone gasket, 0.75 mm thick, overall dimensions of

12.5 x 26 cm, 3 sided of 5 mm width. (Potomac RubberCo., Inc., Washington, DC.)

u. Water bath and incubator/oven capable of maintaining65oC.

v. Centrifuge capable of 9,000 x G maximum. w. Stomacher®

16.3 Procedure

a. Initial Reagent Preparations

i. Fixing solution:

Dissolve 25 g sulfosalicylic acid and 50 g oftrichloroacetic acid in distilled water and diluteto a final volume of 500 ml.

ii. Destaining solution:

Mix 700 ml of ethyl alcohol and 200 ml glacialacetic acid together and dilute to a final volume of2,000 ml with distilled water.

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iii. Staining solution:

Completely dissolve 1 g Coomassie Brilliant BlueR250 dye in 500 ml of destaining solution.

iv. Cathode solution: (1 M NaOH, 100 ml)

v. Anode solution: (0.05 M H2SO4, 100 ml)

b. Sample Preparation

i. Obtain 1 g of diced, raw, muscle tissue and place ina small whirl-pak® bag together with 9 ml ofdistilled water.

ii. Thoroughly macerate the tissue by stomaching for 1-2

minutes and then leave overnight at 4

o

C.

iii. Centrifuge the resulting solution at 9000 x g for 10 minutes at room temperature and apply to samplefilter papers when ready to electrofocus.

c. Apparatus Assembly

i. Set up and align the Lauda K-4/RD circulating water bath, LKB 2117 Multiphor Basic unit, and LKB 2197D.C. Power supply on a laboratory bench such thatthe water bath is adjacent and convenient to the

Multiphor unit and the power supply is on theadjacent side of the Multiphor unit.

ii. When placed on the same table or workbench, theLK-4/RD circulating waterbath causes a vibration problem that may cause the bands on the finalagarose gel plate to be irregular. This problem can be corrected by isolating the waterbath, either by moving the waterbath to a separate table or to thefloor. In cases where the lab has a raised orsuspended floor, the addition of vibration damping

elements (Fisher 01-914045) may be necessary tofurther isolate the vibration.

iii. Install the cooling plate in the Multiphor unitaccording to LKB instruction manual and attachappropriate, insulated, circulation hoses to the water bath and secure to make leak-proof.

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iv. Adjust and calibrate the water bath temperature toassure an adequate supply of water is circulatingthrough the cooling plate at 4oC.

v. Turn on the circulating, calibrated water bath at

least 30 minutes prior to the preparation of a gel plate on the day that an analytical run is to be performed.

d. Agarose Gel-plastic Film Preparation

i. Mix 0.3 g Agarose-IEF (Pharmacia) and 3.6 g sorbitolin a conical flask with 27 ml distilled water and heat with stirring in a boiling water bath until allsolids are dissolved.

ii. Place the flask containing the dissolved ingredientsin a 65oC water bath and allow the solution to cooland equilibrate to 65oC.

iii. Add 1.9 ml of Pharmalyte, pH 5-8 range, ampholytesolution (Pharmacia) with needle and syringe, whilegently swirling the 65oC tempered, liquid agarosesolution. The final agarose solution is 30 ml totalvolume with an ampholine concentration of about 2.5%and agarose concentration of 1%. Leave theliquified agarose solution in the 65oC water bathuntil needed, after completing step (viii).

iv. Obtain a glass plate 125 x 260 mm (LKB 2117-701)Capillary Gel Casting Kit) that has been previouslytreated with the surface wetting agent Prosil-28according to product instructions and place a smallamount of distilled water on the glass surface.

v. Obtain a sheet of gel-bond film and place it on the wet glass plate such that the hydrophobic side ofthe sheet is down and in contact with the water and the hydrophilic side is up. Properly align the

edges of the film sheet with the edges of the glass plate and remove excess water and air bubbles byrolling the surface of the film sheet with a rubberroller. Carefully remove excess water withabsorbent towels.

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vi. Place the three-sided, orange, silicone gasket onthe film sheet and align the gasket edges with theedges of the film sheet.

vii. Place a 125 x 260 mm Prosil-28 treated glass plate

on top of the orange gasket and align the leadingedges with the gasket. Place five clamps around thethree gasket-glass edges (2 each on long sides and 1on the short end). When properly set up you willhave a glass-film sheet sandwich arrangement whichis leak proof on three sides where the gasket is and one open end with a space of about 0.75 mm (equal togasket thickness) between the bottom of the topglass plate and the top of the gel-bond film sheet.

viii. Place this glass-film sheet sandwich arrangement in

a 60-65

o

C oven for 10 minutes to warm up along witha 50 cc syringe and 21 gauge needle.

ix. Remove the warm glass-film sheet sandwich from theoven and set-up on a rack near the water bathcontaining the previously prepared liquid agarosesolution at 65oC. Quickly fill a 50 cc syringefitted with a 1 inch 21 gauge needle with the liquid agarose solution. Insert the needle in the space between the gel bond film sheet and bottom of thetop glass plate. Rapidly but evenly inject theliquid agarose solution to fill this space without

air bubbles before the agarose solution starts togel.

x. Allow the agarose filled sandwich to set undisturbed until the agarose has solidified and then place in arefrigerator for 30 minutes to completely solidifythe agarose.

xi. Carefully remove the five clamps and the top glass plate from the sandwich and obtain the agarosecoated gel-bond film sheet from the bottom glass

plate. When properly executed you will have agel-bond film sheet containing a uniform, bubblefree solidified agarose-ampholine layer ofapproximately 0.75 mm thickness.

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xii. Several agarose gel-bond film plates may be prepared at the same time in order to reduce preparation timefor future runs. The prepared plates must be preserved until needed by storage in the LKBHumidity Chamber (LKB-2117-110). These chambers are

stackable and come in a kit holding up to three gel plates. Plates stored refrigerated for as long as 6 weeks in the humidity chamber show no loss in performance.

NOTE: Do not perform step (xi) above until just prior tostarting step (iii) of section (e) below.

e. Isoelectric Focusing of Samples and References

i. Smear a small amount of reagent grade kerosene(Fisher) on the top of the cooling plate (which has4oC water circulating through it) of the Multiphorunit.

ii. Place an LKB sample position template on top of thekerosene covered cooling plate, position in properalignment with the cooling plate and smooth out sothat no air bubbles are present under the template.Blot excess kerosene from edges of the template withabsorbent towels.

iii. Smear a small amount of kerosene on top of thetemplate and place the previously prepared agarosefilm sheet on top of the kerosene covered template,align edges with the cooling plate, remove anytrapped air bubbles and blot excess kerosene from the edges.

iv. Soak filter paper strips (10 x 5 mm) in sample orreference tissue extracts and apply to the surfaceof the agarose gel near the anode using the visible

template under the agarose-film sheet as a guide. A maximum of 24 samples total (including desired reference extracts) may be placed on the agarosesurface. Be sure that the sample paper strip is incomplete contact with the agarose surface and rinseoff the tweezers between the handling of each samplestrip with distilled water.

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An alternative approach to sample application is tofirst place 24 blank paper strips in the proper position on the agarose surface and then with theuse of a micropipetting device place a standard amount (25 µl) of sample extract on each respective

strip. If it is desirable to employ small paperstrips (10 x 2.5 mm) to accommodate a larger numberof samples (48) for analysis, these strips should have only 10-15 µl sample extract applied to them and care must be taken to not cause overloading and mixing of adjacent samples.

v. Soak electrode filter paper strips with appropriatesolutions for cathode (1 M NaOH) and anode (0.05 M H2SO4), blot excess off on paper toweling and guided by the visible template apply the wet electrode

strips to the surface of the agarose in the properanode and cathode positions and cut to the propersize of the agar.

vi. Place the LKB electrofocusing lid on the Multiphorunit over the cooling plate in the proper alignmentsuch that the platinum electrode wires are centered and make good firm, complete contact with therespective soaked anode and cathode filter paperstrips.

vii. Connect the electrical cables of the electrofocusing

lid to the small pins on the front of the Multiphorunit.

viii. Mount the cover by first introducing the hooks onthe cover into the rectangular holes on the rearside of the Multiphor unit, lower the cover and press the large electrode pins into the holes on thecover.

ix. Connect the electrical leads from the cover to the proper terminals (check for like charge) on the LKB

2197 D.C. power supply.

x. Turn on the power supply and adjust to provide thefollowing conditions: 10 watts constant power, 700 V constant voltage and current unlimited (wide open)for a period of 45 minutes.

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xi. After this period of time, change power to thefollowing conditions: 10 watts constant power, 1000 V constant voltage and current unlimited for a period of 60 minutes.

xii. Turn off power after this period of time, remove thecover and electrofocusing lid and proceed to section(f) below.

f. Fixing, Staining, and Destaining

i. After completing the isoelectric phase of separationin Section 16.3 e, remove the agarose-film sheet,discard the electrode filter paper strips and samplefilter paper strips. Place the agarose-film sheetin the LKB staining tray and immerse in fixing

solution for 30 minutes with occasional gentleagitation. Perform this and all subsequent steps ina chemical fume hood with the exhaust turned on.

ii. Remove the agarose sheet from the first tray and place in a second tray containing destainingsolution. Wash for a 30 minute period changing thefluid once.

iii. Remove the agarose sheet from the destainingsolution and place on a glass plate. Place onesheet of Schleicher and Schuell #577 filter paper

(12.5 x 26 cm) over the agarose surface so that noair pockets are trapped under the paper. Then place2 sheets of Schleicher and Schuell #470 (12.5 x 26cm) on top of the #577 filter paper, followed by asecond glass plate and 1 kg weight. Allow sheets toremain in this manner for 15 minutes to effect aninitial drying of the agarose gel.

iv. Remove the weight, glass plate, and filter papers(discard). Complete the thorough drying of theagarose gel with a draught of hot air from a hand

held hair dryer. The agarose must be completely dryand adhering to the gel-bond sheet as a thin film ofits' own before proceeding to the next step.

v. Place the dried agarose-film sheet in the stainingsolution for 10 minutes.

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vi. Remove, drain, and place in destaining solutionuntil background is sufficiently clear.

vii. Remove, drain, and dry to a final state with thehair dryer.

viii. Examine and compare the isoelectric focused protein patterns of the unknown samples to those of thereference tissue extracts used to identify thesamples in question. The final dry preparation may be kept without further modifications as a permanentrecord of sample analysis.

16.4 Quality Control of Key Reagents or Procedures

In order to assure the integrity and reproducibility of the

previously outlined TLIEF procedure, special attention should begiven to the considerations cited below.

a. Agarose Gel-plastic Film Preparation.

Be sure to maintain the sterility of the stock ampholytesolution by using aseptic techniques and a new sterileneedle and syringe to withdraw the necessary volume ofampholyte needed to prepare the liquified agarosesolution. Ampholytes are susceptible to microbialcontamination and this would destroy their intended function.

b. Do not allow air bubbles to form during the injection ofthe liquid agarose solution into the glass sandwich. Air bubbles at this stage will produce a void in that area onthe solidified agarose sheet. The presence of air bubbles during electrofocusing will cause a discontinuouselectrical resistance between the electrodes. This mayultimately result in improper band migration for theapplied sample at that point.

c. Isoelectric Focusing of Samples and References.

Extracts from reference tissues should be prepared from relatively fresh tissues. Old tissues stored in thefreezer for a period of time beyond 6-12 months begin todemonstrate fewer bands. Reference tissue extracts(controls) should be applied to each agarose sheet used for an analytical determination of unknown samples. Do

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not rely on the use of previously prepared, dried,stained sheets of reference tissues for comparative purposes.

d. Fixing, Staining, and Destaining.

Proper staining contrast of the dried agarose sheet and protein bands depends upon complete removal of ampholytesand total drying of the agarose gel prior to staining.

Care should be given to wash well after the fixing step(step i; Section 16.3 f) and not to reuse the samequantity of fixing solution too many times as this willcause a build-up of ampholytes in it. Complete drying must be accomplished in step iv (Section 16.3 f) bycareful use of the hot air dryer prior to staining (step

v; Section 16.3 f). Destaining (step v; Section 16.3 f) must be accomplished carefully and empirically byfrequent examination of the sheet to insure that under orover destaining is not allowed to occur such that all protein bands are optimumly stained and appear readilyvisible.

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Hamilton, W. D. 1982. Fish species identification by thinlayer agarose isoelectric focusing and densitometric scanning.J. Assoc. Off. Anal. Chem. 65:119-122.

Pharmacia Fine Chemicals Agarose IEF pamphlet #52-1536-01.

Ukishima, Y., M. Kino, H. Kubota, S. Wada, and S. Okada. 1991.Identification of whale species by thin-layer isoelectricfocusing of sarcoplasmic proteins. J. Assoc. Off. Anal. Chem.74:943-950.

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______________________________________________________________________________________________


Notice of Change




This method revision incorporates testing of raw and cooked meat and poultry products for

species determination. Raw species testing was formally included in chapter MLG 21. That

chapter is now obsolete following issuance of MLG 17.02. Chapter 20 described a qualityassurance plan for a piece of equipment that is no longer used. Both obsolete chapters are now

archived.

Cooked species samples continue to be tested via ELISA-TEK ™

Cooked Meat Speciation Kits as

was described in the previous revision of this chapter.

For raw species, the DTEK ™

Immunostick Meat Speciation Testing Kit or Tepnel Biosystems

F.A.S.T. Immunostick Meat Species Screening Kit is now utilized as a screen test for availablespecies. Any sample with a positive screen result for an undeclared species are then cooked and

subjected to confirmatory testing using the ELISA-TEK

™

Cooked Meat Speciation Kits 4-wellconfirmation procedure.

The agar gel diffusion test found in Part C is no longer performed. The test may be useful in the

event that ELISA-TEK ™

Cooked Meat Speciation Kits are unavailable or that speciation of

poultry samples is required.

The methods described in this guidebook are for use by the FSIS laboratories. FSIS does

not specifically endorse any of the mentioned test products and acknowledges that

equivalent products may be available for laboratory use.

______________________________________________________________________________QD-F-Micro-0004.01 Approved: Charles Pixley, 2/25/03

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Title: Identification of Animal Species in Meat and Poultry Products

Revision: 02 Replaces: MLG 17.01 and

MLG Chapter 21 Animal Species

Determination, Immunological

Effective: 2/10/05

Approved by Phyllis Sparling, 1/25/05

Procedure Outline

PART A

Identification of Animal Species in Cooked and Canned Meat and Poultry 17.1 Introduction

17.1.1 General



17.3 Chemicals and Reagents17.4 Sample Preparation and Extraction

17.5 Preparation of Cooked Species Tissue Controls (optional)

17.6 Preparation of Kit Materials17.7 Detailed Enzyme Immunoassay Procedure

17.8 Determination of Test Validity and Sample Status

17.9 Quality Control Procedures17.9 Selected References

PART B

Commercial ELISA Immunostick Screen Test Kit for Raw Species

17.10 Introduction

17.10.1 General

17.10.2 Limits of Detection17.11 Reagents and Equipment

17.12 Raw Sample Preparation

17.13 Test Procedure17.14 Confirmation Sample Preparation

17.15 Quality Control Procedure


PART C

Agar Gel Immunodiffusion Test

17.17 Introduction17.18 Equipment17.19 Reagents

17.20 Preparation of Agar–Gel Immunodiffusion Plates

17.20.1 Agar Plate Preparation

17.20.2 Incubation and Observation

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17.20.3 Charging of Troughs

17.20.4 Charging the Wells17.20.5 Sealing Wells and Troughs

17.20.6 Cutting Pattern of Wells and Troughs

17.21 Interpretation of Precipitin Reactions17.21.1 Staining Reaction Bands (Optional)

17.22 Photographic Recording of Reaction Bands

17.23 Quality Control procedures

17.23.1 Tissue Extracts from Known Species17.23.2 Prepared Agar Gel Immunodiffusion Plates

17.23.3 The Specific Anti-species Sera


PART A

Identification of Animal Species in Cooked and Canned Meat and Poultry

17.1 Introduction

17.1.1 General

This section describes a procedure that can identify the species of animal tissue used as

ingredients in cooked and canned meat foods. A simple water extraction of samples alongwith an amplified double antibody sandwich type enzyme-linked immunosorbent assay

(ELISA) is employed. With this type of ELISA, capture antibody is bound to the

polystyrene plastic of microplate wells. If tissue antigens are present, they are bound bysolid-phase, species-specific, capture antibody in the wells. After washing to remove

unbound material, a biotinylated antibody with the same specificity as the capture antibody

is added. Biotin is a vitamin that can be covalently bound to antibody with relative ease and with virtually no deleterious effect on the specificity of the antibody molecule. The

biotinylated antibody is bound to the solid phase only if antigens of the species in question

were previously captured; otherwise, the unbound biotinylated antibody is removed by

washing. Streptavidin-Horseradish Peroxidase conjugate is then added to the wells.Streptavidin is a glycoprotein which has a remarkable affinity for biotin (K D = 10

-15M

-1).

Any biotin in the solid phase complex will bind the streptavidin-enzyme conjugate.

Unbound conjugate is removed by washing and a substrate for the enzyme is added. If the

species antigens were present in the test sample, a green color will develop as a result of the

action of the bound enzyme on the substrate. This assay allows for high sample output and

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Effective: 2/10/05


same day results. The procedure must be repeated for each species in question using the

appropriate species specific antibodies.

Antigenic molecules, when subjected to high temperatures (such as those encountered in the

production of cooked and canned meat products), will denature. The denaturing processincludes a randomization of the tertiary structure and reduced solubility. These changes

cause a concomitant reduction in the molecules' antigenicity. Hence, standard

immunoassays are usually not possible when samples have been subjected to denaturing

heat.

The present assay was developed by using unheated skeletal muscle tissue that was

fractionated then isolating the antigenic molecules, which proved to be relatively heat stable.These antigens are highly soluble, acidic glycoproteins. Antisera prepared to them are

species specific and are reactive with extracts of cooked and canned meat products

containing that species.

Important note:

One should not attempt to quantify the amount of a particular species tissue in a sample based on this assay. Because the antigens are not completely heat stable, a higher

temperature and longer cooking time will result in a lower reaction. Therefore, the intensity

of the reaction is related to the sample processing as well as to the level of antigen present.

A slight cross reaction occurs among red meat species. The colorimetric detection system of

the ELISA does not allow distinction between a low-level homologous reaction and a cross-

reacting species. This situation has been addressed by establishing an absorbance value thatmust be exceeded for a sample to be considered positive. The selected absorbance value is

significantly higher than any cross-reaction or background color.

Because cross reactions with raw tissue extracts might exceed the cutoff value, raw tissue

can only be analyzed using this test by following the cooking directions in Part B, Section

17.14.


a. This method, as evaluated with a specific lot of ELISA-TEK ™

Cooked Meat

Speciation Kits and extracts of each of the listed species sample extract,

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was found to detect at least the following percentage of adulteration in a

mixed extract of the target and another species:

Beef 1%

Pork 4%Chicken 4% (Poultry kit)

Turkey 4% (Poultry kit)

Sheep 4%

Horse 4%Deer 1%

b. Specificity was 100% for the seven species.

c. Sensitivity and specificity may vary from lot to lot. The manufacturer’s

Certificate of Analysis and laboratory acceptance testing is required for each lot of the test kit.


a. Flow (ICN) Laboratories Titertek Multiskan plate reader, or equivalent

b. Flow (ICN) Laboratories Titertek Microplate Washer, or equivalentmanual or automated washer system

c. Multichannel pipette; 8 channel, adjustable 50-200 ul volume

d. Repeater pipette with accessory of 1.25 and 2.5 ml capacity combitips (optional)

e. A variable-volume pipettor with tips

f. Stomacher ®

g. Whirl-Pak ®

bags; 6 oz. and 18 oz. sizes

h. Centrifuge, capable of operation at 15,600 G and appropriate centrifuge tubes

i. Refrigerator (2-8oC)/Freezer (≤-10

oC)

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j. Aluminum foil

k. Erlenmeyer flasks, 125 ml

l. Disposable Millipore®

filters, 0.45 µm, luer lock

17.3 Chemicals and Reagents

a. ELISA-TEK ™ Cooked Meat Speciation Kits

510621 Detection of Cooked Beef

510631 Detection of Cooked Pork

510641 Detection of Cooked Poultry for Chicken and Turkey510661 Detection of Cooked Deer*

510651 Detection of Cooked Sheep*

510611 Detection of Cooked Horse*

*Note: These species and certain other species are tested by FSIS Laboratories only by

special request.

b. Saline, 0.85%

17.4 Sample Preparation and Extraction

Note: Care must be taken at this stage not to cross-contaminate samples; any equipment or utensils

used must be either disposable or thoroughly washed before the preparation of each sample.

a. Prepare a cooked or canned meat/meat product for sampling by dicing or finely

chopping.

b. Weigh 5 ± 0.5 g of the diced sample in a 6 oz. Stomacher ® or Whirl-Pak ® bag.

c. Add 10 ± 0.5 ml of deionized water or normal saline.

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Note: If the consistency of a product makes it difficult to identify and separate the meat portion, the

sample size in step “b” may be increased with a proportional increase in the amount of diluentadded in step “c” to obtain a representative sample.

d. Place bag and contents into a Stomacher ®

for approximately 60 seconds.Alternatively, for products that tend to emulsify, the mixture may be kneaded or

manually homogenized.

e. Remove homogenized sample from the Stomacher ®

and leave undisturbed for atleast 1 hour at room temperature.

Note: All samples submitted under the cooked program must be cooked. If it is suspected that thesample is not fully cooked, it is advisable to heat the extract (meat/water mix) in a water bath at 95-

100°C for 15 ± 1 minutes prior to mixing and centrifugation/filtration.

f. Pour off some of the sample extract into a centrifuge tube and set the centrifuge at

10,000 X G for 10 minutes (consult centrifuge manual to convert RPM to G).

g. The clear supernatant of the tissue extract is used in the ELISA.

Note: If the sample has a high fat content, the clear supernatant above the settled meat layer may be beneath a layer of fat. Avoid transferring the fat. It may be appropriate to carefully remove a

portion of the aqueous solution using a clean pipette (e.g. Pasteur-type) into a second, clean

container prior to analysis.

Note: Sample extracts may be stored at 2-8°C for up to 36 hours. If prolonged storage is required,

the extracts must be kept frozen. They will remain stable for several months when stored at ≤-10°C.

17.5 Preparation of Cooked Species Tissue Controls

Note: Positive and negative cooked meat species tissue controls are provided with each kit.Cooked species tissue controls, used for lot acceptance of kits, are prepared as follows:

a. Prepare a portion of lean, raw meat by dicing, mincing, or finely chopping tissue.

b. Weigh 20 ± 2 g of the diced tissue in a Stomacher ®

or Whirl-Pak ®

bag. Add 60 ±

0.5 ml of normal saline.

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c. Place bag and contents into a Stomacher ®

for approximately 10 seconds.Alternately, the mixture may be kneaded or manually homogenized.

d. Remove sample from the Stomacher ®

and leave undisturbed for at least 1 hour atroom temperature.

e. Place the contents of the bag in a 125 ml Erlenmeyer flask, seal with aluminum foil,

and place in a boiling water bath (95-100°C) for 15 ± 1 minute.

f. Remove the flask from the water bath and allow it to cool.

g. Transfer a portion of the supernatant (being careful to avoid transferring fat) to a

centrifuge tube and set the centrifuge at 10,000 X G for 15 minutes.

h. Filter the supernatant through a 0.45 micrometer filter, then dispense into clean vials,

label, and store frozen at ≤-10°C.

17.6 Preparation of Kit Materials

Note: Be sure to use a clean pipette tip for each control and sample to be tested. Pipettors must be

verified before use and set to deliver the quantities indicated in the method steps.

a. Antibody Sensitized Microwell Module: Open the foil pouch (label side up) by

cutting between the notches on the end marked “cut here.” Open the sealing strip

and remove the Microwell Module, keeping the wells open side up. Select thedesired number of strips and fit them into a spare frame. Replace the original frame

and remaining strips into the pouch, taking care that the desiccant is present, and

reseal the pouch carefully. If necessary, reseal the pouch with adhesive tape or byheat sealing.

b. Bring the kit positive controls, anti-species biotinylates, streptavidin peroxideconjugate, and “Stop Solution” to room temperature. These reagents are supplied

pre-diluted. Mix each by gentle, repeated inversion.

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c. ABTS Concentrate and Peroxide Citrate Buffer: Mix contents of each separate vial

by inversion. DO NOT SHAKE. ABTS is supplied as a 25 fold concentrate and must be diluted in Peroxide Citrate Buffer to prepare a working ABTS solution.

Note: Dilutions of ABTS Concentrate should be made just prior to use (e.g. during theavidin peroxidase conjugate incubation).

For 96 test wells add 500 µl of ABTS Concentrate to the 12.0 ± 0.1 ml of Peroxide Citrate

Buffer. For any other number of test wells, dilute ABTS Concentrate in a 1:25 ratio with

Peroxide Citrate Buffer. Stopper the vial and mix well by gentle swirling.

d. Wash Solution Concentrate: Wash Solution Concentrate is supplied as a 10 fold

concentrate and requires dilution in purified water to prepare a Working Wash

Solution.

For 96 test wells, use the total contents of the Wash Solution Concentrate (100 ml)

adding deionized water to a total volume of 1L.

For any other number of test wells, dilute the Wash Solution Concentrate in a 1:10

ratio with deionized water.

17.7 Detailed Enzyme Immunoassay Procedure

Sample Extracts are tested using 2 well replicates. Samples containing violative species (see 17.8for sample status) are re-tested for the violative species using a 4 well format.

Plates should be covered during all incubation periods; i.e., with a lab wipe or other protectivecovering.

Number the strips in sequence on the upper frosted edge. A marker, e.g. Sharpie®, works well.

This preserves the identity of the strips should they become detached from the frame. However, if

the strips come detached while fluid are present in the wells then the test should be rerun due to

possible cross contamination and inadequate reagent volumes.

a. Place 100 µl of normal saline in each of the wells selected as blanks.

b. Place 100 µl of each negative control in each of the 4 wells selected.

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c. Place 100 µl of each positive control in each of the 4 wells selected.

d. Place 100 µl of each sample extract in each of the 2 or 4 wells selected. Avoid

transferring fat from sample preparations to the wells.

e. Mix the plate gently by hand. Allow to stand at room temperature for 60 ± 5 min.

f. At the end of the incubation period, empty the wells by flicking into the sink. Filland aspirate the wells three times with Working Wash Solution. Invert the

aspirated plate and rap lightly several times onto a soft paper towel placed on the

lab bench. When inverting the plate, be sure to squeeze the plastic frame at thecenter of the long edges to prevent the strips from falling out of the frame.

g. Add 25 µl of anti-species biotinylate to the bottom of each microwell of the relevant

(same species) antibody sensitized strips. Observe that the bottom of each well is

covered with liquid. If not, gently tap the edge of the plate until this isaccomplished. Avoid getting any antibody on the sides of the wells.

h. Leave at room temperature for 60 ± 5 minutes.

i. Repeat the washing step (step f).

j. Add 25 µl of peroxidase conjugate to the bottom of each well. Again, observe thatthe bottom of each well is covered with liquid and that no conjugate sticks to the

sides of the wells.

k. Leave at room temperature for 30 ± 1 minutes.

l. Repeat the washing step (step f), except wash six times instead of three.

m. Add 50 µl of the Working ABTS Solution to the bottom of each microwell.

n. Cover the plate to protect the ABTS from direct light. Leave at room temperature.

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o. Warm up the plate reader and set the program to use a dual wavelength

measurement mode. The program should read absorbance (OD) using a 414 nmabsorbance filter and a 492 nm reference filter. The results of the second

measurement are subtracted from the results of the first measurement. Set the

instrument to blank on the well in row “A,” column 1, with continuous movement.

p. Observe the microwells containing the positive controls for visual color change.

When color change is observed, place the plate on the reader carriage. Read and

obtain absorbance values for the wells. Continue to read until positive control ODvalues read in a range of 0.450 to 0.500.

q. When positive control OD values read in the desired range, add 50µl of “StopSolution” to each microwell. (If the positive control OD values are already above

0.600, save the reading and print. See section 17.8a for further explanation.)

r. Mix the plate gently by hand to distribute the “Stop Solution” and to prevent further

color development.

s. Return the plate to the reader carriage, read the plate, and obtain a printed copy of

the OD values for the plate. Save the electronic file.

17.8 Determination of test validity and sample status

a. The assay is valid if the mean absorbance value of the positive control is greater than

0.600, with standard deviation of its replicates no more than 0.060, and the mean of the negative controls is less than 0.060. Otherwise, the test is invalid and should be

repeated. Positive controls that are 0.900 or above can cause low level non-

reportable results to become positive. Rerun these samples if species result is inviolation.

b. Samples are classified as presumptive positive if the raw OD value of one or both of

the two replicate wells is greater than 0.250.

c. Test samples determined to be presumptive positive for an undeclared species must

be repeated using 4 well replicates. A test sample analyzed in 4 well replicates is

classified positive if the adjusted mean OD is greater than 0.250. The adjusted mean

OD value is obtained by subtracting 3 times the standard deviation from the raw

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mean OD value of the 4 wells. Computer software, such as Excel®, may be used to

calculate the mean and standard deviations from the raw data.

d. Samples are reported as negative for each species for which the raw OD value of

both of the two replicate wells is less than 0.250.


Each species analysis is run with a positive and negative control supplied in each kit. Lotacceptance should be performed on each lot of species kits received.

a. Control testing should be performed as in Section 17.5 steps “a-h”.

b. Store all kit components at refrigerator temperature (2-8oC) when not in use, to

preserve and maintain reactivity of immunoreagents.

c. Observe the manufacturer's expiration date of all test kit components. Kits should

not be used beyond the expiration date. Do not mix components from one lotwith components from another lot.

d. Dilution of ABTS into the Peroxide Citrate Buffer should be done just prior to use.

e. Kit components should be allowed to equilibrate to room temperature before

commencing test procedure.

f. The 5 gram test sample used for extraction must be representative of the entire

original sample in order to insure that test results accurately reflect the true

composition of the original sample.


Andrews, C. D., R. G. Berger, R. P. Mageau, B. Schwab, and R. W. Johnston. 1992. Detection of beef, sheep, deer, and horse meat in cooked meat products by enzyme-linked immunosorbent assay.J. Assoc. Off. Anal. Chem. Int. 75:572-576.

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Berger, R. G., R. P. Mageau, B. Schwab, and R. W. Johnston. 1988. Detection of poultry and

pork in cooked and canned meat foods by enzyme-linked immunosorbent assays. J. Assoc. Off.Anal. Chem. 71:406-409.

PART B

Commercial ELISA Immunostick Screen Test Kit for Raw Species

17.10 Introduction

17.10.1 General

A commercial ELISA Immunostick Screen Test is employed for presumptive identification

of species composition of raw meat and poultry tissues. This procedure is an Enzyme-Linked Immunosorbent Assay (ELISA) method that incorporates the use of NUNC dip-stick

paddles (immunosticks) as the solid phase and the use of pre-dispensed, standardized

reagents in color coded tubes. It is currently marketed and distributed in the U.S. in acomplete (25 test) kit form and is referred to as a commercial ELISA Immunostick Raw

Meat Species Screening Test Kit.

This raw meat species screen test is a double antibody "sandwich" ELISA procedure withantibody specificity directed against the various species albumins which are contained in

meat tissues. Specific antibody-sensitized immunosticks are allowed to capture ahomologous species’ albumin from sample tissue extracts, then the immunosticks react with

the second peroxidase labeled antibody of the same specificity, followed by a final reaction

step in an ABTS/H202 chromogen/substrate solution. A short incubation period and a brief

tap water rinse are performed between each of the first two steps. A positive reaction,indicating the presence of the test species tissue in the sample, is evidenced by a distinct

green color formation in the last reagent tube. All positive screen test results which

represent sample violations are to be confirmed by the enzyme-linked immunosorbent assay(ELISA) for Cooked Species (See Part A). The heating of raw meat and poultry products

results in the isolation of heat–resistant antigens that are immunoreactive with the antibodiesfound in the Cooked ELISA kits.

Each of the Immunostick Screen Kits contains all the necessary reagents, controls and accessories to easily perform the screen test with the production of very accurate results.

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a. This method, as evaluated with a specific lot of ELISA Technologies,

Inc. Commercial ELISA Immunostick Raw Meat Species Screen Test

Kits for each of the listed species, was found to detect the following percentage or greater of adulteration in a mixed extract of the target

and a nontarget species:

Beef 1%

Pork 1%Chicken 1% (Poultry kit)

Turkey 4% (Poultry kit)

Sheep 1%Horse 1%

b. Specificity was 100% for the six species.

c. Sensitivity and specificity may vary from lot to lot. A manufacturer’s

Certificate of Analysis and laboratory acceptance testing is required for each lot of the test kit.

17.11 Reagents and Equipment

a. Commercial ELISA Immunostick Raw Meat Species Screen Test Kits. Color codes

for individual species kits used are as follows (Table 1):

Table 1. Color Codes for Commercial ELISA Immunostick Screen Test Kits.

Color Code Species

Red Beef

Yellow Pork

Blue Poultry*

Orange Horse**

Green Sheep**

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* NOTE: The ELISA Immunostick Poultry screen test does not differentiate between chicken and turkey. Upon special request this can be accomplished by

performing the traditional agar-gel immunodiffusion procedure (Part C). Since the

AGD procedure has less than the required sensitivity, its use should be limited towhole meat or poultry tissues or a mixed meat/poultry emulsion where the poultry

component is known to constitute over 5% of the final meat block.

**NOTE: These species and others shall be performed in FSIS Laboratories byspecial request only.

Each individual species kit contains the following items:

i. Twenty-five color coded, white plastic immunosticks sensitized with specific

anti-species capture antibody in tubes of preservative buffer solution.

ii. Twenty-five color coded tubes containing species specific antibody-enzyme

conjugate reagent.

iii. Twenty-five tubes (non-color coded) containing color development buffer

reagent.

iv. One vial of concentrated ABTS color reagent.

v. One vial of aqueous sodium fluoride stop solution.

vi. One vial of positive control solution (homologous species albumin).

vii. Product insert test kit instruction pamphlet.

b. Adjustable pipette and appropriate disposable pipette tips.

c. Stomacher ®

400 or 3500, or equivalent.

d. Whirl-Pak ®

polyethylene bag, 6 oz size (7.5 x 17 cm), or equivalent.

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17.12 Raw Sample Preparation

All types of raw meat and poultry product samples are prepared as follows:

a. Weigh out 1 ± 0.1 g of thawed, diced, raw sample product which is a homogeneous,representative portion of the whole sample.

b. Place in a 6 oz Whirl-Pak ®

bag, or equivalent.

c. Add 9 ± 0.1 ml of distilled water.

d. Place the bag and its contents in a Stomacher ®

and stomach for a period of 60seconds. Allow the extract to settle for 2-3 minutes, until a particle-free liquid layer

is formed in the top portion of the bag's contents, or centrifuge at 10,000 X G for 10

minutes (see centrifuge manual for proper speed setting). Use the upper liquid layer as the sample extract in the following test procedure.

17.13 Test Procedure

The following procedure uses minor modifications from the manufacturer’s test kit instruction

insert. These procedural modifications are designed to improve the accuracy, precision, and

reproducibility of test results. The subsequent instructions represent the testing of one sample withone species test procedure. Obviously multiple samples and/or species tests may be performed

simultaneously, as long as one is careful to keep track of reaction times, washing steps, various

reagent steps, etc., relative to each given test sample.

a. Remove the appropriate color coded species immunostick tube, antibody-enzyme

conjugate reagent tube, and color development buffer tube (a set of 3) fromrefrigerated storage and allow equilibrating to room temperature.

b. Label immunostick caps and all tubes with appropriate sample identification codes.

c. Prepare the color development buffer reagent tube (non-color coded) for later use byadding 40 µl of ABTS concentrate to this tube, replacing the cap, then mixing in a

gentle but complete manner.

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d. Obtain the first color coded immunostick tube, unscrew the cap and remove the

immunostick-paddle. Add 200 µl of prepared sample extract to the liquid in thetube, replace the immunostick-paddle in the tube, and mix contents by rotating the

cap rapidly for at least 4-6 times then tighten the cap. DO NOT INVERT tubes to

accomplish mixing at any stage in this procedure. Only handle the paddle by itsattached cap at all times. DO NOT TOUCH paddle with fingers.

e. Allow this tube to stand for 10 minutes at room temperature.

f. Remove the immunostick-paddle and wash the paddle and entire cap completely by

placing it under a gentle stream of cold tap water for a minimum of 10 seconds, then

shake to remove excess water.

Note: Water dispensed from a squeeze bottle can also be used to carefully perform

this wash step.

g. Place the washed immunostick-paddle into the second color coded tube of antibody-

enzyme conjugate reagent. Mix contents by rotating the cap rapidly for at least 4-6times and tighten the cap.

h. Allow this antibody-enzyme reagent tube to stand for 10 minutes at room

temperature.

i. Remove the immunostick-paddle and wash the paddle and entire cap completely by

placing it under a gentle stream of cold tap water for a minimum of 30 seconds, thenshake to remove excess water.

Note in step “f” above also applies here.

j. Place the washed immunostick-paddle into the final, non-color coded, tube of ABTS

prepared (step c) color development buffer reagent, mix contents by rotating the cap

rapidly for at least 4-6 times and tighten the cap.

k. Allow the color development reagent tube to stand for 10 minutes at room

temperature.

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l. Add 200 µl of sodium fluoride stop solution to this color development tube, leave

the paddle in, and mix well to stop the reaction.

m. Observe the above tube with the white paddle in it for the presence of any

discernable green color in the solution or on the paddle surface. A green color indicates a positive test and the presence of the test species in the original meat

sample. A colorless solution around the white paddle indicates a negative test and

the absence of the test species in the sample.

All ELISA immunostick positive species results which represent sample violations shall be

confirmed by performing the Cooked ELISA test found in Part A of this chapter.

17.14 Confirmation Sample Preparation

a. Weigh out 5 ± 0.5 g of thawed, diced, raw sample product which is a homogeneous,representative portion of the whole sample and add 15 ± 0.5mls of normal saline or

distilled water.

b. Stomach for 60 seconds and let stand for 1 hour at room temperature.

c. Transfer contents to a screw cap test tube, loosen cap and autoclave for 15 ± 1

minutes at 100 ± 1oC, or boil for 15 ± 1 minutes. Let cool, then transfer to a

centrifuge tube and centrifuge at 10,000 X G for 10 minutes (see centrifuge manual

for proper speed conversion). Use the supernatant for the Cooked ELISA procedure

(Part A) and analyze using the 4 well confirmation procedure. See Section 17.8.c.


Each species analysis should be run with a negative and positive control of the species of interest.

This should also be performed when receiving a new lot of test kits. Each species test kit is supplied

with a positive control vial (homologous species albumin solution) for these purposes. The negativecontrol for any one particular species test kit may be obtained by using the positive control solutionfrom any of the other heterologous species test kits (e.g., horse albumin solution should always give

negative results in all other species kits except horse).

a. Control testing should be performed as in steps “a-m” in Section 17.13.

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b. Store all kit components at refrigerator temperature (2-8oC) when not in use, to

preserve and maintain reactivity of immunoreagents.

c. Observe the manufacturer's expiration date of all test kit components. Kits should not be used beyond the expiration date. Do not mix components from one lot with

components from another lot.

d. The concentrated ABTS color reagent solution tube should be observed over the kitshelf life. If this ABTS concentrate should start to turn a darker shade of green than

when it was originally received, this indicates decomposition, and a new tube of

ABTS concentrate should be requested from the vendor.

e. Kit components should be allowed to equilibrate to room temperature before

commencing test procedure.

f. The one gram test sample used for extraction must be representative of the entire

original sample in order to insure that test results accurately reflect the truecomposition of the original sample.

g. Preparation of the color development buffer reagent tube by the addition of ABTS

concentrate (step “c” of Section 17.13, Test Procedure) should only be accomplished just prior to commencing the test procedure. Preparation of this reagent tube should

not be done in advance (hours/days) because of the inherent chemical instability of

ABTS in buffered substrate for extended time periods.

h. Accurate timings of washing and reaction steps should be performed.

i. Assure that all surfaces of the white immunostick-paddle and cap are adequately

washed during the two timed wash steps.

j. Do not use hot or warm water for immunostick-paddle washing, only cold.

k. Since all reactions of this solid phase immunoassay occur on the surfaces of the

white immunostick-paddle, it is very important not to touch the paddle surface with

fingers or any other physical objects which might interfere with the

immunoreactions.

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l. When performing different species tests simultaneously on the same sample, be sureto maintain the proper continuity of color coded reagent tubes for each respective

test species as you complete the test procedure (e.g., an anti-beef species

immunostick [red color code] that has reacted with a beef sample extract, if improperly placed in an anti-pork enzyme conjugate reagent tube [yellow color

code], will produce a false negative result).


Anonymous. 1991. Commercial Immunostick Raw Meat Species Screening Kits; productinsert instruction pamphlet

Fukal, L. 1991. Review Article. Modern immunoassays in meat-product analysis. Die Nahrung 35(5):431-448.

Hsieh, Yun-hwa P., B.B. Woodward, S.H. Ho. 1994. Detection of Species Substitution in Raw and Cooked Meats Using Immunoassays. J. Assoc. of Food Protection, Vol.58 (5), pp 555-559.

PART C

Agar Gel Immunodiffusion Test

17.17 Introduction

The test described in this section is included primarily for informational purposes. FSISlaboratories do not routinely perform this analysis.

The agar-gel immunodiffusion procedure described in this section is based upon fundamental

principles established previously by Ouchterlony, 1968, and modified for specific application and Agency use by Fugate and Penn, 1971. Agar-gel immunodiffusion is notable for its qualitativeability to demonstrate similarities and resolve differences in related proteins based upon the

formation of specific immunoprecipitin lines resulting from the diffusion of specific antigens and

antibodies from wells or troughs cut into an agar matrix after they have reached their optimum

proportions. As such, this procedure is ideally suited for meat species protein identification. If any

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false or "non specific" reactions should occur in a double immunodiffusion assay, it is possible to

distinguish them from true positive reactions by carefully observing the immunoprecipitin patternformed and it's relationship to known antigen extracts. The three basic types of reactions usually

observed in double immunodiffusion assays are lines of identity, lines of partial identity, and lines

of non-identity. With a little practice and experience these types of reactions can be easilydistinguished and their interpretation in relation to resolving the identity and/or relationships of

similar proteins can be made in a definitive and reliable manner.

Although several different patterns of wells or troughs may be generally used in an agar-gel to perform double immunodiffusion reactions, the pattern ultimately employed is usually dependent

upon the intended, specific application of the assay. Hvass, 1985, used a relatively simple,

common, 7 well, circular pattern to differentiate raw meat species, while Fugate and Penn, 1971,used a more complicated pattern consisting of 3 antisera troughs and 24 antigen extract wells. The

latter was designed with the intention of demonstrating relationships among more than one species

on a single plate and also to provide several identical reaction areas on the same plate showing theidentity or non-identity relationship of an unknown meat species sample with known reference

species tissue extracts.

17.18 Equipment

a. Dish, Petri, plastic, 15 X 100 mm disposable

b. Pipettes, disposable, capillary, Pasteur type

c. Box, plastic, humidity chamber, or other air tight container used to maintain highhumidity

d. Cutter, agar-gel, or template pattern

e. Flask, side arm

f. Tubing, rubber or neoprene, high vacuum type

g. Tubing, brass (Cork borer), 5/32 x 1-3/4 inch

(3.95 x 44.5 mm)

h. Applicators, wooden, cotton tipped

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i. Pipettes, graduated, serological, assorted sizes

j. Dishes, staining (only if agar is to be dried and stained)

k. Slides, microscope, 1 x 3 inch (2.54 x 7.62 cm); (only if agar is to be dried and

stained)

l. Filter paper, Whatman®

No. 1 and No. 42

m. Pans, plastic, 6 x 12 x 6 inch (15.2 x 30.5 x 15.2 cm), or other suitable containers

(used only if agar is to be air dried and stained)

n. Assorted laboratory flasks, beakers, tubes, etc.

Clean all glassware, rinse in distilled water, and heat a minimum of two hours at 200 ± 2oC

in a dry heat oven to eliminate contamination from prior use.

17.19 Reagents

a. Normal saline, (0.85 percent sodium chloride solution)

b. Buffered saline (0.85 percent sodium chloride solution, pH 7.2 phosphate buffered)

c. Phosphate buffer stock solution - pH 7.2

d. Agar, 1.0 percent (Oxoid ®

Purified Agar, L28)

e. Tissue extracts from known animal species:

Cut muscle tissue collected from animals (known species) into 10 ± 0.5 g of ground

or finely diced portions and freeze until needed. Add 30 ± 1ml normal saline and stomach for 5 – 10 minutes maximum. Let stand a minimum of 90 minutes. Decantliquid and filter through Whatman

®No. 42 filter paper. Use immediately. (Note

Section Quality Control of key reagents or procedures.)

f. Antisera:

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Undiluted anti-horse, beef, pork, sheep, chicken and turkey species serum, or others

as may be required.

g. Tissue extracts (unknown samples to be confirmed):

Extract unknown tissue(s) as in (e) above, using 25 ± 0.5 g tissue and 75 ± 1mlnormal saline.

h. Staining solution:

Dissolve 2 g acid fuchsin in 500 ± 0.5 ml absolute methyl alcohol; add 400 ± 0.5mldistilled water and 100 ± 0.5 ml glacial acetic acid.

i. Destaining solution:To 500 ± 0.5 ml absolute methyl alcohol, add 400 ± 0.5ml distilled water and 100 ±

0.5 ml glacial acetic acid.

j. Acidified Distilled Water:

To 1000 ± 1ml distilled water, add 0.2 ml glacial acetic acid.

k. Mounting fluid:

A commercially available material for mounting cover slips permanently.

17.20 Preparation of Agar-Gel Immunodiffusion Plates

17.20.1 Agar Plate Preparation

Re-melt purified agar prepared above and dispense 18-20 ml into the 15 x 100 mm plastic

Petri dishes. Allow to solidify and refrigerate for a minimum of 30 minutes. Store no more

than 2 weeks under refrigeration in a high humidity atmosphere. Do not use plates showingdesiccation or microbial growth. (Note: Quality Control Section 17.23)

17.20.2 Cutting Pattern of Wells and Troughs

Remove the plates from refrigeration and cut the desired pattern by one of the two methodsdescribed below:

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a. Use a gel cutting tool which has the proper well and trough cutting tubes

and has knife edges permanently embedded in a fixture, such as Plexiglas®

or other solid substance.

Figure 1 illustrates one such tool. Align the tool carefully on the agar surface to obtain a perpendicular cut, and then press down firmly to cut the

agar.

FIG. 1 - Cutting tool used to cut pattern of wells and troughs in agar-gel. (Fugate and Penn, 1971)

b. Using a pattern of the desired arrangement drawn on graph paper, center

the plate over the pattern, agar side up. Press a metal tube of acceptable

diameter, connected to a vacuum source by a vacuum tube and side arm

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flask, through the agar at the indicated places on the pattern. Then cut the

troughs with a razor blade or scalpel along the lines of the pattern; or use atool fashioned with two blades or knife edges the correct distance apart,

and with a downward motion cut the agar.

Remove the agar plugs in the wells with a metal tube connected to a

vacuum source. Experience will dictate how to avoid tearing the agar

surrounding the wells. Remove the trough plugs with an applicator stick

which has one end shaved to present a shovel edge. Gently push theapplicator stick to the dish bottom and guide it along the cut, raising the

strip of agar as a plow would.

Remove the remaining agar in the wells and troughs with a cotton tipped

applicator very carefully so as to not tear the surrounding agar surface.

17.20.3 Sealing Wells and Troughs

With a Pasteur pipette, place a thin layer of agar on the floor of each well and trough,sealing the bottom edges of the cut agar to the plate. Do not add an excess of agar. Repair

torn wells or troughs in a similar way; if necessary, refill the well or trough and recut it.

Caution: An overfilled well will distort the agar and the reaction bands.

17.20.4 Charging the Wells

Mark the outside of the plate to identify the location and contents of each well and trough.Using a Pasteur capillary pipette, partially fill the wells with the known and unknown

extracts prepared in “e” and “g” of Section 17.19, maintaining a concave meniscus.

Overfilling to form a convex meniscus will interfere with diffusion and may cause wells tooverflow. Always place the extract of the unknown between known antigens of two

different species. Like antigens will form continuous reactant bands in the agar media, and

unlike antigens will form discontinuous bands (See Figure 2).

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FIG. 2 - Precipitin pattern resulting from heterologous antigen-antisera reactions: “a” antigens derived from

species A; “b” antigens derived from species B; “u” antigens derived from unknown; “⇒” are lines

of partial identity; “→” are lines of identity. Although atypical, the above pattern results when all

antigens react with antisera used. The identification of unknown antigen “u” is accomplished bylines of identity formed with antigen “a.” Both “a” and “u” form lines of partial identity with lines

formed by antigen “b,” which is indicated by a spur reaction. It can be concluded that antigen “u” is

derived from species A and is similar but not identical to species B. (Fugate and Penn, 1971)

17.20.5 Charging of Troughs

Fill troughs with the antisera. Use one plate to determine two species only (e.g., beef and

sheep, or beef and horse, etc.). Use the top and bottom troughs for one antiserum, and thecenter trough for the other. (See Figure 2)

17.20.6 Incubation and Observation

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Replace the plate covers and allow the plates to remain at room temperature for 1 1/2 to 2 h.

Refill the wells and troughs with the appropriate antigens and antisera. Line the bottom of an airtight chamber with wet filter paper or cotton. Incubate the plates in this high humidity

chamber at room temperature for 18 to 24 h. To read the plates, direct a light source parallel

to the agar surface (i.e., from the side of the plate), and hold the plate over a dark black background. The reactant bands will appear white on a grey surface. If the bands are not

fully developed, refill the wells and troughs, and continue incubation in the chamber for an

additional 24 to 48 h under refrigeration.

Following incubation, remove the plates from the humidity chamber, discard the remaining

reactants and gently wash the plates under a stream of distilled water. Use a soft cotton

applicator to remove any film from the agar surface and precipitated matter from the wellsand troughs. Dry the bottom of the Petri dish with a soft laboratory tissue and observe the

plate for reaction bands. Position the plate in alignment with the worksheet (Figure 3) and

draw the reaction bands observed on the plate onto the worksheet.

FIG. 3- Worksheet showing well and trough arrangement and antigen-antisera placement (Fugate and Penn,

1971)

17.21 Interpretation of Precipitin Reactions

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Interpretation of results depends upon lines formed with known and unknown antigens. Figure 4

(A) illustrates an identity line, i.e., the precipitin line that forms when the antigens are identical.Figure 4 (B) shows partial identity lines, i.e., the lines that form when extracts contain similar but

not identical proteins which react with the same antiserum. Figure 2 (page 25) illustrates a typical

reaction with an unknown and two known antigens, showing lines of identity and partial identity.Since unknown antigen “u” forms a continuous wave pattern with known antigen “a,” lines of

identity form. The lines formed by known antigen “b” appear as spurs of those formed by antigen

“a” and “u,” and are typical lines of partial identity.

FIG. 4 - Precipitin lines of identity and partial identity. Pic A: lines of identity formed with homologous

antigen-antiserum only (antigen “a” vs. antiserum “A”); pic B: lines of partial identity formed whensimilar antigens react with the same antiserum. Note the typical spur formed, indicating lines of partial

identity (antigen “c” and “d” are similar but not identical). (Fugate and Penn, 1971)

Figure 2 also illustrates the pattern of precipitin lines formed when the sample contains tissueantigens from two species (wells “ba”). In the majority of cases, the antisera will not react with

heterologous antigens, and lines of partial identity do not form. This occurs when the animal species

are closely related (such as bovine and ovine).

Figure 5 illustrates areas containing identical antigen alignment. Four of the 6 areas have antigens

reacting with antiserum “A” and 2 of the 4 areas are in position to react with antiserum “B.” The 2

remaining areas (2 and 4) are control as well as indicative sites. The mixtures of antigens “a” and

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“b” in wells marked “ba” are in position to react with both antisera and illustrate precipitin lines that

occur when the sample contains tissues from both species.

FIG. 5 - Position and reaction sites (6 areas) each consisting of 4 antigen wells. With the exception of areas

2 and 4, antigen placement is identical in each area. Areas 2 and 4 utilize one well each for amixture of the 2 known antigens (ba), and illustrate precipitin reactions when sample consists of

tissues from both species. All areas, except 1 and 6, are positioned to react with both antisera.

Interpretation of results from areas 1, 3, 5, and 6 should correlate. Lines enclosing areas indicate portion of plate mounted on slides for preservation. (Fugate and Penn, 1971)

17.21.1 Staining Reaction Bands (Optional)

To keep a permanent record, dialyze to remove free proteins and salts, then dry, stain, and

prepare a mount under a cover slip, as follows:

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Flood the plate with 500 to 1000 ml pH 7.2, buffered saline in a plastic pan. Replace with

fresh buffer twice daily for three days, then once daily for two more days. Finally replacewith acidified distilled water and let stand overnight.

Drain off the acidified distilled water, and cut a block of the reaction areas from the agar,and place it onto a 1 x 3 inch (2.54 x 7.62 cm) marked glass slide. Cover the block with a

strip of filter paper, and dry in the incubator to a very thin film. Wash gently with a cotton

applicator wetted with distilled water to remove adhering bits of the filter paper. Stain the

films in acid fuchsin staining solution for 10 minutes. Remove the excess stain and rinse indestaining solution for a period of 15-20 minutes using 2-3 changes, until the agar is clear.

Allow the slides to dry, and then mount under cover slips with mounting fluid.

17.22 Photographic Recording of Reaction Bands

One of the easiest methods to obtain a permanent record of the immunodiffusion reaction is to photograph the entire unstained plate. Although there are many ways to achieve this, one of the

easiest and quickest is to use a Cordis®

Immunodiffusion Camera. This is an instrument with preset

optics, light source and Polaroid ®

Camera which uses Polaroid ®

Type 084 or 107 black and whitefilm packs. The plate is placed in the instrument, the shutter is tripped, the film tab is pulled from

the camera, and within 25 seconds an excellent quality black and white print of the

immunodiffusion reaction is produced.


17.23.1 Tissue Extracts from Known Species

It is extremely important to establish the authenticity of these reference tissues before theyare used, since the basis for the types of immunodiffusion reactions obtained with unknown

tissue extracts in the agar gel immunodiffusion test depends upon the use of known species

tissue extracts.

17.23.2 Prepared Agar Gel Immunodiffusion Plates

It is usually convenient to prepare a large number of plates at one time for future needs.

Care must be taken to prevent deterioration of these plates during storage in the refrigerator.

It has been found most useful to stack about 10 plates together in double or triple, air tight,

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tightly sealed plastic bags. Any plates showing microbial contamination, desiccation, or salt

crystal formation should not be used as they will adversely effect the formation of immunoprecipitin lines.

17.23.3 The Specific Anti-species Sera

Sera used in the immunodiffusion procedure should always be initially checked for their

proper reactivity against known, authentic reference tissues prior to their routine use as a

diagnostic reagent.


Fugate, H. G., and S. R. Penn. 1971. Immunodiffusion technique for the identification of animal

species. J. Assoc. Off. Anal. Chem. 54:1152-1156.

Hvass, A. 1985. Species differentiation in minced meat products by immunodiffusion, p. 53-64. In

R. L. S. Patterson (ed.), Biochemical Identification of Meat Species. Elsevier Science Publishing

Co., Inc., New York, NY.

Ouchterlony, O. 1968. Handbook of Immunodiffusion and Immunoelectrophoresis. Ann Arbor

Science Publishers, Inc., Ann Arbor, MI.

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Chapter 18. SPECIES IDENTIFICATION FIELD TESTS (SIFT)

Mark E. Cutrufelli and Richard P. Mageau

18.1 Introduction

A series of individual, serological screen tests has been developed for rapid species verification of raw whole/ground meat tissue oremulsified meat products in field environments. They arecollectively referred to as the Species Identification Field Tests(SIFT). The individual tests which comprise SIFT are as follows:ORBIT (Overnight Rapid Bovine Identification Test), PROFIT (PoultryRapid Overnight Field Identification Test), PRIME (Porcine Rapid Identification Method), SOFT (Serological Ovine Field Test), REST(Rapid Equine Serological Test), and DRIFT (Deer Rapid

Identification Field Test).

The basis of these tests is that of an agar-gel immunodiffusiontechnique using stabilized reference antigen and antibody reagentimpregnated paper discs and prepared agar-gel plates that have a printed template for correct placement of test components.Identification of a species tissue is demonstrated by a reaction ofcomplete fusion between sample and reference antigenimmunoprecipitin bands which become plainly visible after overnightincubation of the immunodiffusion plate at room temperature. Keycomponents are stable for at least one year when stored underrefrigerator conditions. Each test has been shown to have adequate

sensitivity and specificity for its intended purpose of the particular species in question. These tests are reliable, practical, economical, and very easy to perform and interpret inany work environment. Individual species tests for beef, pork, poultry and sheep are commercially available as a complete testkit. As a result of an Association of Official Analytical Chemists(AOAC) collaborative study, the method of these tests is anofficial AOAC first action method.

18.2 Materials and Methods

All materials necessary for the performance of SIFT for beef, pork, poultry and sheep species may be commercially purchased asindividual test kits. The method of performing SIFT for beefspecies detection using an ORBIT test kit is described below.Performance of SIFT for other species, using the other SIFT kitsavailable, would be conducted in an identical manner except for thesubstitution of the appropriate dye colored - template marked agar-

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gel plates and species reference antigen and antibody reagent discsrelative to the species being tested. Specific formulations for preparation of the agar-gel plates and the reference antigen and antibody reagent discs for each species SIFT kit are detailed inthe individual references cited at the end of this protocol.

18.21 ORBIT Kit Composition is as Follows:

a. ORBIT agar-filled plates with pink dye; pattern for disc placement silk screened on plate bottom.

b. Vial of Anti-Beef Antibody Discs-A-.c. Vial of Beef Reference Antigen Discs-B-.d. Vial of Blank Discs-S-.e. One piece flat black construction paper.f. Three pieces of white paper.g. One felt-tip marking pen.

h. Polyethylene sample bags.i. Three forceps.j. Hyperion viewer (optional accessory).

18.22 Ground Meat Accessory Kit Composition is as Follows:

a. Wooden applicator sticks - six inches long. b. Sample cups - silk screen printed with two permanent

measurement lines on outside.c. Forceps.

18.3 Procedure

a. Remove prepared ORBIT agar-gel immunodiffusion plates and reagent discs from the refrigerator and allowequilibration to room temperature.

b. Using the forceps carefully place one anti-beef antibodydisc, flat on the agar surface, such that the A lettered circle of the template is completely and evenly covered by the disc.

c. In an identical manner place one beef reference antigen

disc over the B lettered circle of the same plate.

d. Sample discs may be prepared from either thawed whole muscle tissue or from ground/formulated meat products:

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i. If the sample is whole tissue, make a vertical sliceabout 38 mm deep in an area which is free of fat orconnective tissue. With clean forceps place one blank sample disc halfway into the depth of the slitand gently squeeze the slit closed such that both

sides of the disc are in contact with the tissue.Let the disc remain in this position 10 - 30 secondsto absorb tissue fluids and appear obviously wet.

ii. If the sample is of a ground/formulated type, placeabout 1 gram well packed into the sample cup suchthat it is filled level with the bottom black measuring line. Add sufficient quantity of cold tap water to fill the beaker level to the top black measuring line. Mix sample and water with a clean wooden applicator stick such that a uniform emulsion

results. Tilt the cup 45°°

and with clean forcepsimmerse a blank sample disc in the emulsion to adepth necessary for complete saturation. Excessfluid and meat particles are removed from the disc by wiping it on a cup rim during removal.

e. The sample disc, from either type of sample is placed over one of the S lettered circles of the ORBIT platecontaining the reference discs.

f. Treat a second sample in an identical fashion and placethat disc over the remaining unoccupied S lettered circleof the same plate.

g. Tightly seal the lid on the plate and leave undisturbed overnight (15 - 24 h) at room temperature.

h. The plates are then examined with an indirect white lightsource against a flat black background. This may be done with a Hyperion viewer or by using black paper taped toand suspended vertically from the rear part of a desklamp's housing.

i. Examine the plate for the formation of characteristicimmunoprecipitin lines in the agar among the four discsto determine which sample contain beef.

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18.4 Results

Immunodiffusion reactions for the ORBIT test are interpreted as arethose for other SIFT plate reactions. A reference band should always be visible between the reference antigen-B- and reference

antibody-A- discs. Complete fusion of this line with a band formed between the antibody-A-disc and the sample-S-discs is indicative ofa positive reaction for that sample. Absence of a band between thesample and the antibody disc is read as negative. Any lines formed near the sample disc that are not extensions of the reference band are also negative reactions.


a. Maintain storage of unused prepared plates and reagentdiscs at refrigeration conditions (4oC) in order to

assure adequate shelf life and proper reactivity.DO NOT FREEZE.

b. Do not use any kit components beyond their expirationdate.

c. Use separate, clean forceps for each individual disc placement to prevent reagent or tissue fluid carry overand cross contamination.

d. Proper disc placement and positioning is critical toobtaining expected reactions.

e. An immunoprecipitin band must always be produced betweenthe reference antigen and antibody discs, as this servesas the positive control and assures the proper reactivityof the test system. If a reference band is not produced,the test system is invalid, samples should not beinterpreted and the cause of the failure to produce thereference band must be determined and corrected beforesubsequent testing can proceed.

f. Do not attempt to read any immunodiffusion plates that

have reacted for more than 24 h.

g. The normal room temperature for proper incubation ofimmunodiffusion plates is considered to be in the rangeof 70 - 78oF (21.1 - 25.6oC).

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"Changes In Methods". 1987. Beef and poultry adulteration of meat products, species identification test, First Action. J. Assoc. Off. Anal. Chem. 70:389-390. Sec. 24. C01-24. C06.

Cutrufelli, M. E., R. P. Mageau, B. Schwab, and R. W.Johnston. 1986. Development of poultry rapid overnight field identification test (PROFIT). J. Assoc. Off. Anal. Chem. 69:483-487.

Cutrufelli, M. E., R. P. Mageau, B. Schwab, and R. W.Johnston. 1987. Detection of beef and poultry by serologicalfield screening test (ORBIT and PROFIT): collaborative study.J. Assoc. Off. Anal. Chem. 70:230-233.

Cutrufelli, M. E., R. P. Mageau, B. Schwab, and R. W.Johnston. 1988. Development of porcine rapid identification method (PRIME) by modified agar-gel immunodiffusion. J. Assoc.Off. Anal. Chem. 71:444-445.

Cutrufelli, M. E., R. P. Mageau, B. Schwab, and R. W.Johnston. 1989. Development of serological ovine field test(SOFT) by modified agar-gel immunodiffusion. J. Assoc. Off. Anal. Chem. 72:60-61.

Cutrufelli, M. E., R. P. Mageau, B. Schwab, and R. W.Johnston. 1991. Development of a rapid equine serological

test (REST) by modified agar-gel immunodiffusion. J. Assoc.Off. Anal. Chem. 74:410-412.

Cutrufelli, M. E., R. P. Mageau, B. Schwab, and R. W.Johnston. 1992. Development of a deer rapid identificationfield test (DRIFT) by modified agar-gel immunodiffusion. J. Assoc. Off. Anal. Chem. Int. 75:74-76.

Mageau, R. P., M. E. Cutrufelli, B. Schwab, and R. W.Johnston. 1984. Development of an overnight rapid bovineidentification test (ORBIT) for field use. J. Assoc. Off.

Anal. Chem. 67:949-954.

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CHAPTER 19. COMPETITIVE ENZYME-LINKED IMMUNOASSAY (CELIA) FOR THEDETECTION AND QUANTITATION OF CHLORAMPHENICOL

Richard P. Mageau

19.1 Introduction and Principles

Enzyme Immunoassays (EIA) have become increasingly popular todetect and quantitate a wide range of biological molecules ofinterest. The excellent specificity and sensitivity afforded byEIA are two major factors contributing to the development and useof this technique for quantitative detection of low molecular weight haptenic molecules such as antibiotics. The ImmunologySection of the Microbiology Division developed and published anoriginal EIA procedure to detect and quantitate the antibiotic

chloramphenicol (CA).

The specific type of EIA developed was an indirect CompetitiveEnzyme-linked Immunoassay (CELIA) system. The principles of thisassay are as follows. The binding of the limiting number ofspecific rabbit CA antibody molecules in liquid phase to solid phase bound CA antigen is competitively inhibited by free liquid phase CA in the sample under assay. Bound antibody (not displaced)is indicated by using an enzyme linked anti-rabbit antibody preparation which is subsequently reacted with an appropriatesubstrate. Enzyme activity, measured spectrophotometrically, isinversely proportional to the concentration of CA in the sample.

The CELIA procedure for CA when performed on bovine muscle tissueextracts or phosphate buffered saline CA standards has thefollowing characteristics: sensitivity of 1 ng/ml (P<0.05), linearquantitative displacement over the range of 1-100 ng/ml, a mean 50%displacement end point of 15 ng/ml and excellent specificity withrespect to other antibiotics and related chemicals.

The specific procedure subsequently described provides the completeinformation necessary to perform the CELIA for CA. This procedurerepresents a modified version of the originally developed and

published manual method. This modified version is automated and employs 96 well microtiter plates and Flow (ICN) automatic plate washing and optical density reading equipment. This automated version affords the potential opportunity for high volume sampleanalysis and effective cost savings by the reduced use of extremelyexpensive developmental biochemical reagents.

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a. Flow (ICN) Laboratories Titertek Multiskan MC platereader; #78-530-00.

b. Flow (ICN) Laboratories Titertek Microplate Washer;

#78-431-00.c. Flow (ICN) Vacuum pump for above washer; #78-426-00.d. Flow (ICN) Titertek Multichannel pipette; 8 channel,

adjustable 50-200 ul volume; #77-859-00.e. Eppendorf Repeater Pipette (Daigger Scientific

Co.#G20551) with accessory of 2.5 ml capacity Combitips(Daigger #G20552C) and 5.0 ml capacity Combitips (Daigger#G20552D).

f. Dynatech Laboratories Microelisa plates, Immulon I, flat bottom, 96 wells, #11-010-3350 and covers.

g. Incubator, 37oC (any properly operating brand).

h. Stomacher®, Model 80 (Tekmar Co., Cincinnati, OH).i. Whirl-pak® bags; 75 x 180 cm size.j. Centrifuge, capable of operation at 15,600 x g

(Eppendorf, Model 5412; Brinkman Instruments, Inc.), and appropriate centrifuge tubes.

k. Refrigerator (4oC).l. Microtest Manifold, Wheaton, straight, 8 place with Luer

Lock connection (Daigger #G20560A).

19.21 Chemicals and Reagents

a. Na2HPO4 (Fisher, S-374). b. NaH2PO4 (Fisher, S-369).c. NaCl (Fisher, S-271).d. Citric acid, anhydrous (Fisher, A-940).e. Hydrogen peroxide, 30% reagent grade (Fisher, H-323).f. Tween 80 (Fisher, T-164).g. Sodium azide†; NaN3, purified (Fisher, S-227).h. Bovine Serum Albumin, powder, fraction V (Sigma, A-4503),

store in refrigerator.i. Chloramphenicol, crystalline (Sigma, C-0378), store in

refrigerator.j. ABTS substrate indicator; 2,2' azino-di-(3-ethyl

Benzthiazoline Sulfonic acid), (Sigma, A-1888).

19.22 Biochemical Reagents and Supplies

a. Anti-chloramphenicol serum (undilute). b. Chloramphenicol-BSA conjugated antigen (50 µg/ml stock).

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c. Goat anti-rabbit immunoglobulin G horseradish peroxidase(GARP) conjugate; Miles-Yeda, Israel, (undilute).

d. Chloramphenicol negative beef tissue (initial supplyonly; used to set up tissue-CA standards).

e. Normal Rabbit Serum (undilute).

NOTE: The above 5 items must be stored in the frozen state atall times to maintain stability.

19.23 Preparation of Stock Reagent Solutions

a. 0.15 M Phosphate Buffered Saline at pH 7.2 (PBS)

Add 10.35 grams of NaH2PO4 and 4.38 grams of NaCl to 1liter of distilled water and dissolve completely to prepare the "acid" solution. Add 10.65 grams of Na2HPO4

and 4.38 grams of NaCl to 1 liter of distilled water and dissolve completely to prepare the "base" solution. While mixing with a magnetic stirrer and monitoring the pH on a pH meter, add a sufficient quantity of the "acid"solution to the "base" solution to achieve a final,stabilized pH of 7.2. Dispense into glass containers,autoclave at 121oC for 15 minutes and store at room temperature. It is most convenient to make up this buffer in 5 liter quantities at a time.

b. Phosphate Buffered Saline Containing 0.05% Tween 80(PBS-Tween)

To 1 liter of prepared 0.15 M phosphate buffered salineat pH 7.2 add 0.5 ml of Tween-80 and mix (not on magneticstirrer) for several hours at room temperature untilcompletely dissolved. Store this prepared solution inthe refrigerator (4oC).

c. Phosphate Buffered Saline Containing 0.5% Bovine Serum Albumin (PBS-BSA)

To 1 liter of prepared 0.15 M phosphate buffered saline

at pH 7.2, add 5 grams of powdered bovine serum albuminand 1 gram of sodium azide (NaN3) and mix (not on magnetic stirrer) at room temperature until completelydissolved. Store this prepared solution in therefrigerator (4oC).

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d. ABTS - H2O2 Substrate Buffered Solution

Prepare a 0.1 M citric acid solution by dissolving 1.92grams of anhydrous citric acid in 100 ml of distilled water. Prepare a 0.1 M dibasic sodium phosphate stock

solution by dissolving 1.42 grams of Na2HPO4 in 100 mldistilled water. Add sufficient quantities of these twostock solutions together while mixing with a magneticstirrer and monitoring the pH on a pH meter to prepare100 ml of a 0.1 M citrate-phosphate buffer at a finalstabilized pH of 4.0.

To 100 ml of the above prepared 0.1 M citrate-phosphate buffer add 22 mg of ABTS [2,2' azino-di-(3-ethylBenzthiazoline Sulfonic acid)] and 15 µl of stock 30%hydrogen peroxide, mix gently by hand (no magnetic

stirrer) until completely dissolved. Pass this substratesolution through a 0.45 µm Millex® filter, place in asterile glass container, and store in the dark at room temperature until needed. This substrate solution should be prepared 24 h in advance of need and may be used aslong as it retains its original light green color. A solution which has deteriorated to the point where itcannot be used is evidenced by a dark azure-green colorformation.

e. PBS Chloramphenicol (CA) Standards

Prepare a stock 1 mg/ml chloramphenicol (CA) solution by weighing out 10 mg powdered, pure CA on an analytical balance and placing in 10 ml PBS. Allow the CA todissolve thoroughly into solution by occasional mixingover a period of 24-48 h, or longer if necessary, due tolimited solubility of the CA. From this stock 1 mg/ml CA solution make serial ten-fold dilutions in PBS (10 mlquantities) to produce CA standards at concentrations of10,000, 1,000, 100, 10, and 1 ng/ml respectively. Storethese standards in the refrigerator (4oC) until used inthe assay.

f. Tissue Extract CA Standards

Prepare tissue extract from known CA free, raw, bovine muscle tissue by the following manner:

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i. Place 5 grams of diced tissue in a 75 x 180 cm Whirl-pak® bag.

ii. Add 10 ml PBS.

iii. Place bag in Model 80 Stomacher® and stomach for30 seconds.

iv. Remove bag from Stomacher® and leave undisturbed for1 h at room temperature.

v. Pour off the liquid contents from the extraction baginto a centrifuge tube.

vi. Centrifuge at 15,600 x g for 15 minutes.

vii. Collect the clear supernatant tissue extract.If necessary, filter to remove all debris and lipid particulates, and place in a sterile glasscontainer.

Using the PBS-CA standards prepared in (e) above,make ten-fold dilutions of each needed 10X higherconcentration standard into the freshly prepared beef tissue extract to produce CA standards atconcentrations of 1,000, 100, 10, and 1 ng/mlrespectively. These tissue extract CA standardsshould be made fresh each time a standard curve isto be run in the CELIA. The tissue extractoriginally prepared, without CA, should be stored inthe refrigerator and may be used for subsequent CA standards preparation as long as the extract showsno evidence of microbial contamination or protein precipitation. Tissue extracts should always be prepared from tissues similar to those beinganalyzed for the presence of CA with respect tospecies and organ or tissue type.

19.3 Performance of CELIA for CA

a. Obtain a flat bottom, 96 well Dynatech Immulon I microelisa plate and cover from stock supplies.

b. Prepare a sufficient quantity of the Chloramphenicol-Bovine Serum Albumin (CA-BSA) conjugated antigen for plate well sensitization. Make a small volume dilution

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of the stock 50 µg/ml CA-BSA antigen solution in PBS suchthat a final concentration of 50 ng/ml CA is obtained.

c. By using the 8 channel pipette, place 200 µl of the 50ng/ml CA-BSA (in PBS) sensitizing antigen solution into

all wells except those of column 2. Leave these wellsempty for the present time.

d. Place a cover on the plate and allow the CA-BSA antigento passively absorb to the wells by incubating the platefor 3 h at 37oC.

e. Test sample extractions should now be concurrentlystarted at this stage in the following manner:

i. Place 5 grams of diced tissue in a 75 x 180 cm

Whirl-pak® bag.

ii. Add 10 ml of PBS.

iii. Place bag in Model 80 Stomacher® and stomach for 30seconds.

iv. Remove bag from Stomacher® and leave undisturbed for1 h at room temperature.

v. Pour off liquid contents from the extraction baginto a centrifuge tube.

vi. Centrifuge at 15,600 x g for 15 minutes. (Eppendorf Model #5412 centrifuge using 1.5 ml volumecentrifuge tubes is very convenient for this).

vii. Place the clear, test sample supernatant extracts inthe refrigerator (4oC) until called for in step (p)of this assay procedure.

f. Remove the plate from the incubator [continued from step(d)], remove the cover and mount on the carrier of a

Titertek Microplate Washer which has been primed withPBS-Tween and set to deliver 300 µl fluid to each well.

g. Remove the CA-BSA sensitizing antigen solution from the wells by aspiration with the washer and wash the wellsonce with 300 µl of PBS-Tween per well.

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19-7

h. Remove the plate from the washer, invert over a sink,hold the plate tightly in one hand and flick severaltimes to remove any remaining excess liquid from the wells.

i. Tap the plate in an inverted position several times on asoft paper towel (Sorg Laboratory Towels) placed on thesurface of the lab bench and allow the plate to remaininverted for 1-2 minutes to complete the draining process. Place the plate right-side up and cover untilnext reagent addition.

j. Block unwanted reactive sites on the plastic wells byfilling all wells (including those in column 2) with250 µl of PBS-BSA per well, dispensed by using a 8 place microtest manifold attached to a Cornwall syringe.

k. Replace the cover on the plate and incubate for 2 h at37oC.

l. Remove the plate from the incubator, place on the carrierof the washer, aspirate the PBS-BSA blocking solution outof each well and wash the wells twice with 300 µl ofPBS-Tween per well.

m. Repeat steps (h) and (i).

n. With an appropriate pipetting device place 150 µl of PBSin the wells of column 1, 2, 3, and 4 of row A and B.Place 150 µl of CA free tissue extract in the wells ofcolumn 1, 2, 3, and 4 of row C and D and the wells ofcolumn 1 and 2 of row E, F, G, and H. These wells allserve as negative reagent controls (column 1 and 2) or 0level controls (column 3 and 4).

o. Place 150 µl of PBS CA standards at concentrations of 1,10, 100, and 1000 ng/ml in wells of column 5 and 6, 7 and 8, 9 and 10, 11 and 12 respectively of rows A and B.Place 150 µl of tissue extract CA standards at

concentrations of 1, 10, 100, and 1000 ng/ml in wells ofcolumn 5 and 6, 7 and 8, 9 and 10, 11 and 12 respectivelyof rows C and D. These wells serve to produce thestandard CA inhibition curves in PBS (rows A and B) and tissue extract (rows C and D).

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19-8

p. Place 150 µl of each test sample extract [from step (e)]in 2 adjacent wells (duplicates) of an individual row. All wells of column 3-12 of row E-H are available for usefor duplicate analysis of individual test sample extracts(20 test sample capacity/plate). Record in some

appropriate fashion the location of each test sampleextract within the available wells for sample analysisfor future reference.

q. With the use of an Eppendorf Repeater pipette and a 2.5 ml Eppendorf combitip attached, add 50 µl of normalrabbit serum diluted 1:700 in PBS to all wells of column1. The wells of this column serve as zero blank normalrabbit serum controls, producing no visible reactions and are used to blank in the reader making spectrophotometric measurements of the reactions in all subsequent wells in

each row.

r. With the use of the repeater pipette and a new 2.5 mlcombitip attached, add 50 µl of anti-chloramphenicolserum diluted 1:700 in PBS to all remaining wells.

s. Carefully mix and distribute the contents in each well bygently rocking the plate and tapping the ends againstyour fingers. DO NOT allow the contents of any well tospill out as this will invalidate this result.

t. Place the cover on the plate and incubate overnight(16-18 h) in the refrigerator at 4oC.

u. Remove the plate from the refrigerator, allowequilibration to room temperature, place on the carrierof the washer, aspirate the contents out of each well and wash the wells twice with 300 µl PBS-Tween per well.

v. Repeat steps (h) and (i).

w. By using the 8 channel pipette, add 200 µl of goatanti-rabbit immunoglobulin G horseradish peroxidase

(GARP) conjugate diluted 1:5000 in PBS-Tween to all wells.

x. Place the cover on the plate and incubate for 2 h at37oC.

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19-9

y. Remove the plate from the incubator, place on the carrierof the washer, aspirate the contents out of each well and wash the wells three times with 300 µl of PBS-Tween per well.

z. Repeat steps (h) and (i).

aa. With the use of the 8 channel pipette, add 200 µl of ABTS-H202 substrate buffered solution to all wells.

bb. Place the cover on the plate and incubate for 90 minutesat 37oC.

cc. Twenty minutes prior to the end of the above incubation period, turn on the power to the Titertek Multiskan MC plate reader and allow it to warm up.

dd. After the 90 minute incubation period of step (bb) iscomplete, remove the plate from the incubator, remove thecover and place the plate on the carrier of the Multiskan MC plate reader.

ee. Program the reader for the current date, Mode 1 (single wavelength absorbance), Wavelength filter #2 (414 nm), push the carrier and plate into the measuring head and blank the instrument (zero O.D. point set) on column 1.

ff. Press the START button and obtain a printed paper stripof the Optical Density (O.D.) values for all of thereaction wells on the plate.

gg. Remove the plate from the reader and visually examine the plate to see that the obvious colored reactionintensities generally correspond to the numerical valueson the printed data sheet to assure that the instrument properly read the plate.

hh. Turn off the power to the Multiskan MC plate reader and discard the plate (save the cover for reuse) after

completion of the Data Analysis, Plotting, and SampleInterpretation Section described below.

19.4 Data Analysis, Plotting, and Sample Interpretation

a. All wells in column 1, which serve as the zero-blanknormal rabbit serum control, should have no color

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19-10

reaction. This indicates a proper lack of non-specificattachment of rabbit serum or GARP conjugate to the bound CA antigen in the wells. Under these conditionsthese wells are excellent controls to blank in (zero point set) the O.D. reading instrument.

b. All wells in column 2 serve as BSA negative controls toassess non-specific attachment of anti-CA antibody (and also GARP). Since these wells were never sensitized withCA antigen and only blocked with BSA, no positivereactions (high O.D. values) should be observed. Thesecontrols may also be considered as a check on the otherhalf of the primary antigen-antibody component of theassay system initiated in column 1.

c. Wells in columns 3 and 4 of rows A, B, C, and D should

demonstrate maximum binding of anti-CA antibody (zeroinhibition) and have the highest O.D. values. Theserepresent the zero controls for the standard inhibitioncurves produced by subsequently increasing concentrationsof CA.

d. The remaining wells of rows A and B represent thestandard inhibition curve for PBS CA standards and thoseof rows C and D represent the standard inhibition curvefor tissue extract CA standards. The O.D. values in bothof these series of wells should decrease with increasingconcentrations of CA due to inhibition of binding ofanti-CA antibody.

e. The remaining wells of the plate (columns 3-12 of rowsE-H) represent reaction values for test sample extractsrelative to the presence or absence of CA in the originalsamples.

f. For each pair or set of wells containing exactly the sametest materials, calculate the average O.D. value.

g. Obtain a piece of 5 cycle semi-logarithmic graph paper

containing 100 numerical scale divisions. Label theordinate (100 numerical scale divisions) with O.D. valuesfrom 0 to 2.0 in increments of 0.2 units. Label theabscissa (5 cycle logs) with CA concentrations of 0, 1,10, 100, and 1000 ng/ml.

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19-11

h. Plot the average O.D. values generated for the PBS CA standards and tissue extract CA standards from 0 to 1000ng/ml respectively on the graph paper. Draw straightlines from point to point. You will now have twoinhibition (displacement) curves for increasing

concentrations of CA in PBS or tissue extracts.

i. Examine the two inhibition curves and compare the slopesand overall O.D. values. The PBS CA standard displacement curve represents the basic reaction level ofthe primary antigen-antibody system influenced only by pure CA. The tissue extract CA standard displacementcurve represents the influence of CA and interaction ofvarious proteinaceous materials extracted from the testsample. If the tissue extract CA inhibition curve issignificantly different from the PBS CA inhibition curve

(which it usually is) use the former for determining positive CA concentration levels in test samples.

j. Calculate the 50% displacement end point for bothstandard inhibition curves (50% of the 0 standard O.D.). Values in the range of 5 to 20 ng/ml with a mean value ofaround 15 ng/ml should be obtained as an indication of properly operating displacement systems.

k. To determine if a test sample contains CA and toquantitate the amount, if it is present, proceed asfollows:

i. Obtain the O.D. value for the test sample and determine the relationship to the tissue extract CA standard curve.

ii. If this value is between 0 and 1 ng/ml (i.e. O.D.greater than the 1 ng/ml standard), the sample isconsidered to be free of CA.

iii. If the value falls within the linear portion of thestandard curve, from 1-100 ng/ml, the sample is

considered to contain CA. To determine the amountof CA present per gram of tissue, interpolate from the curve the ng/ml CA value on the abscissarelative to the particular O.D. obtained for thatsample and multiply it by 2. This assumes that allof the CA from the original 5 gram of tissue isextracted into the 10 ml PBS volume and the

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19-12

resulting dilution therefore is 1:2 rather than theusual 1:3.

iv. If the O.D. value falls beyond the linear portion ofthe standard curve (ie. O.D. less than the 100 ng/ml

standard), the sample is also considered to containCA but accurate quantitation is not possible from this particular analytical run. More accuratequantitation in this case would be achieved bytaking this sample extract, making serial ten-fold dilutions of it in PBS (101 -106), repeating theCELIA analysis a second time on these dilutions and determining which dilution produced an O.D. value within the linear portion (1-100 ng/ml) of the PBSCA standard curve.

Calculations for this sample would then be reduced to: interpolated CA value of ng/ml from the PBS CA standard curve abscissa x ten-fold dilution factor x2 = ng CA/gram of tissue.


a. The assay reagents have been evaluated for use only withDynatech Immulon I microtiter plates. No other platesshould be used.

b. All stock reagent solutions must be properly prepared and maintained free of contamination or chemical breakdown.

c. All stock immunochemical reagents must be stored in thefrozen state at all times to maintain stability.

d. The stock ABTS-H2O2 substrate buffered solution should not be used if it has turned to a significantly darkershade of green from that of the original preparation.

e. Be sure the stock, commercial preparation of Goat anti-rabbit immunoglobulin G horseradish peroxidase (GARP)

conjugate reagent has not deteriorated to the point of producing improper final O.D. readings. Use only anunexpired lot of this reagent.

f. To insure validity of the quantitative aspects of thisassay, extreme care must be exercised to accurately

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19-13

prepare the standard CA concentrations in PBS and CA freetissue extracts from stock sources of the pure CA drug.

g. The CA free tissue used to prepare extracts forsubsequent preparation of the CA tissue extract standards

should be initially validated as being free of CA by areliable procedure.

h. Standard curves for CA in PBS and CA in tissue extracts must always be run in an analytical determination for the presence of CA in test samples.

i. The tissue source used to prepare the CA tissue extractstandard curve must be of the same species and organ typeas that of the test sample to be quantified.

j. The standard CA inhibition curves should always be quitesimilar from run to run and the 50% displacement end point should always be in the same general range.Drastic deviations in the above indicates an improperlyoperating displacement system due to critical reagentdeterioration or technical error in the assay set-up and must therefore be corrected.

k. A valid test run can only be assured by the demonstrationof proper CA standard inhibition curves for each particular analytical determination.

† Safety Caution: Do not dispose of spent sodium azidePBS-BSA solution by pouring down sinkdrains.

Collect in separate liquid wastecontainer and dispose of as hazardouswaste according to standard wastemanagement procedures for your laboratory.

Accumulation of sodium azide in lead sink

drains may result in an explosion.

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19-14


Campbell, G. S., R. P. Mageau, B. Schwab, and R. W. Johnston.1984. Detection and quantitation of chloramphenicol bycompetitive enzyme-linked immunoassay. Antimicrob. Agents

Chemother. 25:205-211.

Shekarchi, I. C., J. L. Sever, Y. J. Lee, G. Castellano, and D. L. Madden. 1984. Evaluation of various plastic microtiter plates with measles, toxoplasma, and gamma globulin antigensin enzyme-linked immunosorbent assays. J. Clin. Microbiol.19:89-96.

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The methods described in this guidebook are for use by the FSIS laboratories. FSIS does

not specifically endorse any of the mentioned test products and acknowledges that

equivalent products may be available for laboratory use.




______________________________________________________________________________________________


Notice of Change


Title: Detection and Identification of Extraneous Material in Meat and Poultry

Products.



This Microbiology Laboratory Guidebook method chapter was revised to update the

formatting and to meet the requirements of the laboratory’s document control system and

ISO 17025. Additional content includes sections on quality control and safety precautions. The general content has been re-written to clarify procedures and update the

method.

______________________________________________________________________________QD-F-Micro-0004.01 Approved: Charles Pixley, 2/25/03

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SOP No: MLG 32.01 Page 1 of 39

Title: Detection and Identification of Extraneous Material in Meat and Poultry Products.

Revision: 01 Replaces: Original Chapter 32 Effective: 12/30/04

Procedure Outline

32.1. Introduction32.1.1. General Introduction

32.2. General Quality Control and Good Laboratory Practices for theEntomology and Extraneous Materials Laboratory

32.2.1 . Equipment and Reagents

32.2.2. Laboratory Quality Control

Part A: DETECTION OF LIGHT FILTH IN PREPARED INFANT FOODS CONTAININGMEAT AND POULTRY

32.3. Introduction32.4. Equipment and Reagents

32.4.1. Equipment

32.4.2. Reagents32.5. Procedures

32.5.1. Procedure for Meat and Poultry

32.5.2. Procedure for Baby Food Dinners32.6. Results

32.7. Quality Control

32.8. Safety Caution

Part B: DETECTION AND IDENTIFICATION OF EXTRANEOUS MATERIALS IN NON-

MEAT FOOD INGREDIENTS - MACROSCOPIC EXAMINATION

32.9 Introduction32.10 Terms and Concepts

32.11. Equipment and Reagents

32.11.1 Equipment32.11.2 Reagents

32.12. Procedure

32.12.1. Examination of Ground Spices

32.12.2. Examination of Whole Spices, Seeds, and Large Flake Leafy Spices32.12.3. Preparation of Hair and Feather Specimens

32.12.4. Preparation of Small Insect/Arthropod and Fragment Specimens

32.13. Reporting Results32.14. Criteria for Confirmatory Analysis

32.15. Quality Control and Quality Assurance


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Part C: DETECTION OF GLASS AND NON-ALUMINUM METALS IN MEAT AND

POULTRY PRODUCTS

32.16. Introduction

32.17. Reagents and Supplies

32.17.1. Reagents32.17.2. Supplies

32.17.2.1. For Metallic Contaminants

32.17.2.2. For Glass Contaminants32.18. X-ray Screening

32.19. Procedure32.19.1. Procedure for Index Sample

32.20. Result



Part D: METHOD FOR THE ISOLATION OF GLASS FROM PREPARED MEAT AND

POULTRY BABY FOODS

32.23. Introduction32.24. Reagents and Equipment

32.24.1. Reagents

32.24.2. Equipment32.25. Procedure

32.25.1. Cleaning of Exterior of Sample Container

32.25.2. Sample Analysis32.25.3. Characterization of Contaminants


PART E: ANALYSIS OF FECAL MATERIAL32.27. Introduction

32.28. Reagents and Equipment32.28.1. Reagents

32.28.2. Equipment32.29. Procedure32.29.1. Test Response


32.30.1. Blank Preparation32.30.2. Positive Control

32.30. Selected References


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32.1 Introduction

32.1.1 General Introduction

Food Safety and Inspection Service (FSIS) is responsible for insuring that the meat and poultry products offered to the consumer are safe, wholesome, unadulterated and truthfully

labeled. In fulfilling this responsibility, the Agency's laboratories perform sanitation

analyses of the meat and poultry products including investigations for extraneous or foreign

materials. According to law, a meat or poultry product is adulterated if it consists in whole

or in part of any filthy substance, is for any reason unsound or unwholesome, or if the product was prepared or packed under unsanitary conditions where it may have been

contaminated [21 United States Code 601(m)(3)(4), 21 United States Code 453(g)(3)(4)].

Extraneous material is defined as any foreign material found in a food product and

associated with objectionable conditions or practices in production, storage, or distribution.Examples of extraneous materials are: filth, metal, glass, sand, wood, paper or plastic. Filth

is defined as any objectionable matter contributed by animal contamination of a product

such as: rodent, insect or bird matter; or objectionable material contributed by unsanitaryconditions. The presence of extraneous material in a food product is not only unappealing

but represents a breakdown in good manufacturing practices and could pose a serious

health hazard to the consumer. The isolation and identification of extraneous materialssometimes yields evidence that a product was stored or processed under unsanitaryconditions and is unfit as human food.

The study of extraneous materials found in food is called Microanalytical Entomology.The U.S. Food and Drug Administration (FDA) and the Association of Official Analytical

Chemists (AOAC) have published reference articles, books and methods on this subject.

These publications discuss methods of analyses, contaminant identification, and contaminant significance.

This chapter contains the methods developed and used by FSIS Entomology and

Extraneous Materials Laboratories (EEML) to isolate and to identify extraneous materialsfrom meat and poultry products. These methods are intended for the stated product and contaminant. Before using one of these methods on a different product or for a different

contaminant, the method must be thoroughly evaluated for that purpose. Aside from the

methods developed in our laboratories, FSIS EEMLs use many AOAC methods.


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32.2 General Quality Control and Good Laboratory Practices for the

Entomology and Extraneous Materials Laboratory

For extraneous materials analyses it is of the utmost importance to maintain a clean and

contaminant-free laboratory. All possible action must be taken to prevent thecontamination of the sample with insects or extraneous materials. Below are listed general

practices and techniques which must be observed in the Entomology and Extraneous

Materials Laboratories to insure a quality analysis.

32.2.1 Equipment and Reagents:

a. Sieves

i. Each analyst should be assigned a sieve. The analyst is

responsible for maintaining his/her sieve. The sieve should be cleaned immediately after using it to prevent debris from

drying on the sieve.

ii. As specified by the AOAC the #230 sieve should be a plain

weave, not a twill weave.

iii. Before beginning an analysis, the sieve should be examined for rips and tears. Small tears can be mended with a drop of

solder and will not affect the usefulness of the sieve. Sieves

with tears and holes should not be used.

iv. The sieve should be cleaned with a soft brush and liquid

detergent and backwashed by spraying water through the bottom of the sieve to remove any debris in the sieve after

use.

v. Annual Cleaning - The sieves should be cleaned as needed) by the following procedures:

(1) Soaked in a 5% aqueous pancreatin suspension, at pH8.2-8.5 for 4-5 h at 37-40

oC.


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(2) Soaked in a 10% EDTA (tetrasodium

ethylenediaminetetraacetic acid) for 2-3 h at

40-50oC.

(3) Soaked in a 10% NaOH (w/v) for 2-3 h at 80-90oC.

b. Magnetic Stirrers

i. Magnetic stirrers should be stored in a clean plastic container

with lid. This should protect the stirrer from picking upmetal fragments while not in use. The interior of this

container should be kept clean.

ii. Magnetic stirring bars can be cleaned by removing the large

particles with forceps and small filings by soaking in an"aqua regia"† solution (a 1:3 mixture of nitric acid and

hydrochloric acids).

c. Filter Paper

Filter paper should be stored in a container that will protect it fromextraneous materials contamination. A petri dish or a small plastic sandwichcontainer with a tight fitting lid would be ideal. Of course, this precaution is

worthless if the analyst does not replace the lid and leaves the filter paper

container on the lab bench uncovered for extended periods of time. As withthe container for the magnetic stirrers, the container for the filter paper must

be kept clean.

d. Laboratory

The entomologists, technicians and aides will routinely:

i. Wipe the lab bench and the work area with a damp sponge

before beginning an analysis.

ii. Clean the lab and the microscope room thoroughly at least

once a month. This should include wiping down all benches,

table tops and tops of any refrigerators or ovens, and cleaning


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or vacuuming all window sills. DO NOT clean up the

laboratory while analyzing samples.

e. Glassware

i. Avoid use of plastic beakers, funnels, graduated cylinders,

etc. because insect fragments and hairs adhere to plastics.

ii. After cleaning glassware allow it to dry in an inverted

position. Store glassware inverted or cover the opening withaluminum foil. When it is not possible to store the glassware

inverted or to cover it, the analyst should rinse the glasswarewith water prior to use.

f. Trap Rods

i. Clean the trap rod with soap and water after use.

g. Balances

i. All balances should be inspected and serviced by a trained service technician once a year.

ii. Every day the balance is used, the lab analyst should clean

the exterior of the balance, level (if possible) and check theaccuracy of the balance with a 100 g calibration weight (or

other weight as deemed appropriate for that balance).

h. Microscopes

i. All microscopes should be inspected and serviced by a

trained service technician on a yearly basis.

ii. Each analyst should be assigned a microscope and will be

responsible for the daily maintenance of that instrument. Theanalyst will clean the exterior surface of the microscope, the

eyepieces and the illuminators.

i. Reagents


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i. Before mixing reagents, be sure to clean the top of the

reagent bottles to prevent contaminants from falling into thesolution.

ii. Rinse out carboys before preparing solutions.

iii. Label reagents with the "date prepared" and the "expiration

date" (if the latter is applicable).

iv. Request the "Certification of Analysis" for chemicals, such as

paraffin oils.

j. Sample Handling Procedures

When opening the sample container maintain control of the closure

mechanism. Remove rubber bands from bags. Do not cut or otherwise

break rubber bands. Remove the staples from bags and paperwork. Donot pull open bags sealed with staples or rubber bands.

32.2.2. Laboratory Quality Control

a. Air Quality

A petri dish with filter paper wetted with glycerin should be left exposed for 24h in the laboratory to detect any air borne contaminants. Place these petri

dishes on the lab bench, in the fume hood, and near a window. Examine

microscopically at 30X. Perform once a week. Record the results of thisexamination in a bound "Quality Control Notebook".

b. Water Quality

Sample the tap water (hot and cold) by running the water through a #230

sieve for 15 minutes. Wash the trappings from the sieve on to filter paper and

examine microscopically at 30X. Perform this analysis once a week. Record the results in a bound "Quality Control Notebook".

c. Hairs and Fibers


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The analyst should prepare microscope slide mounts of their head, arm, and

eyebrow hair and be able to recognize their own hair from other hair.

Include a slide mount with facial hair, if applicable. Analyst should preparemicroscope slide mounts of fibers from personal clothes which have a loose

knit and could fall into a product. All of these slides should be maintained inthe lab as a record.

d. Alternative/Analytical Point Quality Control

A blank sample consisting of the same amounts of compounds, solvents and solutions used in an analysis is run alongside actual samples whenever a

digestion, light flotation, or other chemical/isolation method is performed.Isolates from the blank sample are enumerated, identified and documented in

a Quality Control notebook. The date of the analysis and actual samples the

blank accompanied are listed with the blank findings for traceability purposes.


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PART A: DETECTION OF LIGHT FILTH IN PREPARED INFANT FOODS

CONTAINING MEAT AND POULTRY

32.3. Introduction

The presence of any objectionable animal material in a food product is defined as filth.

Oleophilic filth is defined as light filth. Examples of light filth include insects, insect

fragments, hairs, and feather barbules. These adulterants can be detected in a food product

by separating them from the food in the oil phase of an oil/aqueous mixture.

The methods described here isolate insect fragments and rodent hairs from prepared baby

food containing meat or poultry. Bovine hairs and feathers can also be recovered from the pure meat and poultry. The product is digested in a hydrochloric acid solution and the

solubilized material is washed through a #230 sieve. In a pure meat/poultry product, the

meat tissue is totally digested and can be washed through the sieve. The material remainingon the sieve can be transferred directly to filter paper. In the baby food dinners, meat

products combined with cereals or vegetables, the plant material is not completely digested

and thus does not pass through the sieve. In this case, a light filth flotation using paraffin oilis necessary to separate the filth material from the plant material. This flotation step will

provide cleaner filter paper, thus easier and more accurate enumeration of the light filth.


32.4.1. Equipment

a. Laboratory Balance,

b. Beaker, 2 L

c. Beaker, 600 mld. Wildman trap flask, 1 L

e. Hot plate, magnetic stirring

f. Sieve, stainless steel, U.S. Standard No. 230

g. Magnetic stirrer bar, Teflon coated (1 X 5 cm)h. Buchner funnel

i. Vacuum Pump

j. Watch Glass for a 2 L beaker k. Petri Dish (2), 100 X 10 mm

l. Filter Paper, S&S #8 Ruled

m. Stereoscopic Microscope, 10 - 30Xn. Trap Rod


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o. Aerator, local hardware store

p. 1 L graduated cylinder

q. 50 ml graduated cylinder r. 25 ml graduated cylinder

32.4.2. Reagents

a. Igepal CO-730 (nonionic detergent, active at low pH) available

through GAF Corporation, 140 W. 51st St. NY, NY 10020

b. Concentrated Hydrochloric acid (HCl) †c. Tergitol #4, Sigma Chemical Co.

d. 40% isopropanol in filtered, distilled water.e. Paraffin oil (Saybolt viscosity 125/135) Sargent - Welch

f. Glycerin/Ethanol mixture (vol:vol 1:1)

g. Sodium Bicarbonate

32.5. Procedures

32.5.1. Procedure for Meat and Poultry

a. Preparation - Wash the exterior of the jar, particularly around the lid to remove any contaminants which may be drawn into the jar uponopening.

b. Quantitatively transfer contents of one jar strained or one jar junior infant food to a two liter beaker with distilled water. Be sure to rinse

the inside of the lid into the beaker.

c. Bring volume to around 800 ml with distilled water.

d. Add 5 ml Igepal CO-730 and 45 ml concentrated HCl with stirring.

Cover with watch glass.

e. Bring to a boil and boil for 30 minutes.

f. Transfer the hot mixture to a 230 mesh sieve and wash with a

forcible stream of hot aerated tap water until washings are clear and

acid is removed. Wash the remaining material to one side of sieve.Retain the washings in a pan to neutralize at a later time with sodium


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bicarbonate.

g. Add 2 ml of "Tergitol #4" to contents of sieve and wash with forciblestream of hot aerated water, until foaming subsides. Wash all of the

remaining residue to one side of sieve.

h. Wash the contents of the sieve onto lined filter paper in a buchner

funnel with isopropyl alcohol. Wash down the sides of the filter

paper. Aspirate the paper to near dryness.

i. Add a small amount of glycerine/ethanol (32.33 f) to a petri dish.

Using forceps, remove the filter paper from the buchner funnel and place in the petri dish.

j. Examine microscopically at 30X. (See Section 32.36)

32.5.2. Procedure for Baby Food Dinners

a. As an initial preparation, wash the exterior of the jar, particularly

around the lid, to remove any contaminants which may be drawn into

the jar upon opening.

b. Quantitatively transfer contents of one jar strained or one jar junior

infant food to a two liter beaker with distilled water. Be sure to rinse

the inside of the lid into the beaker.

c. Bring volume to around 800 ml with distilled water.

d. Add 5 ml of Igepal CO-730 and 45 ml of concentrated HCl with

stirring. Cover with watch glass.

e. Bring to a boil and boil for 30 minutes.

f. Transfer the hot mixture to a 230 mesh sieve and wash with a

forcible stream of hot aerated water until washings are clear and acid is removed. Wash the remaining material to one side of sieve.

Retain the washings in a pan to neutralize at a later time.


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g. Add 2 ml of Tergitol #4 to contents of sieve and wash with a forcible

stream of hot water, until foaming subsides. Wash the remaining

material to one side of sieve.

h. Transfer contents quantitatively to a 1 L Trap Flask with 40%isopropanol.

i. Bring volume of the liquid in the flask to 500 ml with 40%

isopropanol.

j. Bring to a boil and continue simmering boil for 5 minutes with

magnetic stirring.

k. Remove from heat and let stand for 1 minute. Insert trap rod into

flask. With disc held just below liquid surface slowly add paraffinoil (29.33 e) by pouring it slowly down the trap rod.

l. Stir magnetically for 3 minutes at a speed sufficient to draw a vortexto the stirring bar without splashing and without introducing air into

the liquid.

m. Allow the mixture to stand for 1 minute.

n. Fill the flask to the neck with 40% isopropanol by pouring slowly

down the trap rod with the disc just below the oil layer.

o. Allow the mixture to stand for 20 minutes. Resuspend the material at

the bottom of the flask by turning the flask in a clock-wise or counter clock-wise direction on the bench at 5 and 10 minutes to release any

trapped oil, taking care not to disturb the oil layer.

p. Trap off the oil layer into a 600 ml beaker. With 40% isopropanol,rinse the neck of flask and stem of trap rod and pour rinsings into

same beaker. Repeat rinsing procedure as necessary.

q. Pour the contents of the above beaker on to lined filter paper in a

buchner funnel. Rinse the beaker with 100% isopropanol until all the

oil is gone.


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r. Wet a petri dish with a small amount of glycerine/ethanol and place

the filter paper on this dish.

s. Examine microscopically at 30X. (See Section 32.36)

t. Add 25 ml of paraffin oil to the flask.

u. Slowly push the oil into the aqueous phase with the trap rod.

Continue to slowly plunge up and down, about 1/2 the height of the

flask, for 1 minute. Be careful not to introduce any air into theliquid.

v. Fill the flask up to the neck with 40% isopropanol and let stand for

15 minutes. Turn the flask at 5 and 10 minute intervals to release

any trapped oil drops.

w. Trap off the oil layer into a 600 ml beaker. With 100% isopropanol,

rinse the neck of flask and stem of trap rod and pour rinsings into thesame beaker. Repeat rinsing procedure as necessary.

x. Continue as in Steps q & r. Examine at 30X. (See Section 32.36)

32.6. Results

The lined filter paper should be examined line by line at 30X magnification. Identify and count any hairs and insect fragments observed. Report the following:

a. whole or equivalent insects (adults, pupae, maggots, larvae, cast skins)

b. insect fragments, identified

c. insect fragments, unidentified

d. aphids, scale insects, mites, spiders, psocids, thrips, etc. and

fragments of the above

e. rodent hairs (state the length of the hairs)



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See Section 32.2.2.


a. Do not dispose of hazardous waste by pouring down sink drains.

b. Collect in separate containers and dispose of this waste according to

standard waste management procedures for your laboratory.

c. Use caution when working with hydrochloric, other acids and strong bases.

d. Wear goggles and gloves to protect eyes and skin when preparing thesolution and when moving and wet sieving the sample.

e. Digest and wet sieve samples under a safety hood.


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Part B: DETECTION AND IDENTIFICATION OF EXTRANEOUS MATERIALS IN

NON-MEAT FOOD INGREDIENTS - MACROSCOPIC EXAMINATION

32.9. Introduction

A food is considered adulterated if "it consists in whole or in part" of any filth or

decomposed substance or if the food is "otherwise unfit for human food." Extraneous

materials detected in the ingredients indicate the product was prepared under unsanitary

condition where it may have become contaminated. The presence of extraneous materials inthe product ingredients would render the final product adulterated. The purpose of this

procedure is to presumptively determine the presence of rodent excreta, insects, insectwebbing, mold and other extraneous materials in the dry non-meat food ingredient. This

method is intended as a screening procedure. A vast majority of samples analyzed by this

procedure will be free of extraneous materials.

This method will allow for prompt examination of samples by all FSIS laboratories and

insure that compliant samples are reported promptly to the operating inspectors. This procedure will also reserve the analytical time the analyst has for the smaller number of non-

compliant samples that will require more time consuming analyses. This method is

recommended only for screening; all positives or apparently violation samples are to beconfirmed by AOAC or other accepted microscopic methods.

32.10. Terms and Concepts

The following terms are used in the macroscopic examination and reporting results:

a. Thrus: Any material going through the sieve.

b. Overs: Any material remaining on the sieve after sieving.

c. Animal Contaminated: Any material showing animal excreta or evidence of rodentor other animal chewing or gnawing.

d. Insect Infested: Any non-meat ingredient that contains live or dead insects, webbing, excreta, or definite evidence of insect feeding.

e. Miscellaneous Extraneous Material: Includes stones, dirt,wire, string, non-toxic foreign seeds, etc.


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f. Moldy: A product bearing any evidence of mold.

g. Rodent Excreta: Excretory pellets of the black rat (Rattus rattus), Norway rat (Rattus

norvegicus) or the house mouse (Mus musculus), or pieces/fragments thereof, asdetermined by the presence of murine rodent hairs in the matrix of the fecal material.

h. Other Animal Excreta: Any excretory product, other than

rodent, as identified by microscopic examination.

i. Whole Insects: Includes an adult insect, a pupa, a larva, or

a major portion thereof.


32.11.1. Equipment:

a. Jones Riffle Sampler (8261-C10 Arthur H. Thomas)

b. Balance, Top-loading, 1 kg capacity

c. Balance, analytical, 500 g capacity

d. Sieves, U.S. Standard Series (4-881 Fisher Scientific Co.) 3 1/2

through 20

e. Magnifier-Lamp (L6039-2 Scientific Products) (LUXO-LFM2FE)

f. Trays, Cutting, (62686-363, VWR Scientific)

32.11.2. Reagents:

i. Isopropyl Alcohol-Propylene Glycol Solution

1. Isopropyl alcohol, 100 ml

2. Propylene glycol, 300 ml

3. Picric acid, 160 mg (optional; follow safety precautions)


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Preparation: Mix isopropyl alcohol and propylene glycol. If yellow

staining is desired in preparation of specimens, add picric acid and

stir until dissolved. Heat hair specimens in the solution to deaeratefor microscopic examination. The solution can be stored at room

temperature.

ii. Hydrogen Peroxide-Sodium Lauryl Sulfate Solution

1. 30% Hydrogen peroxide, 1-5 ml

2. 5% sodium lauryl sulfate solution, 1-2 drops

Preparation: Mix two reagents in a small crucible, or in a glass or

porcelain serological plate well just before use. Heat dark hair

specimens in the solution to remove enough pigment to allowmicroscopic examination. Reagents should be stored in separate

containers in the refrigerator.

iii. Glycerin Jelly

1. Gelatin, 10 g

2. Glycerin, 70 ml

3. Distilled water, 60 ml

4. Phenol, 1 g

Preparation: Soak gelatin for 1-2 hours in water; add glycerin and

phenol. Warm for 10-15 minutes (not above 75oC) and stir until

homogeneous. Pour into petri dishes and store in refrigerator. Or

microwave gelatin and water in bottle until it starts to boil. Cap bottle and shake until homogenous. Add phenol and glycerin and

shake bottle to mix. Remove small portions as needed. Melt small

lump on a microscope slide for mounting hair, feather, and insect/arthropod specimens. Coverslip, let set (horizontally) for

several hours, and ring with nail polish (or similar substance) for

semi-permanent slides.


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iv. Hoyer’s Chloral Hydrate (Berlese’s Fluid)

1. Water, 50 ml

2. Gum arabic (ground-up crystals or powder), 30 g

3. Chloral hydrate, 200 g

4. Glycerine, 20 ml

Preparation: Stir until homogenous. Filter through glass wool; store

at room temperature. Dehydrate insect/arthropod specimens inincreasing concentrations of alcohol (70, 95, and 100%) and then in

xylol before mounting in medium on microscope slides. Coverslip

and let harden (horizontally) for several hours for permanent slides.

v. Nesbitt’s Solution

1. Chloral hydrate, 40 g

2. Concentrated HCl acid, 2.5 ml

3. Distilled water, 25-50 ml (more for lightly sclerotized

specimens)

Preparation: Stir. Use cold to clear insect/arthropod specimens

(takes several hours to a day or more).

vi. Potassium Hydroxide Solution (10-15% aqueous KOH solution)

Preparation: Stir until homogenous. Use cold, warm, or boiling to

clear insect/arthropod specimens (cold solution takes several hours toa day or more; boiling takes a few minutes but may distort the

specimen). Rinse specimen in water (preferably with small amount

of acetic acid added) after clearing to remove excess KOH.

32.12. Procedure

32.12.1. Examination of Ground Spices


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a. Mix sample received by passing through a riffle sampler 4 times,

recombining separations before each pass.

b. Separate approximately 200 g of sample and weigh. NOTE: Retainexcess sample for use in confirmatory analysis, if needed.

c. Sift sample portion-wise through a #20 sieve, retain "thrus".

d. Transfer "overs" to a cutting tray and spread evenly so that allmaterial can be observed.

e. If sufficient material is present to preclude spreading, place material

on one side of tray and move portion-wise to middle.

f. Examine the middle portion at 3 to 5 magnifications with strong,

even light. (A bench-top magnifier-lamp is suitable for this purpose).

g. Note and identify (if possible) all categories of extraneous material

observed.

h. If confirmatory analysis is needed, place "thrus" and "overs" into a plastic bag along with the excess sample.

i. Send all portions for confirmatory analysis to an advanced referencelaboratory, if available and necessary.

j. A written report of the extraneous material observed should accompany samples submitted for confirmation.

32.12.2. Examination of Whole Spices, Seeds, and Large Flake Leafy Spices

a. Mix sample received by passing through a riffle sampler 4 times,

recombining separations before each pass.

b. Separate approximately (200 g for whole spices and seeds; 50 g for

leafy spices and herbs).


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c. Sift sample portion wise through a sieve of such size that more of the

whole spices are retained as "overs". NOTE: The sieve should never

be of a smaller opening size than "Tyler Standard #8" or USAStandard 2.36 mm".

d. Transfer both "thrus" and "overs" separately on to a cutting tray and

spread evenly so that all material can be observed.

e. Examine both portions at 3 to 5X magnification under strong, even

light. (A bench-top magnifier lamp is suitable for this purpose). NOTE: If sufficient material is present to preclude obtaining a single

layer, place all material on one side of the tray and move portion-wise to middle and examine.

f. Note and identify (if possible) insects, insect damage, other filth and extraneous material observed.

g. If confirmatory analysis is needed place "thrus" and "overs" into a plastic bag along with the excess sample.

h. Send all sample portions for confirmatory analysis to an advanced reference laboratory, if available and necessary.

i. A written report of the extraneous material observed should

accompany samples submitted for confirmation.

32.12.3. Preparation of Hair and Feather Specimens

a. Remove with fine forceps hairs and feathers observed in the

sample isolates and place in a small porcelain crucible or in a well

of a glass or porcelain serological plate for deaeration. Add

sufficient amount of isopropyl alcohol-propylene glycol (1:3)solution (picric acid is optional; use 40 mg per 100 ml solution for

staining, if desired) to cover the specimen(s), and heat to sub-

boiling for ten minutes to two hours. Mount the specimen inglycerine jelly on a microscope slide, coverslip, and seal with clear

nail polish.


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b. Alternately, place the specimen in one or more drops of glycerine

on a microscope slide, coverslip, and heat the slide on a slide

warmer set at 55-65oC for one to 18 hours or until the specimen is

deaerated. For storage, remount the specimen in glycerine jelly as

described in step (a).

c. For hair specimens too dark for microscopic examination,

substitute for or follow the deaeration process in step (a) with

bleaching of the hair. Bleaching is accomplished by heating the

hair to sub-boiling in 1-5 ml of 30% hydrogen peroxide with 1-2drops of 5% sodium lauryl sulfate solution added (in crucible or

serological plate). Add more hydrogen peroxide as needed duringheating – do not let the hair dry and burn. The specimen must be

observed during the heating process and removed when a light red

to colorless state is reached (one to 30 minutes). Heating for toolong will result in a dissolved specimen.

32.12.4. Preparation of Small Insect/Arthropod and Fragment Specimens

a. Remove with fine forceps specimens observed in the sampleisolates and place on a microscope slide for mounting. Temporary

mounts for microscopic examination can be made by adding

glycerine and coverslip.

b. Semi-permanent aqueous mounts can be made by transferring the

specimen directly to glycerine jelly on a slide, coverslipping,warming the slide, then allowing the medium to firm up (at room

temperature), and ringing the coverslip with nail polish or other

suitable sealant.

c. Permanent resin mounts can be made with Hoyer’s chloral hydrate

(Berlese’s fluid). Specimens must first be dehydrated withsolvents before mounting and coverslipping. The slide mounts

need to set/dry.


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d. Clearing of dark or thick-bodied specimens can be done in

potassium hydroxide (KOH) or Nesbitt’s solution before mounting

on a slide.

32.13. Reporting Results

a. Identify all categories of extraneous materials observed and record the quantity in

each category.

b. To report the results as "percent extraneous material by weight", transfer theextraneous material to a tared dish and weigh.

Use the following formula to calculate the percentage:

Percent (%) = extraneous material (gm) in category X 100

Sample Weight

32.14. Criteria for Confirmatory Analysis

These criteria are presented as internal guidelines to assist the analyst trained for

macroscopic analysis in determining whether or not a sample should be subjected to a more

extensive examination. Each type of contamination observed should be considered both onits own sanitation significance and in conjunction with other observations reported by thefield or seen by the analyst.

Any sample exhibiting the following characteristics must be confirmed by analysts trained in more sensitive microscopic or chemical analytical techniques:

a. Any sample showing evidence of active or current infestation withinsects and/or other animal contamination.

b. Any sample of whole seeds, herbs or other spice material that exhibits

evidence of mold and/or insect damage.

c. Any sample that appears to contain in excess of 0.5% by weight of any

non-hazardous extraneous material (stones, soil and non-toxic seeds).

d. Any sample appearing to contain animal excreta, including insect

excreta identified during macroscopic examination.


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32.15. Quality Control and Quality Assurance

See Section 32.2.2.


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Part C: DETECTION OF GLASS AND NON-ALUMINUM METALS IN MEAT

AND POULTRY PRODUCTS

32.16. Introduction

Meat and poultry products are exposed to a wide variety of materials during processing and

packaging. Due to faulty processing, breakage in machinery or improper handling, pieces

of the processing equipment or packaging material can be introduced into the finished

product. The presence of extraneous materials in a finished product may pose a serious

health risk to all consumers.

This method provides a fast, simple, and reliable means for isolating glass or metalcontaminants from meat and poultry products. The sample is digested in an alkaline

solution. The glass and non-aluminum metals are unaffected by the digestion. These

contaminants are separated from other undigested material in a brine solution.

The laboratory equipment used in the analysis will depend on the type of contamination.

When the suspected contaminant is glass, use of laboratory glassware in analysis must beavoided. Similarly, when the contaminant is suspected to be a metal, use of metal utensils

and containers should be avoided. This will serve to protect the integrity of the sample

during analysis.

32.17. Reagents and Equipment

32.17.1. Reagents

a. 7% Alcoholic Potassium Hydroxide (KOH) †

Dissolve 7 g of KOH in 100 ml of 95% Ethyl Alcohol NOTE: KOH pellets can be used.

b. Sodium Chloride (NaCl) Solution

Prepare 2 L of NaCl solution at room temperature by adding 300 g of NaCl/L of distilled water.

c. Tergitol #4

d. Glycerol/Ethanol Mixture (vol:vol 1:1)

32.17.2. Supplies


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32.17.2.1. For Metallic Contaminants

a. Heavyweight Plastic Picnic knives and forks or 40 lb. test

Monofilament

b. Magnetic Stirring Hot Plate and Magnetic Bar (AOAC XIV

44.002n)

c. #230 Sieve (AOAC XIV 44.002 r)

d. Filter Paper (AOAC XIV 44.002 i)

e. Hirsch Funnel with Screen (AOAC XIV 44.002 k)

f. 2 L Beaker, glass

g. 600 ml "tall" beaker, i.e. Pyrex #1060

h. 2 L Graduated cylinder

i. Watch Glass for a 2 L beaker

32.17.2.2. For Glass Contaminants

The equipment is the same as above except do not use glassware in

analysis and substitute with the following for glass beakers:

a. Stainless Steel Beaker with 2 L capacity

b. Reusable Plastic Beaker with 600 ml capacity (Nalgene

Polypropylene #1201)

c. Polypropylene Graduated Cylinder

d. Plastic Basin to cover 2 L beaker, Nalgene #69010040 or

equivalent


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32.18. X-ray Screening

a. Follow safety procedures pertinent to the use of the cabinet X-ray system. In thiscase, a ‘Radioactive Material Use Permit’ must be provided by the Radiation Safety

Staff under the United States Department of Agriculture. The steps outlined here aregenerally those followed for use of a cabinet X-ray system which emits ionizing

radiation at a controllable range of 10-110 kVp.

b. Turn the X-ray unit on with a key and allow warming up for a minimum of 15

minutes. Set to an emission of 50 kVp for five minutes (turn on by pressing the“START” button). Once the emission has stopped, turn the kVp setting back to zero

before opening the door.

c. Place the product sample (in its retail container, plastic bag, or spread out on an open

plastic tray) on a film negative or on a film holder loaded with a film negative.Center the film on the marked surface of the shelf inside the X-ray unit.

d. Close the door to the X-ray unit. Set the desired emission and exposure time, and press the “START” button.

e. After exposure, remove the sample. Develop the film following directions for thetype of film used. Examine the radiograph for dark, sharply outlined areas. Isolatethese portions of the product sample for further analysis by other methods. The

X-ray unit is most applicable in screening for metal, glass, and bone particles.

f. Turn off the cabinet X-ray unit with the key after use.

32.19. Procedure

a. Cut sample to be digested into 1" x 1" pieces to facilitate the digestion process (Use

plastic utensils or monofilament if examining for suspected metal contamination).

b. Weigh 225 g sample into a 2 L beaker.

c. Add 1.5 L 7% Alcoholic KOH. (see Section 32.56, Safety Caution)

d. Cover with a watch glass. Heat to a boil while stirring on a stirring hot plate until the

sample is completely digested (approximately 1 h). Initially, it will be difficult to stir the sample magnetically but after 10 min at medium to low heat the sample will be


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sufficiently digested to permit magnetic stirring.

e. Transfer sample to a No. 230 sieve. Apply a moderately forceful stream of hot water to push digested residues through sieve. Retain washings in a pan for hazardous

chemical disposal.

f. If there is only a little residue present, transfer this directly to filter paper and examine

microscopically.

g. If large amounts of undigested material remain, add 2 ml of Tergitol to help solubilizeremaining residues. Repeat washing until suds subside. Transfer sample to a "tall"

600 ml beaker with distilled water.

h. Add 400 ml NaCl solution.

i. Wait 30 seconds, and then pour off suspended material. Be careful not to disturb or

pour off material on bottom.

j. Repeat steps h and i.

k. Wash the material remaining on bottom of beaker onto ruled filter paper with distilled water and examine microscopically.

NOTE: Check the magnetic stirring bar for metal contamination.

32.19.1. Procedure for Index Sample

a. If an index sample of the contaminant is available, put a portion of the indexsample in the 7% Alcoholic KOH Solution. (see Section 32.56 Safety

Caution)

b. Bring the solution to a boil and examine the index sample noting anychemical reaction it may have undergone.

c. Repeat the boiling and examine again. If the sample reacts with the solution,do not use an alkaline digestion. Use an acid or enzymatic digestion instead.


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32.20. Result

The lined filter paper should be examined line by line at 30X magnification. Report thefollowing:

a. Metal

1. Count the number of pieces of metal recovered.

2. Record the size or the number of contaminants within a size range.

3. Provide a general description of the contaminants recovered. Note theshape, thickness, color or discoloration, magnetism, and surface markings.

b. Glass

1. Count the number of fragments recovered.

2. Record the size or the number of contaminants within a size range.

3. Provide a general description of the contaminants. Note the presence or absence of the following characteristics: very thin, cube shaped, mold markings, rounded edges, smooth curved surfaces, color, conchoidal

fractures.

4. Examine suspected fragments (immersed in mineral oil or other immersion

oil) under polarized light to determine if they are isotropic (glass is isotropic

or non-birefringent; i.e., fragments will disappear when polarizing filter is positioned under the microscope stage to create a dark field).

5. If refractive index is to be measured, take the following steps:

i. Compare the refractive index of any suspect glass fragments with

refractive index oils by immersing in oil on a microscope slide,

coverslipping, and examining microscopically (immersion method).A bright white “halo” (the Becke line) appearing along the edges of

the fragment moves in the direction of higher refractive index as the

focal distance is increased (and toward lower refractive index whenfocal distance is decreased).


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ii. Rinse the fragment with benzene (or other suitable solvent), let dry,

immerse in oil with a lower or higher refractive index than the oil just used, and reexamine.

iii. Repeat the procedure until the edges of the fragment are

indistinguishable (i.e. no Becke line is visible) from the oil it is

immersed in; yellow and blue may be observed as a refractive index

match is approached. These observations indicate that the refractive

index of the fragment is similar to that of the oil used. [Morecomplex measurements can be made at different temperatures or

different wavelengths of light using more elaborate instrumentation, but the method just described is simple and sufficient for general

purposes.]


See Section 32.2.2.

a. In step 32.19.e, be sure to wash the heavy contaminants from the bottom of the

beaker to the sieve. Heavy contaminants settle quickly to the bottom of the beaker and an ample stream of water is needed to wash them from the beaker. The beaker should be inverted over the sieve and the material in the beaker should be washed

into the sieve with a gentle stream of water.

b. Check the magnetic stirring bar for small magnetic filings before beginning analysis.


† Do not dispose of hazardous waste by pouring down sink drains. Collect in separate

containers and dispose of as hazardous waste as per standard waste management procedures

for your laboratory.Use caution when working with potassium hydroxide. Wear goggles and gloves to protect

eyes and skin when preparing the solution and when moving and wet sieving the sample.

Digest and wet sieve samples under a safety hood.


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Part D: METHOD FOR THE ISOLATION OF GLASS FROM PREPARED MEAT

AND POULTRY BABY FOODS

32.23. Introduction

The recommended procedure for isolating glass from a food product is the heavy sediment

procedure for that product. There is no heavy sediment procedure for meat/poultry baby

food products. The method outlined below was developed in response to the need for a

standard procedure for isolating glass from meat/poultry baby food products.

Bottled food can become contaminated with glass in a number of ways. The container may

already be contaminated when it arrives at the food processors. The finished food productmay become contaminated by glass breakage during processing. Containers can break

during storage, shipping, retail, and consumer handling and fragments from broken

containers can contaminate the exterior of other containers. If these exterior contaminantsare in or around the jar opening, they could contaminate the product when the jar is opened.

This method is quick and easy. The sample is washed in a #60 sieve. The bulk of thesample is washed through the sieve. The remaining material is transferred to a beaker and

mixed with a brine solution. In a brine solution the heavy contaminants, such as glass,

settle to the bottom of the beaker. The brine solution and the suspended food material are poured off and discarded. These two steps, adding and pouring off the brine solution, arerepeated three times. The heavy contaminants remaining on the bottom of the beaker are

washed on to a filter paper which is examined microscopically. This isolation procedure

takes less than 15 minutes. Suspect particles must be tested to confirm that they are in factglass.

To protect the integrity of the sample, no glass should be used in any part of this method.After adding the brine solution to the plastic beaker containing the sample, any glass

fragments will settle to the bottom within seconds. The longer the settling step, the more

food material settles to the bottom which creates dirty plates. Ten seconds is plenty of time

for glass fragments to settle to the bottom.

32.24 Regents and Equipment

32.24.1. Reagents

a. Sodium Chloride (300 gm/L)


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Add 2 L of distilled water to 2 L plastic beaker. Add 600 g of NaCl while

magnetically stirring. The above recommended plastic beaker will

accommodate 2 L of salt solution. Cover beaker with plastic basin and continue stirring until NaCl is completely dissolved.

32.24.2. Equipment

NOTE: DO NOT USE ANY GLASS APPARATUS DURING THE

ANALYSIS.

a. Plastic Beaker w/ 600 ml capacity, ie. Nalgene #1201

b. Plastic Beaker w/ 2 L capacity, ie. Nalgene #1201

c. Plastic Basin to cover 2 L beaker, ie. Nalgene #69010040

d. Plastic Graduated Cylinder

e. #60 Sieve (AOAC 16.1.01(B)(r) 16th Ed.)

f. #230 Sieve (AOAC 16.1.01(B)(r) 16th Ed.)

g. Magnetic Stirrer and Bar (AOAC 16.1.01(B)(n) 16th Ed.)

h. Ruled Filter Paper (AOAC 16.1.01(B) (i) 16th Ed.)

i. Disposable Petri Dish (100 X 10 mm)

j. Lab Spatula

k. Hirsch Funnel

l. Side arm trap flask connected to vacuum pump

m. Laboratory Balance, 1 kg capacity

n. Compound Microscope with polarizer

o. Aerator, Water (AOAC 16.1.01(B)(a) 16th Ed.)


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32.25. Procedure

32.25.1. Cleaning of Exterior of Sample Container

a. Thoroughly rinse exterior of jar and around lid on to a #230 sieve using

hot water. Use the spatula to clean food residues from lid and jar

threads. This step is included to be sure no glass is on exterior of the jar.

b. Wet filter paper with water and center it in Hirsch funnel. Turn on

vacuum and fit filter paper to sides of funnel.

c. Transfer material on the sieve to filter paper with distilled water. Do not

aspirate the paper to dryness otherwise the glass fragments will "pop" off the paper. If needed, wet the paper with a drop or two of water. The

paper should be moist enough so that it adheres to the petri dish but it

should not be soaked. If the paper is too wet, the water will hide small,flat pieces of glass.

d. Transfer filter paper to petri dish and examine paper microscopically for glass fragments.

e. Confirm any suspect particles using a compound microscope with

polarized light.

f. Count, measure and describe all glass fragments found on the exterior of

container. Report any particles of glass as contaminants found on theexterior of the sample container. Report number of fragments found

within a size range. Fragments less than 1 mm can be reported as "Less

than 1 mm." An excessive number of fragments can be reported as "Too

Numerous To Count."

32.25.2. Sample Analysis

a. Quantitatively transfer contents of jar to #60 sieve. A spatula can be

used to remove the bulk of the sample. Use water from a squirt bottle to

thoroughly rinse interior of jar. Retain jar for further examination at stepone.


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b. Thoroughly wash sample in sieve with hot aerated water. When no more

material passes thru sieve, wash remaining material to one side of sieve.

c. Quantitatively transfer contents of sieve to a plastic beaker w/ distilled water. Use no more than 200 ml of water.

d. Dilute to 400 ml with NaCl solution.

e. Let material settle 10 seconds, then pour off suspended material. Morethan 10 seconds is not needed. Glass will settle to bottom in 10 seconds.

Waiting longer than 10 seconds allows more food material to settle to the bottom. Be careful not to disturb or pour off residues on bottom.

f. Repeat Steps d & e to remove excess plant material, usually twice more.

g. Wet filter paper with water and center it in Hirsch funnel. Turn on

vacuum and fit filter paper to sides of funnel.

h. Wash residues remaining on bottom of beaker to ruled filter paper. Do

not aspirate the paper to dryness otherwise the glass fragment will "pop"off the paper. If needed, wet the paper with a drop or two of water. The paper should be moist enough so that the paper adheres to the petri dish

but the paper should not be soaked. If the paper is too wet, the water will

hide small, flat pieces of glass.

i. Transfer paper to petri dish and examine microscopically.

j. Count, measure, and describe all glass fragments found in the food

product. Report number of fragments found within a size range.

Fragments less than 1 mm can be reported as " Less than 1 mm." An

excessive number of fragments can be reported as "Too Numerous ToCount."

k. Confirm any suspected particles using a compound microscope and polarized light. Glass is an isotropic compound and will not transmit

crossed polar light. Sand or quartz is birefringent, thus will transmit

crossed polar light.


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l. Examine all retail sample jars for chips, fractures, or other defects if any

glass fragments are found within product.

m. Maintain in reserve all glass fragments and all jars from which glass

fragments were removed.

32.25.3. Characterization of Contaminants

If classification or comparison of glass contaminants is needed to identify a possible source, determine the refractive index of the glass contaminants.


See Section 32.2.2.

a. Do not use any glassware in this analysis.

b. Before beginning an analysis, wipe down or wash the entire work area.

c. Rinse the beakers and graduated cylinder before using them.

d. Backwash the sieve by spraying water through the bottom to remove any debris

in the sieve before using it.

Part E: Analysis of Fecal Material

32.27. Introduction

Diagnostic characteristics of excreta pellets and the alkaline phosphatase test provide a

method to aid in the identification of the excreta pellets of animals and insects. The alkaline

phosphatase isoenzyme is in the intestinal tract of most mammals, including mice and rats.In this procedure, the alkaline phosphatase will split the phosphate radical from

phenolphthalein diphosphate in the Working Test Media to produce a reddish free

phenolphthalein.

32.28. Reagents and Equipment

32.28.1. Reagents


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a. Magnesium Chloride Solution--Dissolve 0.203 g MgCl2.6H2O in H20

and dilute to 500 ml.

b. Stock Test Reagent--Dissolve 4.75 g borax (Na2B4O7.10H2O) and

1.57 g anhydrous Na2CO3 in 250 ml H2O with stirring. Add 0.235 g

phenolphthalein diphosphate and stir while adding 0.5 ml MgCl2

solution. Preparation is stable.

c. Work Test Media (WTM)--Measure equal volumes stock test reagentand H2O. Place cold H2O into a media bottle or beaker on a stirring

hot plate, add stirring bar, and, with rapid stirring, add sufficient agar to yield a 2% agar dispersion. Heat solution to boil on stirring hot

plate or in microwave. Pour equal amount of stock test reagent into

container of boiled agar solution. Stir rapidly or cap bottle and shakefor one minute. Add ca 1 ml portions of WTM to wells before

cooling to 40-41oC. WTM must be cooled to 40-41

oC before

contacting samples.

Short Term Storage: WTM may be held ca 48 h at 40-41oC if

covered snugly with foil or plastic.

Long Term Storage: WTM may be stored up to 12 months if

refrigerated and protected from direct sunlight. Discard if pink color

and/or volume loss is observed.

32.28.2. Equipment

a. Pasteur pipets--1 ml capacity

b. Water bath--set at 40-41oC

c. Tissue culture multi-well plate with cover-capacity

d. Laboratory Balance, 1 Kg capacity

e. Beakers, 100 ml and 800 ml or 1 L capacity


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32.29. Procedure

a. Measure suspect pellet.

b. Moisten suspect pellet with H2O and examine for hair and other diagnostic

characteristics.

c. Place small part of particle into WTM well.

d. Cover particle with ca 1 ml portion of cooled (40-41oC) WTM.

e. Place plate in 40-41oC H2O bath.

f. Check for development of red color near particles.

32.29.1. Test Response

Time varies according to species from 2-3 min for most mouse samples, up to 4

hours for samples from some grass eaters, such as deer or rabbit.


32.30.1. Blank Preparation

a. Autoclave pellets 15+ min. at 15 psi.

b. Store in closed container in freezer.

c. Alternatively, place 100 ml beaker in 800 ml or 1 L beaker and add

ca 25 mm depth H2O to each.

d. Place small test tube with 1 ml WTM in smaller beaker.

e. Heat H2O to boiling ca 2 min.


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f. Remove small tube and quickly transfer crushed pellet material to

tube.

g. Return tube to rapidly boiling H2O ca 2 min.

h. Remove tube and with small glass rod work all particles from side

wall of tube down into liquid.

i. Replace tube in small beaker, cover large beaker with watch glass

and continue to boil ca 5 min.

j. Remove small tube, mix contents quickly and transfer to negativetest well.

32.30.2. Positive Control

Store known rat and mouse excreta pellets in closed container in freezer.


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32.31. Selected References

Boese, J. L., and R. Bandler (ed.). 1990. Extraneous materials: isolation, Chapter 16. InOfficial Methods of Analysis of the Association of Official Analytical Chemists, 15th

Edition. AOAC International, Inc., Gaithersburg, MD 20877.

Borror, D. J., D. M. De Long, and C. A. Triplehorn. 1981. An Introduction to the Study of

Insects, 5th Edition. Saunders College Publ., Philadelphia, PA.

Borror, D. J., C. A. Triplehorn, and N. F. Johnson. 1989. An Introduction to the Study of Insects, 6

thEdition. Saunders College Publ., Fort Worth, TX.

Brickey, P. M., J. S. Gecan, J. J. Thrasher, and W. V. Eisenberg. 1968. Notes on

microanalytical techniques in the analysis of foods for extraneous materials. J. Assoc. Off.

Anal. Chem. 51(4):872-876.

Gecan, J. S., S. W. Cichowicz, and P. M. Brickey. 1990. Analytical techniques for glass

contamination of food: A guide for administrators and analysts. J. Food Prot. 53(10):895-899.

Gentry, J. W., K. L. Harris, and J. W. Gentry Jr. 1991. Microanalytical Entomology for Food Sanitation Control, Vol. 1 & 2. Published by J. W. Gentry and K. L. Harris,Melbourne, FL.

Gorham, J. R. (ed.). 1977. Training Manual for Analytical Entomology in the Food Industry, FDA Technical Bulletin #2. U.S. Dept. of Health, Education and Welfare, Public

Health Service, Food and Drug Administration, Washington, DC.

Gorham, J. R. (ed.). 1981. Principles of Food Analysis for Filth, Decomposition, and

Foreign Matter, FDA Technical Bulletin #1, 2nd

Edition. U.S. Dept. of Health and Human

Services, Public Health Service, Food and Drug Administration, Washington, DC.

Gorham, J. R. (ed.). 1991. Insect and Mites Pest in Food - An Illustrated Key. U. S. Dept.

of Agriculture Agricultural Handbook #655. U.S. Dept. of Agriculture, Agricultural

Research Service and U.S. Dept. of Health and Human Services, Public Health Service,Food and Drug Administration, Washington, DC.

Humason, G. L. 1962. Animal Tissue Techniques, 3rd

Edition. W. H. Freeman and Company, San Francisco, CA.


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Kurtz, O. L., and K. L. Harris. 1962. Microanalytical Entomology for Food Sanitation

Control. Published by Association of Agricultural Chemists, Arlington, VA.

Miller, E. T. 1982. Forensic glass comparisons, p. 152-154. In R. Saferstein (ed.), ForensicScience Handbook. Prentice-Hall, Englewood Cliffs, NJ.

Olsen, A. R., T. H. Sidebottom, and S. A. Knight. 1995. Fundamentals of Microanalytical

Entomology. Published by CRC Press, New York, NY.

Peace, D. McClymont. 1985. Key for the Identification of Mandibles of Stored-Food

Insects. Health and Welfare of Canada. Association of Official Analytical Chemists,Gaithersburg, MD 20877.

Personal communication from Don J. Vail (retired), Food and Drug Administration, AtlantaLaboratory, 1988.

Sheehan, D. C. and B. B. Hrapchak. 1980. Theory and Practice of Histotechnology, 2nd

Edition. The C. V. Mosby Company, St. Louis, MO.

Stehr, F. W. (ed.). 1987 and 1991. Immature Insects, Vol.I & II. Kendall/Hunt Pub. Co.,Dubuque, IA.

U. S. Food and Drug Administration. 1984. Macroanalytical Procedures Manual, FDA

Technical Bulletin #5. U.S. Dept. of Health and Human Services, Public Health Service,Food and Drug Administration, Washington, DC.


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CHAPTER 33. DETECTION OF ANTIMICROBIAL RESIDUES IN MEAT ANDPOULTRY TISSUE BY SCREEN TESTS

B. P. Dey, Clarence A. White, Richard H. Reamerand Nitin H. Thaker

33.1 Introduction

Rapid microbiological screen tests are used in slaughterestablishments to detect the presence of antimicrobial residues infood animal tissues. The Swab Test on Premises (STOP) is used forall red meat species except bob veal calves, where the Calf Antibiotic and Sulfa Test (CAST) is used. The Fast AntimicrobialScreen Test (FAST) developed recently and tested on bovine tissue,has been found to have greater sensitivity than STOP and CAST. Thetest is being conducted in bovine slaughter establishments on alimited basis. The FAST procedure is presently being tested inswine.

These microbial inhibition tests are simple to perform, costeffective and allow routine testing and release of large numbers offood animal carcasses in the shortest possible time. Use of thesescreen tests permit FSIS to analyze only those carcasses which werefound to contain antimicrobial compounds by in-plant tests.

PART A

33.2 DETECTION OF ANTIMICROBIAL RESIDUES BY SWAB TEST ON PREMISES(STOP)

Clarence A. White, B. P. Dey and Richard H. Reamer

33.21 Background

The Swab Test on Premises (STOP) was developed for tentativedetection of antimicrobial residues in carcasses. It is performed by inserting a sterile cotton swab into the kidney sample of acarcass. After 30 minutes, the tissue fluid soaked top, one-fourth portion of the swab is transferred to an agar plate seeded with

Bac i l l us subt i l i s spores. After incubation for 16-18 h at 29°°C, plates are examined for a zone of inhibition (ZI) around the swab.If no inhibition is seen, the carcass is free of antimicrobial

residue at detectable levels. In case of inhibition, presence ofantimicrobial residues is suspected and muscle, liver and kidneytissues from the suspect carcass are collected and submitted forconfirmation and identification at FSIS laboratories.In 1980, a modified version of the original test was introduced inslaughter establishments. The agar plates and vials of spores are

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separately supplied. In the modified version, prior to performingthe test, the plates are surface streaked with the spore suspension by sterile swabs. The rest of the procedure is similar to theoriginal test. The supplies are now commercially available and arestable for 6 months when stored at either room or refrigerated temperatures.

Initially when the test was developed, it used tissues from kidney,liver, muscle, and injection site. However, at present kidney is

the target tissue. The sensitivity of the STOP test forsulfonamide detection is unsuitable for regulatory purposes.

33.22 Equipment, Reagents and Supplies

33.221 Equipment

a. Laminar Flow Hood or equivalent clean room b. Sorvall RC5C Refrigerated Centrifuge, Sorvall Rotor

SS-34, and Sorvall Swinging Bucket Rotor HB-4 or

equivalent. Centrifuge must be able to operate at20,000 x G at a constant 5°°C. It should also operate with a swinging bucket rotor at 1,500 x G at room temperature or equivalent.

c. Virtis homogenizer, Model 60K or equivalentd. Sterile Virtis jarse. Vortex mixer or equivalent

f. Incubators, one maintaining 37°°C and the other 29°°Cg. Precision water bath (48 ± 1

oC) with cover (Model 183) or

equivalenth. Quebec Colony Counter or equivalenti. Fisher-Lilly Antibiotic Zone Reader (Fisher Scientific,

Cat. No. 07-906) or equivalent

33.222 Reagents

a. Distilled water:

The distilled water must be prepared using an all glassstill (Corning Megapure 6L or equivalent) and stored ina glass or any acceptable reservoir which is not a partof the system. All spore lots must be prepared usingglass distilled water.

* Do not use deionized water.

* Resins of some systems produce quaternary ammonium

compounds which interfere with the analysis. b. Phosphate buffer (3 M, pH 7.1)

Dissolve 306.9 g of K 2HPO4 and 168.6 g KH2PO4 per liter of

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distilled water. If necessary, adjust pH by dropwiseaddition of either 0.1 N HCl or NaOH depending upon pH

reading. Sterilize by autoclaving at 121°°C for 15

minutes or filtering through a 0.2 µµ m filter.

c. Ethyl alcohol (USP grade, 200 proof)

Dehydrated Alcohol, USP, Ethyl Alcohol, 200 ProofPunctilious

R , (Ethyl Alcohol [Ethanol] CAS #64-17-5,

Warner-Graham Company, 160 Church Lane, Cockeysville, MD21030). For a 50% solution, mix 1 part of ethylalcohol with 1 part glass distilled water. Prior to

use, filter sterilize through a 0.2 µµ m filter.

d. Polyethylene glycol, Mol. Wt. 4000 (Baker Chemicals)Sterilize in a covered beaker by autoclaving prior touse.

e. Butterfield's Phosphate Buffer, sterile

33.223 Supplies

a. Sterile Roux bottles b. Sterile glass beads, 4 mm diameterc. Sterile 100 ml graduated glass stoppered cylinders or

volumetric flasksd. Sterile centrifuge tubes, 40 ml (Nalgene 3118 or

equivalent)e. Sterile pipettes, 10 ml and 1 ml graduated to the tipf. Sterile, clear glass vials 51 x 15 mm with deep seated

screw caps

g. Pressure sensitive labels not to exceed 2" x 1/2"h. Acetate shrink-wrap material for sealing 51 x 15 mm

glass vials or equivalent closure materiali. Forcepsj. Permanent marking penk. Antimicrobial sensitivity discs containing 5 mcg of the

antibiotic neomycin (N5)l. Sterile cotton swabs on hollow plastic tubes m. Sterile, plastic 60 mm diameter X 15 mm petri plates

(Falcon Cat. # 1007 or equivalent)

33.23 Media

a. Brain Heart Infusion broth (BBL or equivalent);reconstitute according to manufacturer's directions,dispense 10 ml/tube and sterilize (121

oC for 15 minutes)

b. Blood agar plates (Columbia Blood Agar Base, 5% HRBC)c. Antibiotic Agar No. 5 (Streptomycin Assay Agar)d. Mueller-Hinton Agar

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e. A-K Sporulating Agar No. 2

i. Agar slants - reconstitute A-K Sporulating Agar No. 2 according to manufacturer's directions withan extra 0.5% Purified Agar (Difco or equivalent),sterilize by autoclaving at 121

oC for 15 minutes

and prepare as slants.

ii. Roux bottles - add 300 ml reconstituted A-K Sporulating Agar No. 2 with an extra 0.5% Purified Agar. Sterilize (121

oC for 15 minutes) and allow

medium to harden in Roux bottles placed in ahorizontal position.

33.24 Test Organism

Bac i l l us subt i l i s ATCC 6633 (American Type Culture Collection,Rockville, MD)

33.241 Purity and Biochemical Properties of Bac i l l us subt i l i s

a. Reconstitute a lyophilized culture in Brain Heart

Infusion broth and incubate at 37°°C for 18 h. Streak blood agar plates with the broth culture, incubate at

37°°C for 18 h and check for culture purity.

b. For isolation, streak the culture onto two Columbia Agar plates with 5% defibrinated horse blood. Incubate at

37°°C for 18 h.

c. Prepare a Gram stain of three well isolated colonies.

All cultures should be Gram positive.

d. Stain a drop of the spore suspension with malachitegreen and counterstain with carbol-fuchsin solution.The spores will appear green, whereas the vegetativecells will appear red or pink.

e. Use one Columbia Agar plate with 5% defibrinated horse blood from the culture to test for the presence ofcatalase. Bac i l l us sp. are catalase positive.

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f. Use colonies from the other plate to check biochemicalcharacteristics of the culture by inoculating O-Fglucose, Voges-Proskauer, and mannitol broths. Incubate

at 35°°C for 18 h. The biochemical patterns of B.subt i l i s should agree with the following chart:

Catalase Gram stain

Sporeforming

O-F glucose Voges-Proskauer

Mannitol

+ + + O + V

(+) = positive; (-) = negative; (F) = fermentative;(O) = oxidative; (A) = acid; (V) = variable.

g. If the organism does not meet all the above criteria,replace with a new ATCC culture of the organism.

33.242 Preparation of B. subt i l i s spores

a. After the culture meets all biochemical criteria, pickseveral well isolated colonies from the plates and streak A-K Sporulating Agar No. 2 slants (one per Roux

bottle) and incubate the slants at 37°°C for 18 h.

b. To each agar slant, add 4-6 sterile glass beads and 2-3 ml sterile distilled water and gently shake for 2 minutes to dislodge bacterial growth.

c. Aseptically transfer the slant suspensions to a Roux

bottle containing A-K Sporulating Agar No. 2 and spread with the help of the glass beads. Multiple cultures may be prepared and pooled for transferring.

d. Incubate the Roux bottles horizontally for 18-24 h at

37°°C and then at room temperature for the remainder of 1 week (6 days).

e. Harvest the growth from the Roux bottles by adding 20-30sterile glass beads and approximately 25 ml of steriledistilled water per bottle. Gently agitate bottles todislodge bacterial growth. (Care must be taken not to break the agar during harvesting).

f. Aseptically transfer the bacterial suspension intosterile centrifuge tubes (40 ml volume) and heat the

tubes in boiling water (100°°C) for 10 minutes.

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g. Wash the heated suspension three times with steriledistilled water by centrifuging and decanting in thefollowing manner:

i. Centrifuge at 5°°C for 20 minutes at 20,000 x G.ii. Pour off supernatant.

iii. Resuspend the pellet in 20 ml sterile distilled water.

iv. Repeat Steps i, ii and iii two more times.

h. Wash and coat a Virtis jar with a mixture of sterile phosphate buffer and sterile polyethylene glycol in thefollowing manner:

Mix 34.1 ml of sterile phosphate buffer and 11.8 g ofsterile polyethylene glycol in a 100 ml sterile glassstoppered volumetric flask and shake vigorously. Bringto volume with sterile distilled water. Pour the mixture into a Virtis jar and place the jar on the

homogenizer. Blend for 5 minutes at 5,000 RPM. Discard the mixture. Repeat the process.

i. Prepare a fresh solution of sterile buffered polyethylene glycol (34.1 ml of phosphate buffer and 11.8 g of polyethylene glycol) in a 100 ml glassstoppered sterile volumetric flask. Add 25 ml of the washed spore mixture and bring to volume with distilled water. Shake vigorously. Pour the mixture into a coated Virtis jar and homogenize for 5 minutes at 5,000 RPM.

j. Dispense the mixture equally into four sterilecentrifuge tubes and centrifuge in a swinging bucketrotor at 1,500 x G (3,000 RPM in H-4 Rotor in SorvallRC5C) for 2 minutes at room temperature.

k. A two-phase system with an interface will be formed inthe centrifuge tube. Being careful not to disturb ordisperse the interface layer, transfer the sporecontaining, upper phase using a 10 ml pipette to asecond set of sterile centrifuge tubes.

l. Centrifuge the tubes at 20,000 x G for 20 minutes at 5°°C.Pour off the supernatant. Resuspend the pellet in each

tube with 20 ml sterile distilled water and pool the

contents of all tubes into a sterile container.

m. Pipette 25 ml aliquots of spore suspension into eachsterile centrifuge tube. Centrifuge tubes at 20,000 x G

for 20 minutes at 5°°C. Repeat the process five timesafter decanting the supernatant and re-suspending the

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pellet in 20 ml of sterile distilled water.

n. After the last wash step, resuspend each spore pellet in20 ml 50% ethyl alcohol. Pool all spore suspensionsinto a sterile bottle containing 15-20 sterile glass

beads. Store the stock suspension at 35-40°°F (2-4.4°°C).

(Properly preserved stock spore suspension may be used indefinitely).

33.243 Enumeration of B. subt i l i s Spores in Working Suspension

a. To determine the number of spores/ml in each new sporestock suspension, prepare tenfold serial dilutions(10

-2-10

-10) of the suspension using Butterfield's

Phosphate Buffer. (Pipet 1.0 ml of well mixed sporestock suspension (use vortex mixer) into 9 ml buffer and then make serial dilutions up to 10

-10.).

b. Using separate pipettes, pipette 1.0 ml of each dilution

into triplicate 100 x 15 mm plates.

c. Pipette 15 ml molten Plate Count Agar (cooled to 50 +

1°°C) into each plate. Mix by swirling or tilting platesto evenly disperse the inoculum throughout the medium.

Incubate for 48 h at 37 + 1°°C.

d. Count colonies (30-300) in triplicate plates on a QuebecColony Counter. Record and average the number ofcolonies/ml in each dilution. Determine the number ofcolony forming units (cfu)/ml of the stock solution.

e. To prepare the final spore suspension at a concentrationof 1 x 10

6cfu/ml in 50% ethyl alcohol from the stock

spore suspension, use the following formula:

Concentration Desired of stock spore Dilution concentration ofsuspension = factor X working spore(cfu/ml) suspension (cfu/ml)

Example:

Stock spore suspension = 1 x 109spores/ml

Desired concentration of working spore suspension

= 1 x 106 spores/ml:

(1 x 109cfu/ml) = (x) (1 x 10

6cfu/ml)

(1 x 109cfu/ml) = x

(1 x 106cfu/ml)

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x = 1000

In this example, the stock spore suspension must bediluted 1:1000 (1 part stock spore suspension plus 999 parts diluent) in 50% ethyl alcohol to prepare the 1 x

10

6

spore/ml concentration.

33.244*Packaging of B. subt i l i s Spore Suspension (for Field Use)

a. Dispense 4.0 ml of the final (working) spore suspension(1 x 10

6cfu/ml in 50% ethyl alcohol) into sterile

51 x 15 mm clear, glass vials with deep seated,leak-proof screw caps.

b. After securely capping spore vials, seal withshrink-seal, or equivalent closure material, to preventleakage or dehydration.

c. Label the vials with the following information on atransparent mylar pressure sensitive label, orequivalent:

i. "STOP spores"ii. B. subt i l i s ATCC 6633iii. Lot Numberiv. Packaging Date

NOTE: B. subt i l i s spores (1 x 106or 1 x 10

7cfu/ml) can

also be obtained from EDITEK, Burlington, NC, byspecial order.

33.25*Preparation of STOP Plates (for Field Use)

a. Add 25.5 g of Antibiotic Agar No. 5 (Streptomycin Assay Agar) powder into 1 L of glass distilled water. Heat while stirring and bring to a boil. Sterilize at 121

oC

for 15 min. Cool and mix the medium thoroughly in a 48°°C water bath. Continue mixing during cooling and dispensing.

b. Aseptically add 6.0 ml of the agar to each 60 x 15 mm plate and distribute evenly. Place plates on a flat

level surface and allow agar to harden.* NOTE: Under FSIS contract, STOP spores (1 x 10

6cfu/ml)

and plates are now produced commercially and areroutinely available for use. After they meet allquality control specifications they are used in

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slaughter plants.

c. Label the lid of each plate with the followinginformation:

i. "STOP PLATE"

ii. Lot Numberiii. Expiration Date

d. Refrigerate plates in sealed double plastic bags to prevent moisture evaporation. These plates can be used for a period of 90 days.

33.251 Preparation of STOP Plates (Used in Laboratory)

a. Add 25.5 g of Antibiotic Agar No. 5 (Streptomycin Assay Agar) powder to 1 L of distilled water. Heat whilestirring and bring to a boil. Sterilize at 121

oC for 15

minutes. Cool and mix the medium thoroughly in a 48°°C

water bath.

b. Aseptically add 1 ml of 1 x 107cfu/ml B. subt i l i s spore

suspension per 100 ml of the agar. Mix thoroughly.Pipette 8 ml of the agar into each 100 x 15 mm plate and tilt plates to insure even distribution. Allow the plates to harden on a flat, level surface.

c. Label the lid of each plate with the followinginformation:

i. "STOP PLATE"ii. Date

d. Refrigerate plates in sealed double plastic bags to prevent moisture evaporation. These plates can be used for a period of 10 working days.

33.26 Performing the STOP Test

33.261 Sample Condition

a. Assure that the samples are cold, 4°°C or below.

b. Identify samples according to standard operating

procedures.

NOTE: Presently STOP is used only on kidney tissue of allclasses of animals, i.e., bovine, swine, sheep/goat,

and horses with the exception of bob veal calves.

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33.262 Procedure

a. Allow frozen samples to thaw completely at room temperature for a sufficient period of time such thatice crystals are no longer present within the sample.

b. Open a sterile cotton swab pack, remove one swab, and insert the sharp end of the swab shaft about 1/2" to3/4" into each kidney tissue.

c. Move the swab shaft back and forth several times to macerate the tissue, disrupting tissue cells and releasing tissue fluid. Remove the swab shaft.

d. Reverse the swab and insert the cotton tip into thetissue opening, twisting to make sure that the cottontip is in good contact with the macerated tissue.

e. Allow swabs to remain in the tissue for a minimum of 30

minutes.

f. Allow refrigerated plates to warm to room temperaturefor about 10 minutes before streaking. Check each platefor absence of contamination, cracking of agar ordryness.

g. Lift the plate cover slightly and mark an "X" reference mark on the outer side wall of the plate. Place thecovered plate bottom side down on the work place surface with the reference mark at the 12 o' clock position. With a fine-tip permanent marking pen, start at the "x"and draw a line across the bottom of the plate dividing

it into two equal sections.

h. Check for seal integrity of vials containing spores.

i. Shake the B. subt i l i s spore vial (1 x 106cfu/ml) and dip

a sterile swab in the solution. Gently touch the swabto the side of the vial to remove excess fluid. Replacethe screw cap on the vial.

j. Streak the surface of the agar plates with the swab from a point marked on the side of the plate moving up and down and from left to right. Turn the plate 1/4 turnand streak again.

k. Repeat this streaking process 2 more times. Finally turnthe plate 1/2 turn and streak. (Use a separate swab foreach plate)

NOTE: Above applies only for plates used in the plant. The plates used in laboratories are seeded at a different

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concentration level and therefore should not be surfacestreaked.

l. Place a neomycin 5 µµg disc on the agar surface near thevertical line on a plate.

m. Remove the swab from the tissue, break the shaftapproximately two inches from the swab end.

NOTE: If the swabs appear dry, reinsert them in the tissueand squeeze the tissue around the swab to absorb tissuefluids. For small portions of dry muscle tissue,

moisten swab with distilled water prior to inserting.

n. Gently place the swab on the surface of the plate with

the broken end of the shaft near the neomycin 5 µµg disc making sure not to break the agar surface. Make surethe swab has uniform contact with the agar.

NOTE: Swabs from two kidney tissues from two differentcarcasses can be placed on each plate provided they are

properly identified on the plate.

If two tissue swabs are used per plate, place cottontips in "rabbit ears" configuration (Fig. 1)

Figure 1. Swab placement on plate

o. Incubate the plates upright at 29 + 1°°C for 16-18 h.

p. Store samples under refrigeration until the test iscompleted.

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33.27 Results and Interpretation

a. Remove the incubated plates from incubator and removeswabs.

b. Measure the ZI by the N5 disc with a mm ruler or with an

antibiotic zone reader. The zone should be 20-26 mm wide. If the zone is not 20-26 mm in width, the test isinconclusive and should be repeated.

c. Observe the plates for inhibition of B. subt i l i s growthsurrounding the swabs.

i. If a zone of inhibition is observed, the test is positive. Measure the length and the width of thezone and record results.

ii. If no zone of inhibition is observed, the test isnegative. Record the result.

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Johnston, R. W., R. H. Reamer, E. W. Harris, H. G. Fugate, and B. Schwab. 1981. A new screening method for the detection ofantibiotic residues in meat and poultry tissues. J. Food Prot.44:828-831.

Kramer, J., G. G. Carter, B. Arret, J. Wilner, W. W. Wright,and A. Kirshbaum. 1968. Item 344-837 (4008). AntibioticResidues in Milk, Dairy Products and Animal Tissues: Methods,Reports and Protocols. Food and Drug Administration,Government Printing Office, Washington, DC.

Read, R. B., J. G. Bradshaw, A. A. Swatzentruber, and A. R. Brazis. 1971. Detection of sulfa drugs and antibioticsin milk. Appl. Microbiol. 21:806-808.

United States Department of Agriculture. 1982. The shelfstable swab test system for detecting antibiotic residues in

tissues. Laboratory Communication No. 31. Food Safety and Inspection Service, S&T, Microbiology Division, Washington,D.C.

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PART B

33.3 DETECTION OF ANTIMICROBIAL RESIDUE IN CALVES BY CALFANTIBIOTIC AND SULFONAMIDE TEST (CAST)

Clarence A. White, B. P. Dey and Richard H. Reamer

33.31 Introduction

The Calf Antibiotic and Sulfa Test (CAST) is a modified form of theSulfa Swab Technique (SST). Sulfonamides are frequently used in bob veal calves, a class of animals weighing under 150 pounds and less than three weeks old. This test is used to detect antibioticand sulfonamide residues in bob veal calves at slaughter.

The inspectors performing the test at slaughter plants are supplied with agar plates and vials containing an alcohol suspension ofspores. To perform the test, a sterile cotton tipped applicator

(swab) is inserted into the kidney sample of a bob veal calf and left for 30 minutes to absorb tissue fluids. The agar plates aresurface streaked by sterile swabs with the supplied Bac i l l us mega t e r i um spore suspension. The swab is removed from the kidney, broken as close to the cotton tip as possible, and placed on to theagar plate streaked with spores. After 16-18 h incubation at

44°°C, plates are examined for a zone of inhibition (ZI) around theswab. If no inhibition is seen, the carcass is free ofantimicrobial residues at a detectable level. All carcasses presenting inhibition are subjected to laboratory confirmation.

33.32 Equipment, Reagents and supplies

33.321 Equipment


SS-34, and Sorvall Swinging Bucket Rotor HB-4 orequivalent. Centrifuge must be able to operate at

20,000 x G at a constant 5°°C. It should also operate witha swinging bucket rotor at 1,500 x G at room temperature.


f. Incubators 37°°C and 44 + 1°°Cg. Precision water bath (temperature 48 ± 1

oC) with cover

(Model 183) or equivalenth. Quebec Colony Counter or equivalenti. Fisher-Lilly Antibiotic Zone Reader (Fisher Scientific,

Cat. No. 07-906) or equivalent

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33.322 Reagents

a. Distilled water:

The distilled water must be prepared using an all glass

still (Corning Megapure 6L or equivalent) and stored ina glass or any acceptable reservoir which is not a partof the system. All spore lots must be prepared usingglass distilled water. * Do not use deionized water.

b. Phosphate buffer (3 M, pH 7.1)

Dissolve 306.9 g of K 2HPO4 and 168.6 g KH2PO4 in 1 Ldistilled water. If necessary, adjust pH by dropwiseaddition of either 0.1 N HCl or NaOH depending on pH

reading. Sterilize at 121°°C for 15 minutes or filtering

through a 0.2 µµ m filter.




Warner-Graham Company, 160 Church Lane, Cockeysville, MD21030). For a 50% solution, mix 1 part of ethylalcohol with 1 part glass distilled water. Prior to use,

filter sterilize through a 0.2 µµ m filter.

d. Polyethylene glycol, Mol. Wt. 4000 (Baker Chemicals).Sterilize (121

oC for 5 minutes) in a covered beaker prior

to use.

e. Butterfield's Phosphate Buffer, sterile

33.323 Supplies




screw capsg. Pressure sensitive labels not to exceed 2" x 1/2"h. Acetate shrink-wrap material for sealing 15 x 51 mm

glass vials or equivalent closure material * Resins of some systems produce quaternary ammonium compounds

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which interfere with the analysis.

i. Forcepsj. Permanent marking pen

k. Antibiotic discs: Neomycin - 5 µµgl. Sterile cotton swabs on hollow plastic tubes

m. Sterile, plastic 60 X 15 mm petri plates (Falcon Cat. No. 1007 or equivalent)

33.33 Media

a. Brain Heart Infusion broth (BBL or equivalent);reconstitute according to manufacturer's directions,dispense 10 ml/tube and sterilize at 121

oC for 15

minutes.

b. Blood agar plates (Columbia Blood Agar Base, 5% HRBC).

c. A-K Sporulating agar No. 2.

i. Agar slants - reconstitute A-K Sporulating Agar No.2 according to manufacturer's directions with extra0.5% Purified Agar (Difco or equivalent), sterilize by autoclaving at 121

oC for 15 minutes and prepare

slants.

ii. Roux bottles - add 300 ml reconstituted A-K Sporulating Agar No. 2 with extra 0.5% purified Agar. Sterilize (121

oC for 15 minutes) and allow

medium to harden in Roux bottles placed in ahorizontal position.

d. Mueller-Hinton Agar (Acumedia Manufacturers Inc.,Baltimore, MD); reconstitute according to manufacturer'sdirections, dispense 100 ml/flask and sterilize (121

oC

for 15 minutes).

33.34 Test Organism

Bac i l l us megat er i um ATCC 9885 (American Type CultureCollection, Rockville, MD)

33.341 Purity and Biochemical Properties of Bac i l l us megat er i um

a. Reconstitute a lyophilized culture in Brain HeartInfusion broth and incubate at 37°°C for 18 h. Streak blood agar plates with the broth culture, incubate at

37°°C for 18 h and check for culture purity. b. Streak the culture for isolation onto two Columbia Agar

plates with 5% defibrinated horse blood. Incubate at 37°°C

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for 18 h.

c. Prepare a Gram stain of three well isolated colonies. All cultures should be Gram positive.

d. Stain a drop of the spore suspension with malachite

green and counterstain with carbol-fuchsin solution.The spores will appear green, whereas the vegetativecells will appear red or pink.

e. Use one Columbia Agar plate with 5% defibrinated horse blood from the culture to test for presence of catalase. Bac i l l us are catalase positive.

f. Use colonies from the other plate to check biochemicalcharacteristics of the culture by inoculating O-Fglucose, Voges-Proskauer, and mannitol broths. Incubate

at 35°°C for 18 h.

The biochemical patterns of B. mega t e r i um should agree with the following chart:

Catalase Gram stain

Sporeforming

O-F glucose Voges-Proskauer

Mannitol

+ + + O - A

(+) = positive; (-) = negative; (F) = fermentative;(O) = oxidative; (A) = acid.

g. If the test organism does not meet all the above

criteria, replace with a new ATCC culture of the testorganism.

33.342 Preparation of Bac i l l us megat er i um Spore Suspension

a. After the culture meets all biochemical criteria, pickseveral well isolated colonies from the plates and streak A-K Sporulating Agar No. 2 slants (one per Roux

bottle) and incubate the slants at 37°°C for 18 h.

b. After incubation, put 4-6 sterile glass beads and 2-3 mlsterile distilled water into each tube and gently shake

for 2 minutes to dislodge organisms from agar slants.

c. Aseptically transfer the suspension from slants to aRoux bottle containing A-K Sporulating Agar No. 2 and spread with the help of glass beads. (Multiple cultures may be prepared and pooled for transfer to Roux

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bottles).

d. Incubate the Roux bottles horizontally for 18-24 h at

37°°C and then at room temperature for the remainder of 1 week (6 days).

e. Harvest the growth from the Roux bottles by the use of20-30 sterile glass beads and approximately 25 ml ofsterile distilled water per bottle. Gently agitate bottles to dislodge bacterial growth. (While harvestingcare must be taken not to break the agar).


tubes in boiling water (100°°C) for 10 minutes.


i. Centrifuge at 5°°C for 20 minutes at 20,000 x G.ii. Pour off supernatant.iii. Resuspend the pellet in 20 ml sterile distilled

water.iv. Repeat Steps i, ii and iii two more times.


Mix 34.1 ml of sterile phosphate buffer and sterile 11.8

g of polyethylene glycol in a 100 ml glass stoppered volumetric flask and shake vigorously. Bring to volume with sterile distilled water. Pour the mixture into a Virtis jar and place the jar on the homogenizer. Blend for 5 minutes at 5,000 RPM. Discard the mixture.Repeat the process.

i. Prepare a fresh solution of sterile buffered polyethylene glycol (34.1 ml of phosphate buffer and 11.8 g of polyethylene glycol) in a 100 ml glassstoppered sterile volumetric flask. Add 25 ml of the washed spore mixture and bring to volume with steriledistilled water. Shake vigorously. Pour the mixture

into a coated Virtis jar and homogenize for 5 minutes at5,000 RPM.

j. Dispense the mixture equally into four sterilecentrifuge tubes and centrifuge in a swinging bucketrotor at 1,500 x G (3,000 RPM in H-4 Rotor in Sorvall

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RC5C) for 2 minutes at room temperature.

k. A two-phase system with an interface will be formed inthe centrifuge tube. Being careful not to disturb ordisperse the interface layer, transfer the sporecontaining, upper phase using a 10 ml pipette to a

second set of sterile centrifuge tubes.


tube with 20 ml sterile distilled water and pool thecontents of all tubes into a sterile container.


for 20 minutes at 5°°C. Repeat the process five timesafter decanting the supernatant and re-suspending the pellet in 20 ml of sterile distilled water.


beads. Store the stock suspension at 35-40°°F (2-4.4°°C).(Properly preserved stock spore suspension may be used indefinitely).

33.343 Preparation of Working Spore Suspension of B. mega t e r i um

a. To determine the number of spores/ml in each new sporestock suspension, prepare tenfold serial dilutions (10

-2-

10-10) of the suspension using Butterfield's Phosphate

Buffer. (Pipet 1.0 ml of well mixed spore stocksuspension (use vortex mixer) into 9 ml buffer and then make serial dilutions up to 10

-10.).

b. Using separate pipettes, pipette 1.0 ml of each dilutioninto triplicate 100 x 15 mm plates.


1°°C) into each plate. Mix by swirling or tilting platesto disperse the inoculum evenly throughout the agar.

Incubate for 48 h at 37 + 1°°C.

d. Count colonies (30-300) in triplicate plates on a QuebecColony Counter. Record and average the number ofcolonies/ml for each dilution. Determine the number ofcolony forming units (cfu)/ml of the stock solution.



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Concentration Desired of stock spore Dilution concentration ofsuspension = factor X working spore

(cfu/ml) suspension (cfu/ml)

Example:


Desired concentration of working spore suspension= 1 x 10

6spores/ml:

(1 x 109cfu/ml) = (x) (1 x 10

6cfu/ml)

(1 x 109cfu/ml) = x

(1 x 106cfu/ml)

x = 1000

In this example, the stock spore suspension must bediluted 1:1000 (1 part stock spore suspension plus 999 parts diluent) in 50% ethyl alcohol to prepare the 1 x10

6spore/ml concentration.

33.344 Packaging of B. mega t e r i um Spore Suspension a. Dispense 4.0 ml of the working spore suspension (1 x 10

6

cfu/ml in 50% ethyl alcohol) into each (51 x 15 mm)clear glass vial with leak-proof screw caps.

b. After capping the vials, seal with shrink-seal, orequivalent material to prevent leakage or dehydration.

c. Label the vials with the following information on atransparent mylar pressure sensitive label, orequivalent:

i. "CAST Spores"ii. B. mega t e r i um ATCC 9885iii. Lot Numberiv. Packaging Date

NOTE: Under FSIS contract, CAST spores are produced commercially. After these spores meet all quality

control specifications they are used in slaughter plants.

33.35 Preparation of CAST Plates

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a. Weigh and add 38 g of Mueller-Hinton Agar powder to 1 Ldistilled water. Heat while stirring and bring to a boil. Sterilize the medium at 121

oC for 15 minutes and

then mix it thoroughly. Allow agar to cool to 48°°C in a water bath. Continue mixing during cooling and

dispensing.

b. Using a sterile agar delivery system, deliver 6.0 ml ofthe agar to each 60 x 15 mm plate. Distribute the agarevenly to cover entire surface of the plate. Allow theagar to harden on a flat, level surface.

c. Label the lid of each plate using a label containing thefollowing information:

i. "CAST PLATE"ii. Lot Numberiii. Expiration Date


NOTE: Under FSIS contract, CAST plates are produced commercially. After these plates meet all quality


33.36 Performing the CAST Test


a. Assure that the samples are received at a temperature of

4°°C or below.

b. Identify samples according to standard operating procedures.

NOTE: CAST test should only be used on kidney tissue of bobveal calves.

33.362 Procedure


b. Open a sterile cotton swab pack, remove one swab, and

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insert the sharp end of the swab about 1/2" to 3/4" intothe kidney tissue.


d. Reverse the swab and insert the cotton tip into thetissue opening, twisting to make sure that the cottontip is in good contact with the macerated tissue.

e. Allow swabs to remain in the tissues for a minimum of 30 minutes.

f. Allow refrigerated plates to warm to room temperaturefor about 10 minutes before streaking. Check each platefor absence of contamination, cracking of agar ordryness.

g. Lift the plate cover slightly and mark an "X" reference mark on the outer side wall of the plate. Place thecovered plate bottom side down on the work place surface with the reference mark at 12 o' clock position. With afine-tip permanent marking pen, start at the "x" and draw a line across the bottom of the plate dividing itinto two equal sections.

h. Check for seal integrity of vials containing spores.

i. Shake the B. mega t e r i um spore vial and dip a sterileswab in the solution. Gently touch the swab to the sideof the vial to remove excess fluid. Replace the screw

cap on the vial.

j. Streak the surface of the agar plates with the swab from a point marked on the side of the plate moving up and down and from left to right. Turn the plate 1/4 turnand streak again.

k. Repeat this streaking process 2 more times. Finally turnthe plate 1/2 turn and streak. (Use a separate swab foreach plate)

l. Place a neomycin 5 µµg disc on the agar surface near the

vertical line on a plate.

m. Remove the swab from the tissue, break the shaftapproximately two inches from the swab end.

NOTE: If the swabs appear dry, reinsert them in the tissue

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and squeeze the tissue around the swab to absorbtissue fluids. For small portions of dry muscletissue, moisten swab with distilled water prior to

insertion.

n. Gently place the swab on the surface of the plate with

the broken end of the shaft near the neomycin 5 µµg disc making sure not to break the agar surface. Make surethe swab has uniform contact with the agar.

NOTE: Swabs from two different tissues or carcasses may be placed on each plate provided they are properlyidentified as to location on the plate.

If two tissue swabs are used per plate, place cottontips in a "rabbit ears" configuration (Fig. 2).


o. Incubate the plates upright at 44 + 1°°C for 16-18 h.

p. Store samples under refrigeration until the test iscompleted.


a. Remove plates from the incubator and remove the swabs.

b. Measure the zone of inhibition around the N5 disc with a mm ruler. The ZI should be 24-29 mm wide. If the ZI isnot 24-29 mm in width, the test is inconclusive and

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should be repeated.

c. Observe the plates for inhibition of B. mega t e r i um growth surrounding the swabs.

i. If a zone of inhibition is observed, the test is

positive. Measure the width of the zone and record results.

ii. If no zone of inhibition is observed, the test isnegative. Record the result.

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Johnston, R. W., R. H. Reamer, E. W. Harris, H. G. Fugate, and B. Schwab. 1981. A new screening method for the detection ofantibiotic residues in meat and poultry tissues. J. Food Prot.44:828-831.



United States Department of Agriculture. 1982. The shelfstable swab test system for detecting antibiotic residues in

tissues. Laboratory Communication No. 31. Food Safety and Inspection Service, S&T, Microbiology Division, Washington,D.C.

United States Department of Agriculture. 1984. Performing theCalf Antibiotic and Sulfa Test. Food Safety and InspectionService, Administrative Management, Training and DevelopmentDivision, College Station, TX.

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PART C

33.4 TENTATIVE CONFIRMATION OF CAST RESULTS FOR SULFONAMIDERESIDUES IN MEAT AND POULTRY TISSUE

B. P. Dey, Sandra L. Kamosa and Clarence A. White

33.41 Background

The Calf Antibiotic and Sulfa Test (CAST) is presently being used for detecting sulfonamide residues in bob veal calves. The test as performed by inspectors is as follows: a sterile cotton tipped applicator (swab) is inserted into the kidney sample of an animaland left for 30 minutes to absorb tissue fluids. A Bac i l l us mega t e r i um spore suspension is applied to CAST agar plates by asterile swab. The swab from the kidney is then placed on the agar

plate and incubated at 44°°C for 16-24 h. The plate is thenexamined for a zone of inhibition (ZI) around the swab. In the

case of an 18 mm or greater zone of inhibition, the carcass issubjected to further laboratory analysis. The muscle, liver and kidney tissues from the suspect carcass are sent to thelaboratories for analysis. This procedure describes a modified CAST method with sensitivity equal or better than commercial CASTfor verifying field results in 5-6 h with inclusion of another plate for confirming the presence of sulfonamide residues insuspected samples at the same time.

33.42 Equipment, Reagents and supplies

33.421 Equipment


SS-34 and Sorvall Swinging Bucket Rotor HB-4 or

equivalent. Must operate at 20,000 x G at a constant 5°°Cand also with a swinging bucket rotor at 1,500 x G atroom temperature or equivalent.


f. Incubators: one capable of maintaining a constant 37°°Cand the other 44 ± 1

oC

g. Precision water bath (48 ± 1oC) with cover (Model 183) or


Cat. No. 07-906)

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33.422 Reagents

a. Distilled water:

The distilled water must be prepared using an all glass

still (Corning Megapure 6L or equivalent) and stored ina glass or any acceptable reservoir which is not a partof the system. All spore lots must be prepared usingglass distilled water. * Do not use deionized water.


Dissolve 306.9 g of K 2HPO4 and 168.6 g KH2PO4 in 1 Ldistilled water. If necessary, adjust pH by dropwise

addition of 0.1 N HCl or NaOH. Sterilize at 121°°C for 15

minutes or filter through a 0.2 µµ m filter.




Warner-Graham Company, 160 Church Lane, Cockeysville, MD21030). For a 50% solution, mix 1 part of ethylalcohol with 1 part glass distilled water. Prior to use,

filter sterilize through a 0.2 µµ m filter.



to use.

e. Bromcresol Purple (0.04%) solution. Dissolve 0.1 gBromcresol Purple dye with 18.5 ml of 0.01 N sodium hydroxide, add 231.5 ml of distilled water.

f. Bacto-Dextrose (Difco, Detroit, MI; Cat. No. 0156-17-4)

g. p-aminobenzoic acid (Fisher Scientific Co. NJ;Cat. No A-41-70522)

h. Butterfield's Phosphate Buffer, sterile

33.423 Supplies


volumetric flasks

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*Resins of some systems produce quaternary ammonium compounds which interfere with the analysis.

d. Sterile centrifuge tubes, 40 ml (Nalgene 3118 orequivalent)

e. Sterile pipettes graduated to the tip, 10 and 1 mlf. Sterile, clear glass vials 51 x 15 mm with deep seated screw caps

g. Pressure sensitive labels not to exceed 2" x 1/2"h. Acetate shrink-wrap material for sealing 51 x 15 mm

glass vials or equivalent closure materiali. Forcepsj. Permanent marking pen

k. Antibiotic discs: Neomycin - 5 µµgl. Sterile cotton swabs on hollow plastic tubes m. Sterile, plastic 60 X 15 mm plates (Falcon Cat. # 1007

or equivalent)

33.43 Media

Proceed exactly as that described in Section 33.33.

33.44 Test Organism




33.442 Preparation of B. mega t e r i um Spore Suspension

Proceed exactly as that described in Section 33.342

33.443 Enumeration of B. mega t e r i um Spores in Working Suspension

Proceed exactly as that described in Section 33.343 except prepare the final spore suspension such that it contains 1x 10

7cfu/ml.

33.444 Packaging of B. mega t e r i um Spore Suspension

a. Dispense 4.0 ml of the working spore suspension(1 x 107cfu/ml in 50% ethyl alcohol) into sterile 51 x

15 mm clear, glass vials with deep seated, leak-proofscrew caps.

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c. Label the vials with the following information on atransparent mylar pressure sensitive label:

i. "CAST Spores"ii. B. mega t e r i um ATCC 9885iii. Date

NOTE: B. mega t e r i um spores (1 x 107cfu/ml) can be obtained

from EDITEK, Burlington, NC, by special order.

33.45 Preparation of Plates

33.451 Preparation of Modified CAST (M-CAST) Plates

a. Weigh and add 38 g of Mueller-Hinton Agar (Acumedia)

powder to each liter of glass distilled water. Weighand add 8 g dextrose to the mixture. Add 70 mlBromcresol Purple solution (0.04%) to the mixture. Heat while stirring and bring to boil.

b. Cool to 48oC and adjust the pH to 7.2 ± 0.1. Sterilize

at 121oC for 15 minutes and mix thoroughly. Allow the

agar medium to cool to 48oC in a water bath.

c. Continue mixing during cooling.

d. Add 1 ml of B. mega t e r i um spore suspension (1 x 107

cfu/ml) to every 100 ml of the medium and mix

thoroughly.

e. Aseptically dispense 8 ml of the seeded agar to each 100x 15 mm plate.

f. Distribute the agar evenly to cover entire surface ofthe plate. Place plates on a flat, level surface and allow the agar to harden.

g. Label the side of each plate with a marker with thefollowing information:

i. "M-CAST PLATE"ii. Date

h. Refrigerate plates in sealed double plastic bags to prevent moisture evaporation. These plates can be used for a period of 15 working days.

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33.452 Preparation of Modified CAST Plus (M-CAST+) Plates

a. Weigh and add 38 g of Mueller-Hinton Agar (Acumedia) powder to 1 L glass distilled water. Weigh and add 8 gdextrose to the mixture. Add 70 milliliters ofBromcresol Purple solution (0.04%) to the mixture. Heat

while stirring and bring to a boil.

b. Add 200 mg of p-aminobenzoic acid to the medium.

c. Cool to 48oC and adjust pH to 7.2 ± 0.1. Sterilize at

121oC for 15 minutes and mix thoroughly. Cool the medium

in a 48oC water bath.

d. Continue mixing during cooling.

e. Add 1 ml of B. mega t e r i um spore suspension (1 x 107/ml)

to every 100 ml of the medium and mix thoroughly.

f. Aseptically dispense 8 ml of the seeded agar to each 100x 15 mm plate.

g. Distribute the agar evenly to cover entire surface ofthe plate. Place plates on a flat surface and allow theagar to harden.

h. Label the side of each plate with a marker with thefollowing information:

i. "M-CAST+ PLATE"ii. Date

i. Refrigerate plates in sealed plastic (Ziplock®) bags to prevent moisture evaporation. These plates can be used for a period of 15 working days.

33.46 Performing the Test



4°°C or below.

b. Identify samples according to standard operating

procedures.

33.462 Procedure

a. Allow frozen samples to thaw completely at room temperature for a sufficient period of time such that

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ice crystals are no longer present within the sample.

b. Open a sterile cotton swab pack, remove both swabs, and insert the sharp end of the swabs shaft 1/2" to 3/4"into the kidney tissue.

c. Move the swab shafts back and forth several times to macerate the tissue, disrupting tissue cells and releasing tissue fluid. Remove the swab shafts.

d. Reverse the swabs and insert the cotton tips into thetissue opening, twisting to make sure that the cottontip is in good contact with the macerated tissue.

e. Allow swabs to remain in the tissue for a minimum of 30 minutes.

f. Leave refrigerated plates (M-CAST and M-CAST+) at room temperature for about 20-30 minutes to warm up. Discard

plates which are contaminated, dried or cracked.

g. Place a neomycin 5 µµg (N5) disc and a sulfamethazine 2

µµg (S2) disc on separate M-CAST and M-CAST+ plates(control plates) in use each day the test is performed. Make sure that the distance between the two discs is 35-40 mm.

h. Remove the swabs from the tissue, break the shaftsapproximately two inches from the swab end.

NOTE: If the swabs appear dry, reinsert them in the tissue

and squeeze the tissue around the swab to absorb tissuefluids. For small portions of dry muscle tissue, moisten swab with distilled water prior to insertion.

i. Gently place one of the swabs on an M-CAST plate and the other swab on an M-CAST+ plate making sure not to

break the agar surface. Make sure the swab has uniform contact with the agar.

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NOTE: Properly identified, four (4) swabs from 4 samples can be placed on each plate provided the cotton tip end ofone lies next to the shaft of another as shown in

Fig. 1.

M-CAST Plate M-CAST+ Plate

Fig. 1 . Placement of swabs on M-CAST and M-CAST+ plate

j. Incubate plates with sample swabs and the control discs

(N5 and S2) upright at 44 + 1°°C for 5-6 h.

k. Refrigerate sample until the test is complete.


a. Remove the plates from incubator and remove swabs.

b. Measure the ZI around the N5 and S2 discs on the control plates with a mm ruler or by a zone reader. The N5 zoneshould measure between 20-26 mm on both M-CAST and M-CAST+ plates. There should be a 16-19 mm zone by the S2disc on the M-CAST plate only, where as there will be nozone by the S2 disc on the M-CAST+ plate. If theobserved ZI are not in agreement with the above, repeatthe test.

c. Measure the zone of inhibition surrounding each swabcorresponding to a sample on each plate (from right toleft).

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NOTE: It is essential to read test results within 6 h. Asthe inhibitory effect by bacteriostatic drugs such assulfonamides diminishes, organisms temporarilyinhibited recover over time causing reduction in the

zone of inhibition as incubation time increases.

d. i. Samples with sulfonamide residue appear asillustrated below:

M-CAST plate: Zone of inhibition (Samples B and C) M-CAST+ plate: No zone of Inhibition (Samples B and C)


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ii. Samples free of sulfonamide residue, but containingantibiotics, appear as illustrated below:

M-CAST plate: Zone of inhibition (Samples A and D) M-CAST+ plate: Zone of Inhibition (Samples A and D)


33.48 Quality Control

a. Test organism must be evaluated for purity and proper biochemical patterns.

b. Freshly prepared plates must be tested with the N5 and S2 discs to assure proper performance.

c. Plates must not be used for more than 15 working days past preparation.

d. Extreme caution should be taken in adding para-amino benzoic acid because the chemical at a higherconcentration than the recommended level is toxic to thetest organism.

e. New chemicals/reagents and agar should be checked to

assure quality.

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Dey, B. P., S. Kamosa, and Clarence White. 1995. Tentativeconfirmation of CAST results for sulfonamide residues in meatand poultry tissue. Laboratory Communication No. 78. USDA,Food Safety and Inspection Service, S&T, Microbiology

Division, Washington, D.C.

Johnston, R. W., R. H. Reamer, E. W. Harris, H. G. Fugate, and B. Schwab. 1981. A new screening method for the detection

of antibiotic residues in meat and poultry tissues. J. Food Prot. 44:828-831.



United States Department of Agriculture. 1982. The shelfstable swab test system for detecting antibiotic residues intissues. Laboratory Communication No. 31. Food Safety and Inspection Service, S&T, Microbiology Division, Washington,D.C.

United States Department of Agriculture. 1984. Performing theCalf Antibiotic and Sulfa Test. Food Safety and InspectionService, Administrative Management, Training and Development

Division, College Station, TX.

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PART D

33.5 DETECTION OF ANTIMICROBIAL RESIDUE BY FAST ANTIMICROBIALSCREEN TEST (FAST)

B. P. Dey, Clarence A. White and Nitin H. Thaker

33.51 Introduction

The Fast Antimicrobial Screen Test (FAST), an in-plant screen test, was developed in 1989 to improve the capability of the AntibioticResidue Detection Program. FAST has higher sensitivity and candetect a wider range of antibiotics and sulfonamides than STOP and CAST. The test has been introduced in 50 bovine slaughterestablishments. It is also being evaluated in swine species. Iffound suitable in both bovine and swine, it may be used in allspecies of food animals for detecting antimicrobial residues.Besides improving efficiency, this test would be used uniformly for

detecting antibiotic and sulfonamide residues in food animalcarcasses.

The test as performed by inspectors is as follows: a sterile cottontipped applicator (swab) is inserted into the kidney sample of ananimal and left for 30 minutes to absorb tissue fluids. The agar plates are surface streaked with Bac i l l us megat er i um sporesuspension on a sterile cotton swab. The swab from the kidney isremoved, broken as close to the cotton tip as possible, and placed

onto the agar plate and incubated at 44°°C. The plate is examined for a zone of inhibition (ZI) around the swab at 6 and 18 h. Inthe case of inhibition at 6 h, the plate is further examined at 18h for confirmation. If there is clear inhibition, muscle, liverand kidney tissues from the suspect carcass are collected and further analyzed for confirmation at an FSIS laboratory. When noinhibition is seen at 6 h, the carcass is free of antimicrobialresidues at detectable levels. The test allows screening and releasing a large number of residue free carcasses within a workshift. 33.52 Equipment, Reagents and Supplies

33.521 Equipment

a. Laminar Flow Hood or equivalent clean room

b. Sorvall RC5C Refrigerated Centrifuge, Sorvall RotorSS-34 and Sorvall Swinging Bucket Rotor HB-4 or


c. Virtis homogenizer, Model 60K or equivalent

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d. Sterile Virtis jarse. Vortex mixer or equivalent

f. Incubators: one capable of maintaining a constant 35°°C

and the other at 44 ± 0.5°°Cg. Precision water bath (with cover (Model 183) or

equivalent

h. Quebec Colony Counter or equivalenti. Fisher-Lilly Antibiotic Zone Reader (Fisher Cat.#

07-906)

33.522 Reagents

a. Distilled water:

The distilled water must be prepared using an all glassstill (Corning Megapure 6L or equivalent) and stored ina glass or any acceptable reservoir which is not a partof the system. All spore lots must be prepared usingglass distilled water. * Do not use deionized water.



addition of 0.1 N HCl or NaOH. Sterilize at 121°°C for 15

minutes or filtering through a 0.2 µµ m filter.




Warner-Graham Company, 160 Church Lane, Cockeysville, MD21030). For a 50% solution, mix 1 part of ethyl alcohol with 1 part glass distilled water. Prior to use, filter

sterilize through a 0.2 µµ m filter.



to use.


f. Bacto-Dextrose (Difco, Detroit, MI; Cat. # 0156-17-4) orequivalent

g. Butterfield's Phosphate Buffer, sterile

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* Resins of some systems produce quaternary ammonium compounds which interfere with the analysis.

33.523 Supplies





glass vials or equivalent closure material

i. Forcepsj. Permanent marking pen

k. Antibiotic discs: Neomycin - 5 µµgl. Sterile cotton swabs on hollow plastic tubes m. Sterile, plastic 60 X 15 mm plates (Falcon Cat. No. 1007

or equivalent)

33.524 Media

a. Brain Heart Infusion broth (BBL or equivalent);reconstitute according to manufacturer's directions,dispense 10 ml/tube and sterilize (121

oC for 15 min).

b. Blood agar plates (Columbia Blood Agar Base, 5% HRBC).

c. A-K Sporulating Agar No. 2.

i. Agar slants - reconstitute A-K Sporulating Agar No.2 according to manufacturer's directions with extra0.5% purified Agar (Difco or equivalent), sterilize by autoclaving at 121

oC for 15 minutes and prepare

slants.

ii. Roux bottles - add 300 ml reconstituted A-K Sporulating Agar No. 2 with extra 0.5% purified

agar. Sterilize (121

o

C for 15 minutes) and allow medium to harden in Roux bottles placed in ahorizontal position.

d. Mueller-Hinton Agar (Acumedia Manufacturers Inc.,Baltimore, MD); reconstitute according to manufacturer's

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directions, dispense as desired and sterilize (121oC for

15 minutes).

33.53 Test Organism

Bac i l l us megat er i um ATCC 9885 (American Type Culture

Collection, Rockville, MD)


a. Reconstitute a lyophilized culture in Brain Heart

Infusion broth and incubate at 37°°C for 18 h. Streak blood agar plates with the broth culture and incubate

plates at 37°°C for 18 h. After incubation check forculture purity.

b. Streak the culture for isolation onto two Columbia Agar plates with 5% defibrinated horse blood. Incubate at

37°°C for 18 h.

c. Prepare a Gram stain of three well isolated colonies. All cultures should be Gram positive.

d. Stain a drop of the spore suspension with malachitegreen and counterstain with carbol-fuchsin solution.The spores will appear green, whereas the vegetativecells will appear red or pink.

e. Use one Columbia Agar plate with 5% defibrinated horse blood from the culture to test for presence of catalase. Bac i l l us are catalase positive.

f. Use the other plate to check biochemical characteristicsof the culture by inoculating O-F glucose, Voges-

Proskauer, and mannitol broths. Incubate at 35°°C for18 h.

The biochemical patterns of B. mega t e r i um should agree with the following chart:

Catalase Gram stain

Sporeforming

O-Fglucose

Voges-Proskauer

Mannitol

+ + + O - A

(+) = positive; (-) = negative; (F) = fermentative;(O) = oxidative; (A) = acid.

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g. If the organism does not meet all the above criteria,replace with a new ATCC culture of the test organism.

33.532 Preparation of Bac i l l us megat er i um Spore Suspension

a. After the culture meets all biochemical criteria, pickseveral well isolated colonies from the plates and

streak A-K Sporulating Agar No. 2 slants (one per Roux bottle) and incubate the slants at 37°°C for 18 h.

b. Add 4-6 sterile glass beads and 2-3 ml sterile distilled water to each slant and gently shake for 2 minutes todislodge organisms.

c. Aseptically transfer the slant suspensions to a Roux bottle containing A-K Sporulating Agar No. 2 and spread with the help of sterile glass beads. Multiple cultures may be prepared and pooled.

d. Incubate the Roux bottles horizontally for 18 h at 37°°C

and then at room temperature for the remainder of 1 week(6 days).

e. Harvest the growth from the Roux bottles by adding 20-30sterile glass beads and approximately 25 ml of steriledistilled water per bottle. Gently agitate each bottleto dislodge bacterial growth. (Care must be taken notto break the agar during harvesting).


tubes in boiling water (100°°C) for 10 min.


i. Centrifuge at 5°°C for 20 minutes at 20,000 x G.ii. Pour off supernatant.iii. Resuspend the pellet in 20 ml sterile distilled

water.iv. Repeat Steps i, ii and iii two more times.


Mix 34.1 ml of sterile phosphate buffer and 11.8 g of polyethylene glycol in a 100 ml glass stoppered sterilevolumetric flask and shake vigorously. Bring to volume with sterile distilled water. Pour the mixture into a

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Virtis jar and place the jar on the homogenizer. Blend for 5 minutes at 5,000 RPM. Discard the mixture.Repeat the process.

i. Prepare a fresh solution of sterile buffered polyethylene glycol (34.1 ml of phosphate buffer and

11.8 g of polyethylene glycol) in a 100 ml glassstoppered sterile volumetric flask. Add 25 ml of the washed spore mixture and bring to volume with distilled water. Shake vigorously. Pour the mixture into acoated Virtis jar and homogenize for 5 minutes at 5,000RPM.

j. Dispense the mixture equally into four sterilecentrifuge tubes and centrifuge in a swinging bucketrotor at 1,500 x G (3,000 RPM in H-4 Rotor in SorvallRC5C) for 2 minutes at room temperature.

k. A two-phase system with an interface will be formed in

the centrifuge tube. Being careful not to disturb ordisperse the interface layer, transfer the sporecontaining, upper phase using a 10 ml pipette to asecond set of sterile centrifuge tubes.


tube with 20 ml sterile distilled water and pool thecontents of all tubes into a sterile container.


for 20 minutes at 5°°C. Repeat the process five timesafter decanting the supernatant and re-suspending the pellet in 20 ml of distilled water.


beads. Store the stock suspension at 35-40°°F (2-4.4°°C).(Properly preserved stock spore suspension may be used indefinitely).

33.533 Enumeration of B. mega t e r i um Spores in Stock Suspension

a. To determine the number of spores/ml in each new sporestock suspension, prepare tenfold serial dilutions (10

-

2-10

-10) of the suspension using Butterfield's Phosphate

Buffer. (Pipet 1.0 ml of well mixed spore stocksuspension (use vortex mixer) into 9 ml buffer and then make serial dilutions up to 10

-10.).

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b. Using separate pipettes, pipette 1.0 ml of each dilutioninto triplicate 100 x 15 mm plates.


1°°C) to each plate. Mix by swirling and tilting platesfor even dispersal of the inoculum. Incubate the plates

at 37 + 1°°C for 48 h.

d. Count colonies (30-300) of triplicate plates on a QuebecColony Counter. Record and average the number ofcolonies/ml in each dilution. Determine the number ofcolony forming units (cfu)/ml of the stock solution.




Concentration Desired of stock Dilution concentration ofsuspension = factor X working spore(cfu/ml) suspension (cfu/ml)

Example:


Desired concentrationof spore suspension = 1 x 10

6spores/ml:

(1 x 10

9

cfu/ml) = (x) (1 x 10

6

cfu/ml)

(1 x 109cfu/ml) = x

(1 x 106cfu/ml)

x = 1000

In this example, the stock spore suspension must be diluted 1:1000 (1 part stock spore suspension plus 999 partsdiluent) in 50% ethyl alcohol to prepare the 1 x 10

6

spore/ml concentration.

33.534 Packaging of B. mega t e r i um Spore Suspension (Field Use)

a. Dispense 4.0 ml of the working spore suspension (1 x 106

cfu/ml in 50% ethyl alcohol) into sterile (15 mm diameter x 51 mm height) clear, glass vials with deepseated, leak-proof screw caps.

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NOTE: Under FSIS contract, FAST spores are produced commercially. After they meet all quality control

specifications they are used in slaughter plants.



i. "FAST Spores"ii. B. mega t e r i um ATCC 9885iii. Date

33.54 Preparation FAST Plates (Used in the Plant)

a. Weigh and add 38 g Mueller-Hinton Agar (Acumedia) powderto 1 L glass distilled water. Add 7 g dextrose to the mixture. Add 70 ml Bromcresol Purple solution (0.04%)to the mixture. Heat while stirring and bring to boil.After sterilizing at 121

oC for 15 minutes, mix the

medium thoroughly, and cool it in a 48oC water bath.

Continue mixing during cooling and dispensing.

b. Using a sterile agar delivery system, deliver 6.0 mlagar to each 60 x 15 mm plate. Distribute the agarevenly to cover entire surface of the plate. Place plates on flat level surface and allow the agar toharden.

c. Label the lid of each plate using a label, containingthe following information:

i. "FAST PLATE"ii. Lot Numberiii. Expiration Date


NOTE: Under FSIS contract, FAST plates are produced commercially. After these plates meet all quality


33.55 Performing the FAST Test

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33.551 Tissue Sample and Conditionsa. The kidney is the target tissue for FAST

b. The kidney and other tissue samples should be received

at 4°°C or below and identified properly.

33.552 Procedure


b. Open a sterile cotton swab pack, remove one swab, and insert the sharp end of the swab shaft about 1/2" to3/4" into the kidney tissue.


d. Reverse the swab, insert the cotton tip into the tissueopening and twist to make sure that the cotton tip is incontact with the macerated tissue.

e.* Allow swabs to remain in the tissues for a minimum of 30 minutes.

f. Allow refrigerated plates to warm to room temperaturefor about 10 minutes. Check plates for contamination,cracking or dryness of agar.

g. Lift the plate cover slightly and mark an "X" reference mark on the outer side wall of the plate. Place thecovered plate bottom side down on the work place surface with the reference mark at 12 o' clock position. With afine-tip permanent marking pen, start at the "x" and draw a line across the bottom of the plate dividing itinto two equal sections.

h. Shake the B. mega t e r i um spore vial and dip a sterileswab in the solution. Gently touch the swab to the sideof the vial to remove excess fluid. Replace the screwcap on the vial.

i. Streak the surface of the agar plates with the swab from a point marked on the side of the plate moving up and down and from left to right. Turn the plate 1/4 turnand streak again.

j. Repeat this streaking process 2 more times. Finally turn

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the plate 1/2 turn and streak. (Use a separate swab foreach plate)

* NOTE: If the swabs appear dry, reinsert them in the tissue

and squeeze the tissue around the swab to absorbtissue fluids. For small portions of dry muscle

tissue, moisten swab with distilled water prior toinsertion.

k. As a control, place a neomycin (N5) 5 µµg disc one halfinch from the edge of the plate on the agar surface.

l. Remove swab from the tissue and break the shaftapproximately two inches from the swab end.

m. Gently place the swab on the agar without breaking thesurface. Make sure that swab has uniform contact withthe surface.

NOTE: Two (2) swabs from two samples can be placed on one plate asillustrated below in (Figure 1).


n. Incubate the plates upright at 44 ± 0.5°°C for 6 h, up toa maximum of 16-18 h.

o. Store samples in refrigerator until the test is

completed.


a. Remove plates from the incubator and remove the swabs.

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b. Measure the ZI around the N5 disc with a mm ruler or azone reader. The zone should be 20-26 mm wide. If not,the test must be repeated.

c. Observe the plates for inhibition of B. mega t e r i um

growth surrounding the swabs (Figure 2).

i. Samples with Antimicrobial Chemical Residue

Zone of inhibition around swab "A": Sample A may containantimicrobial residue, and must be subjected toconfirmatory testing procedures.

ii. Samples without Antimicrobial Chemical Residue

No Zone of Inhibition around swab "B" : Sample B is

free of antimicrobial residue.

Figure 2. Inhibition of microorganism by swab

33.57 Quality Assurance

a. The FAST plates can be stored at room temperature protected from extremes of heat, cold and moisture.

b. Store spore suspensions under refrigeration condition with cap tightly closed.

c. Store neomycin disc vial in a plastic bag inrefrigerator.

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d. Do not use outdated plates, spores or N5 discs.e. Shake the spore vial for even dispersal of spores.

f. Check plates before use for contamination, cracking ordrying of agar.

g. Do not to break the agar surface while placing theneomycin disc and the swab.

h. Allow swabs to remain in the tissues for 30 minutes.

i. Read plates any time after 6 h of incubation, up to a maximum of 18 h.

j. Make sure that the incubator temperature is 44 ± 0.5°°C.

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Bright, S. A., S. L. Nickerson, and N. H. Thaker. 1989.Fast Antibiotic Screen Test-A preliminary evaluation. Proc. AOAC Ann. Mtg. St.Louis., MO.

Dey, B. P., and C. A. White. 1995. FAST AntimicrobialScreen Test (FAST) for antimicrobial residue detection in meat. Laboratory Communication No. 79. USDA, Food Safety and Inspection Service, S&T, Microbiology Division, Washington,D.C.

Johnston, R. W., R. H. Reamer, E. W. Harris, H. G. Fugate,and B. Schwab. 1981. A new screening method for the detectionof antibiotic residues in meat and poultry tissues. J. Food Prot. 44: 828-831.

Kramer, J., G. G. Carter, B. Arret, J. Wilner, W. W. Wright,and A. Kirshbaum. 1968. Antibiotic Residues in Milk, Dairy

Products and Animal Tissues: Methods, Reports and Protocols.Item 344-837 (4008). Food and Drug Administration, GovernmentPrinting Office, Washington, DC.

United States Department of Agriculture. 1982. The shelfstable swab test system for detecting antibiotic residues intissues. Laboratory Communication No. 31. Food Safety and Inspection Service, S&T, Microbiology Division, Washington,D.C.

United States Department of Agriculture. 1994. Fast Antimicrobial Screen Test (FAST): For Detection of Antibioticand Sulfonamide Residues in Livestock Kidney Tissue. A Self-

Instructional Guide. 1994. Food Safety and InspectionService, Administrative Management, Human Resource and Development Division, College Station, TX.

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PART E

33.6 EVALUATION OF ANTIMICROBIAL RESIDUES IN MEAT AND POULTRYTISSUE BY A MODIFIED FAST ANTIMICROBIAL SCREEN TEST(M-FAST)

B. P. Dey, Richard H. Reamer and Sandra L. Kamosa

33.61 Introduction

The Fast Antimicrobial Screen Test (FAST) developed in 1989, is presently being used in selected bovine slaughter plants. It isexpected that the test will be used universally in plants for thedetection antimicrobial residue in all species of food animalcarcasses. The test as performed by inspectors is as follows: asterile cotton tipped applicator (swab) is inserted into the kidneysample of an animal and left for 30 minutes to absorb tissuefluids. The agar plates are surface streaked with Bac i l l us

mega t e r i um spore suspension using a sterile cotton swab. The swabfrom the kidney is removed, broken as close to the cotton tip as

possible, and placed onto the agar plate and incubated at 44°°C.The plate is examined for a zone of inhibition (ZI) around the swabat 6 and 18 h. In the case of inhibition at 6 h, the plate isfurther examined at 18 h for confirmation. If there is noinhibition at 6 h, the carcasses is released. The test allowsscreening and releasing a large number of residue free carcasses within a work shift. If there is clear zone of inhibition, muscle,liver and kidney tissues from the suspect carcass are collected and further analyzed for confirmation at an FSIS laboratory. The method described here is a modified FAST procedure for verifyingfield test results in 6 h with sensitivity equal to the commercialFAST at comparable incubation times.

33.62 Equipment, Reagents and Supplies

33.622 Equipment


SS-34 and Sorvall Swinging Bucket Rotor HB-4 or



f. Incubators: one capable of maintaining a constant 37°°C

and the other 44 ± 0.5°°Cg. Precision water bath (48 ± 1

oC) with cover (Model 183) or

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Cat. No. 07-906)

33.623 Reagents

a. Distilled water:

The distilled water must be prepared using an all glassstill (Corning Megapure 6L or equivalent) and stored ina glass or any acceptable reservoir which is not a partof the system. All spore lots must be prepared usingglass distilled water. * Do not use deionized water.



addition of 0.1 N HCl or NaOH. Sterilize at 121°°C for 15 minutes or filtering through a 0.2 µµ m filter.




Warner-Graham Company, 160 Church Lane, Cockeysville, MD21030). For a 50% solution, mix 1 part of ethyl alcohol with 1 part glass distilled water. Prior to use, filter

sterilize through a 0.2 µµ m filter.



to use.


f. Dextrose (Bacto Dextrose-Difco, Detroit, MI; Cat. No.0156-17-4) or equivalent.

g. Butterfield's Phosphate Buffer, sterile

33.624 Supplies

a. Sterile Roux bottles. b. Sterile glass beads, 4 mm diameter

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*Resins of some systems produce quaternary ammonium compounds which interfere with the analysis.

c. Sterile 100 ml graduated glass stoppered cylinders orvolumetric flasks

d. Sterile centrifuge tubes, 40 ml (Nalgene 3118 orequivalent)e. Sterile pipettes graduated to the tip, 10 and 1 ml.f. Sterile, clear glass vials 51 x 15 mm with deep seated


glass vials or equivalent closure materiali. Forcepsj. Permanent marking pen

k. Antibiotic discs: Neomycin (N5)- 5 µµgl. Sterile cotton swabs on hollow plastic tubes m. Sterile, plastic 60 X 15 mm plates (Falcon Cat. No. 1007

or equivalent)

33.63 Media


33.64 Test Organism




33.642 Preparation of B. mega t e r i um Spore Suspension


33.643 Enumeration of B. mega t e r i um Spores in Stock Suspension

Proceed exactly as that described in Section 33.533 except prepare the final spore suspension such that it contains 1x 10

7cfu/ml.

33.644 Packaging of B. mega t e r i um Spore Suspension

a. Dispense 4.0 ml of the working spore suspension(1 x 10

7cfu/ml in 50% ethyl alcohol) into sterile

(51 x 15 mm) clear, glass vials with deep seated,leak-proof screw caps.

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NOTE: B. mega t e r i um spore (1 x 107cfu/ml) can be obtained

by special order from EDITEK, Burlington, N.C.


i. "FAST Spores"ii. B. mega t e r i um ATCC 9885iii. Date

33.65 Preparation of Plates

a. Weigh and add 38 g Mueller-Hinton Agar (Acumedia) powderto 1 L glass distilled water. Add 8 g dextrose to the mixture. Add 70 ml Bromcresol Purple solution (0.4%) to

the mixture. Heat while stirring and bring to boil.Cool (48

oC waterbath). Adjust the pH to 7.2 ± 1. After

the medium has been sterilized at 121oC for 15 minutes,

mix the medium thoroughly.

b. Keep mixing the medium while cooling in a 48oC water

bath.

c. Add 1 ml of B. mega t e r i um spore suspension (1 x 107/ml)

to every 100 ml of the medium and mix thoroughly.

d. Aseptically dispense 8 ml of the seeded agar to each100 x 15 mm plate.

e. Distribute the agar evenly over the entire plate. Place plate on a flat, level surface and allow agar to harden.

f. Label the lid of each plate using a label containing thefollowing information:

i. "M-FAST PLATE"ii. Expiration Date

g. Refrigerate plates in sealed double plastic bags to prevent moisture evaporation. These plates can be used for a period of up to 15 working days.

33.66 Performing the Test



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4°°C or below.

b. Identify samples according to standard operating procedures.

NOTE: FAST test is used on the kidney tissue of all bovine

species where implemented.

33.662 Procedure


b. Open a sterile cotton swab pack, remove one swab, and insert the sharp end of the swab shaft about 1/2" to3/4" into kidney tissue.

c. Move the swab shaft back and forth several times to

macerate the tissue, disrupting tissue cells and releasing tissue fluid. Remove the swab shaft.

d. Reverse the swab, insert the cotton tip into the tissueopening and twist to make sure that the cotton tip is incontact with the macerated tissue.

e. Leave the swab in the tissues for a minimum of 30 minutes.

f. Allow the plates to warm at room temperature for about20 minutes. Check plates for contamination, crackingand dryness of agar.

g. As a positive control place a neomycin 5 µµg (N5) disc inthe center of a plate from the same batch used in theanalysis.

h. Remove the swab from the tissues, break the shaftapproximately two inches from the swab end.

NOTE: If a swab appears dry, reinsert and squeeze the tissuearound the swab to absorb fluid. For a dry muscletissue, moisten the swab with distilled water prior toinsertion.

i. Place the swab on the agar surface gently with uniform contact with the surface.

NOTE: Properly identified, four (4) swabs from 4 samples can

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be placed on each plate provided the cotton tip end ofone lies next to the shaft of another as shown in

Fig. 1.

j. Incubate the plates upright at 44 + 0.5°°C for 6 hours, upto a maximum of 16-18 h.

k. Store samples in refrigerator until the test iscompleted.


a. Remove the control and test plates with swabs from incubator and remove swabs.

b. Measure the ZI around the N5 disc on the control plate with a mm ruler or a zone reader. The zone should be20-26 mm wide. If not, the test should be repeated.

c. Observe the plates for inhibition of B. mega t e r i um growth surrounding the swabs (Figure 2).

i. Samples Free of Antimicrobial Chemical Residue

Figure 1. Swab placement on plate.

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If no zone of inhibition is observed around a swab,the test is negative, .i.e. the samples (A, B, Cand D) do not contain an antimicrobial residue(Fig. 2).

Figure 2. Swabs with no zone of inhibition.

ii. Samples with Antimicrobial Residue

If a zone of inhibition is observed around a swab,the test is positive, i.e. the samples (A and D) may have an antimicrobial residue. Measure the width of the zone.

Figure. 3. Positive samples illustrating zone ofinhibition around the swabs (samples) A and D.

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d. Record and compare result with the field result.Positive samples must be subjected to confirmatorytesting.

33.67 Quality Assurance

a. The M-FAST plates wrapped in plastic bags should bestored at refrigerator temperature (4-8oC).

b. Spore suspensions in tightly closed container should bestored at refrigerator temperature (4-8

oC).

c. Neomycin disc vial wrapped in a plastic bag should bestored at refrigerator temperature (4-8

oC).

d. Observe expiration date of plates. More than 2 week old plates should be discarded.

e. The spore vial should be shaken thoroughly before use.

f. Incubate 1 plate each day at 44oC as control.

g. Check plates before use for contamination, drying orcracking of agar.

h. Allow enough room for each swab placed on a plate.

i. Be careful not to break the agar surface while placingthe neomycin disc and the swabs.

j. Leave swabs in the tissues for a minimum of 30 minutes.

k. Read plates any time after 6 h of incubation, up to a maximum of 18 h.

l. Stabilize the incubator temperature at 44 ± 0.5°°C.

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Dey, B. P., Richard Reamer, and S. Kamosa. 1995. Evaluationof antimicrobial residues in meat and poultry tissues by a modified Fast Antimicrobial Screen Test (M-FAST). LaboratoryCommunication No. 80. USDA, Food Safety and Inspection

Service, S&T, Microbiology Division, Washington, D.C.

Dey, B. P., and C. A. White. 1995. FAST Antimicrobial ScreenTest (FAST) for antimicrobial residue detection in meat.Laboratory Communication No. 79. USDA, Food Safety and Inspection Service, S&T, Microbiology Division, Washington,D.C.

Johnston, R. W., R. H. Reamer, E. W. Harris, H. G. Fugate, and B. Schwab. 1981. A new screening method for the detection

of antibiotic residues in meat and poultry tissues. J. Food Prot. 44:828-831.


United States Department of Agriculture. 1981. Performing theSwab Test on Premises (STOP) for Detection of AntibioticResidues in Livestock Kidney Tissue. Handbook. Food Safetyand Inspection Service, Administrative Management, Trainingand Development Division, College Station, TX.

United States Department of Agriculture. 1982. The shelf

stable swab test system for detecting antibiotic residues intissues. Laboratory Communication No. 31. Food Safety and Inspection Service, S&T, Microbiology Division, Washington,D.C.

United States Department of Agriculture. 1994. Fast Antimicrobial Screen Test (FAST): For Detection of Antibioticand Sulfonamide Residues in Livestock Kidney Tissue. A Self-Instructional Guide. Food Safety and Inspection Service, Administrative Management, Human Resource and DevelopmentDivision, College Station, TX.

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The methods described in this guidebook are for use by the FSIS laboratories. FSIS does not

specifically endorse any of the mentioned test products and acknowledges that equivalent

products may be available for laboratory use.________________________________________________________________________________________




________________________________________________________________________________


Notice of Change


Title: Bioassay for the Detection, Identification and Quantitation of Antimicrobial

Residues in Meat and Poultry Tissue


This MLG chapter has been revised to incorporate the following updated material.

• The reclassification of Micrococcus luteus to Kocuria rhizophila

• Extension of the shelf life of prepared bioassay plates from five to seven days

• The addition of instructions for detecting and quantifying residues of ampicillin

dihydrostreptomycin, gentamicin and tylosin

• A modified recommended standard curve for gentamicin

• References to chemical methods of analysis for residues of clindamycin, variousfluoroquinolones, florfenicol, and chloramphenicol

Additional changes include:

• minor formatting and wording changes

• correction of errors found in Sections 34.5.2 and 34A.3.3.2 Tables

• expanding the concentration of usable commercial vegetative cell preparations down to 5 x

106

cfu/ml

• an additional provision for adjusting standard curve concentrations

• the addition of a caution to keep samples cold


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Title: Bioassay for the Detection, Identification and Quantitation of Antimicrobial Residues in

Meat and Poultry Tissue



Procedure Outline

34.1 Introduction34.1.1 General34.1.2 Limits of Detection

34.2 Safety Precautions34.3 Quality Control (QC) Practices

34.4 Equipment, Reagents and Media34.4.1 Equipment and Supplies34.4.2 Reagents34.4.3 Media

34.5 Test Organisms34.5.1 Microorganisms Used for the Bioassay System34.5.2 Confirmation of Properties of Test Organisms

34.6 Use of Bacterial Culture Preparations34.6.1 Use of Commercial Lyophilized Pellets Containing Vegetative Cells34.6.2 Spore Suspensions of Bacillus cereus ATCC 11778 and B. subtilis ATCC 663334.6.3 Determination of Number of Colony Forming Units (cfu)34.6.4 Optimization of Bioassay Plate Bacterial Lawn

34.7 Preparation of Bioassay Plates34.7.1 General Instructions and Plate QC34.7.2 Preparation of Specific Plates

34.8 Standard Curves34.8.1 Standard Curve QC Requirements34.8.2 Antibiotic Working Standards34.8.3 Standard Curves

34.9 Bioassay Procedures for Detection and Quantitation of Antibiotic Residues in Animal Tissues34.9.1 Sample Preparation and Storage34.9.2 Preparation of Spiked Tissues34.9.3 Performing the Seven-Plate Bioassay

34.10 Individual Antibiotic Assays34.10.1 Tetracyclines – Plate 134.10.2 Penicillin and Antibiotics Giving the Penicillin Pattern – Plates 2 and 334.10.3 Streptomycin and Dihydrostreptomycin - Plate 434.10.4 Erythromycin and Tylosin – Plates 5 and 634.10.5 Neomycin and Gentamicin – Plate 7

34.11 Reading the Bioassay

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34.12 Additional Information on Test Interpretation34.13 Calculating the Concentration of Antibiotic Residue in Tissue34.14 Summary of Antibiotic Reference Standards34.15 Additional Testing Procedures

34.15.1 ELISA Tests34.15.2 Chemical Methods of Antibiotic Residue Confirmation and/or Quantitation

34.16 References

34.A Appendix

34.A.1 General Information34.A.2 Safety Precautions34.A.3 Preparation, Storage, and Use of Vegetative Cell Cultures for Assay

34.A.3.1 Equipment, Materials, Reagents and Media (for 34.A.3 and 34.A.4) 34.A.3.2 Test Organisms34.A.3.3 Determination of Purity and Properties of Vegetative Test Organisms34.A.3.4 Propagation of Test Organisms (Non-spore Formers)34.A.3.5 Preparation and Preservation of Stock Cultures34.A.3.6 Use of Fresh Vegetative Cultures in Assay

34.A.4 Preparation of Suspensions of Spore Forming Test Organisms34.A.4.1 Determination of Purity and Properties of Spores34.A.4.2 Preparation of Spore Suspensions of Bacillus cereus ATCC 1177834.A.4.3 Preparation of Spore Suspensions of Bacillus subtilis ATCC 663334.A.4.4 Preparation of Working Spore Suspensions

34A.5 Manual Calculation of a Standard Curve

34.1 Introduction

34.1.1 General

Antibiotics are used in food animals for the prevention and treatment of infectious diseasesand for growth promotion. When antibiotics are used properly, they should not leavedetectable residues in edible tissues above levels allowed by Federal regulations.Inappropriate use of antibiotics is undesirable for two main reasons. Residues may producetoxic or allergic reactions in susceptible individuals who eat meat or poultry that containsantibiotic residues; and microorganisms may develop resistance to frequently used antibiotics.The US Food and Drug Administration (FDA) antibiotic assay procedures have long beenused to detect antibiotics in milk, other dairy products, animal feeds, and animal tissuesi.

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These methods were developed to quantitate individual antibiotic residues. However, they did not identify the specific antibiotic residue present. Using the principles of these assays, theUnited States Department of Agriculture (USDA), Food Safety and Inspection Service (FSIS)developed a Bioassay System which is a seven plate agar diffusion assay that can detect and quantitate a range of antibiotic residues found in meat and poultry products. Later, this assaysystem was further modified to replace bilayer plates with monolayer plates.

This Bioassay System utilizes bacteria that are relatively sensitive or resistant to a particular classof antibiotic. The bacteria are used in combination with specific antibiotic test agars and four pH-specific, buffered sample extracts. If a detectable antibiotic residue is present in a sample, it produces a zone of clearing (inhibition) on one or more of the test plates. Certain antibioticresidues can be identified according to their characteristic patterns of inhibition. A chart called an antibiogram was developed that depicts expected patterns of inhibition that specific antibioticsare expected to produce on the 7 plates. However, some patterns correspond to multipleantibiotic residues; these samples are then subjected to further testing. Once the antibioticresidue has been identified, the concentration of the residue present may be determined from thestandard curve by comparing the zone sizes produced by the sample to that produced by areference antibiotic standard of known concentration. The identity and concentration of certainantibiotics are determined using chemical tests after they are detected in the bioassay. Chemicalmethods are not covered in this chapter but are referenced in Section 34.15.2


The lowest concentration of a known reference standard that produces a zone of inhibition atleast 8 mm on a particular bioassay plate is referred to as the minimum inhibitory concentration(MIC). The MIC of each bioassay antibiotic defines the limits of detection for that antibioticresidue.


The microorganisms used in the bioassay are Biosafety Level 1 and 2 organisms. Safe laboratory practices should be followed in working with all organisms. Exercise caution in working with hazardouschemicals by wearing appropriate protective safety clothing, gloves, and eyeglasses or face shields.Consult a Material Safety Data Sheet (MSDS) for each hazardous chemical before working with it.Collect hazardous chemical wastes in separate containers and dispose of them in accordance with thestandard chemical waste management procedures for your laboratory. FSIS personnel should follow theFSIS Laboratory Environmental, Health, and Safety Handbook.

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34.3 Quality Control (QC) Practices

All glassware must be chemically clean. Spiders must be carefully cleaned so that no organic,antibiotic, or chemical residues remain. All media used shall be prepared and tested in accordance withthe quality control practices described in the FSIS Quality System documents: the MicrobiologyLaboratory Guidebook Appendix I Media and Reagents, the FSIS Lab-Wide SOPs, and the individual

laboratory’s Work Instructions. Additional quality control practices and critical control points aredescribed in this chapter under the appropriate sections.

34.4 Equipment, Reagents, and Media

34.4.1 Equipment and Supplies

a. Stainless steel bioassay plates or cylinders with 6 wells spaced at 60° intervals (referred to in this chapter as ‘spiders’).

b. Plastic Petri dishes, 100 x 15 mm, high quality with flat bottomsc. Timer

d. Stomacher® Model 80, model 400, or equivalente. Water bath, 45 to 55°C range, capable of maintaining constant temperature± 2°Cf. Zone reading device capable of reading in millimeters, such as a Fisher- Lilly antibiotic

zone reader or calipersg. Sterile forceps

h. Incubators set at 29± 1°C, 35 ± 1°C, and 37 ± 1°Ci. Forceps for stainless steel bioassay spiders

j. Vacuum drying oven, capable of maintaining a constant temperature in the range of 60±

4°C to 110 ± 4°C and vacuum of 6 mm atmospheric pressure or as needed for antibioticstandard preparation.

k. Sterilizing oven (dry-heat, sterilizing ovens must be capable of maintaining a temperature

of 160 ± 10°C for at least 180 to 240 minutes, depending on capacity.)l. Vacuum desiccator m. Micropipettors and suitable tipsn. Flasks, volumetric, 10, 25, 50, 100, and 200 ml, or equivalento. Media bottles, 250-300 ml and other sizes as needed, or equivalent p. Bags for stomaching samples (Whirl-Pak bags, Tekmar filter bags or equivalent)

(Optional)q. Pipettes, graduated, assorted sizes

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r. Refrigerator (2-8°C)

s. Freezer (-10°C or lower)t. Balance, analyticalu. Balance, 0.1 to 200 gram capacity

. v. Colony counter

w. Blender, Tissuemizer ™ or similar x. Vortex mixer

y. Positive displacement pipettor, Hamilton syringe, or similar devise for dispensing smallvolumes of viscous solutions

z. Semi-logarithmic graph paper or computer with spreadsheet programaa. Magnetic stirrer (optional)

34.4.2 Reagents

Recipes are given in the MLG Appendix 1, Media and Reagents except as noted.

a. 0.1 M phosphate buffer, pH 4.5 + 0.1 b. 0.1 M phosphate buffer, pH 6.0 + 0.1

c. 0.1 M phosphate buffer, pH 8.0 + 0.1d. 0.2 M phosphate buffer, pH 8.0 + 0.1e. Butterfield's Phosphate Buffer, pH 7.2f. Reference antibiotics as needed - Minimum: Tetracycline hydrochloride, penicillin G

potassium salt, streptomycin sulfate, erythromycin, neomycin sulfateg. 0.1 N HClh. 0.1 N NaOHi. 1.0 N HCl j. 1.0 N NaOHk. 0.85% NaCl solution (sterile saline)l. Penicillinase Concentrate, equivalent to at least 10,000,000 Kersey Units/ml

m. Specific antibiotics as required for quantitation, USP or equivalentn. Antibiotic sensitivity disks (e.g. BD Sensi-Discs: BD Biosciences, 7 Loveton Circle,Sparks, MD 21152): penicillin P2 or P10, tetracycline Te30, streptomycin S10,erythromycin E15, and neomycin N5

o. Isotonic saline with 0.01% Tween as described in Section 34.6.1.2 p. Dextrose for use in preparation of antibiotic agar No. 2

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34.4.3 Media

Media Recipes are given in the MLG Appendix 1, Media and Reagents.

a. Antibiotic Medium 2, with 0.1% dextrose b. Antibiotic Medium 5c. Antibiotic Medium 8

d. Antibiotic Medium 11e. Plate Count Agar

f. AOAC approved biochemical identification systems such as API Staph Trac®, API

strips® or VITEK ® (bioMérieux, Inc., Durham, NC 27717), MicroID® (OrganonTeknika Corp., Durham, NC); or biochemical reagents as needed to identify themicroorganisms per SOP LW-0015 or Certificate of analysis for cultures.

34.5 Test Organisms

34.5.1 Microorganisms Used for the Bioassay System

The following test organisms are used in the preparation of agar plates used for the standard curves and the seven-plate bioassay system. Use aseptic technique when working with all bacterial cultures.

a. Kocuria rhizophila (formerly Micrococcus luteus), ATCC 9341a (KR) (see Tang et al) b. Kocuria rhizophila, ATCC 15957 (KRER)c. Staphylococcus epidermidis, ATCC 12228 (SE)d. Bacillus cereus var. mycoides spores, ATCC 11778 (BC)e. Bacillus subtilis spores, ATCC 6633 (BS)

Preparations of all cultures are available commercially. The vegetative cultures above are

available as: LYFO-DISK lyophilized organisms from MicroBioLogics, Inc., 217 Osseo Avenue North, St. Cloud, MN 56303-4452:K. rhizophila ATCC 9341a, K. rhizophila ATCC 15957 and S. epidermidis ATCC 12228. Spore suspensions of B. subtilis ATCC 6633 and B. cereus ATCC11778 in 50% ethanol are available from MedTox Diagnostics, Inc, Burlington, NC 27215.Other suppliers may be used if suitable preparations are available.

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34.5.2 Confirmation of Properties of Test Organisms

Culture identity and purity must be verified. A certificate of analysis verifying identity and purity is acceptable for new lots of bacterial cultures. If a certificate of analysis is not available,or if culture identity and purity must be confirmed, follow the procedures described in theAppendix to this chapter.

The antibiotic sensitivity patterns of the test organisms must be confirmed. Prepare bioassay plates listed in the table below as described in Section 34.7with the exception that penicillinase

should not be added to the agar. Place a ‘spider’ or 5 cylinders on each plate. Add 200microliters of the antibiotic concentrations specified in the table below to individual wells. Each prepared plate will be tested with all five of the antibiotics listed in the table. Antibiotics with a‘>’ should not produce a zone of inhibition greater than 8 mm at the indicated concentration onthat plate. For the other antibiotic-plate combinations, a measurable zone of inhibition of 8 to 15mm should be produced at the concentrations listed. Replace any test organism that is notconsistently capable of meeting these criteria with a new ATCC culture.

Antibiotic Sensitivity Patterns of Cultures Table

Antibiotic concentration in µg per ml Test Organism Medium No. Erythromycin Neomycin Penicillin Streptomycin Tetracycline

K. rhizophila9341a

2 0.05 5.0 0.0125 > 200 0.5

K. rhizophila9341a

11 0.025 1.5 0.0125 250 0.4

K. rhizophila15957

11 > 200 1.5 0.0125 1.0 0.2

S. epidermidis12228

11 0.075 0.25 0.8 1.5 250

B. cereus11778

8 0.25 2.00 20 1.00 0.08

B. subtilis6633

5 0.04 0.25 0.0125 0.2 0.08

34.6 Use of Bacterial Culture Preparations

The following material assumes the use of the specified commercial bacterial preparations. Instructionsfor the preparation of fresh and preserved vegetative cell cultures are located in the appendix to this

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chapter. Those instructions may be followed as an alternative approach to the use of commercial preparations.

34.6.1 Use of Commercial Lyophilized Pellets Containing Vegetative Cells

Individual pellets for all three lyophilized test organisms (K. rhizophila ATCC 9341a, K.

rhizophila ATCC 15957, and S. epidermidis ATCC 12228) are now available commercially.

The use of lyophilized pellets reduces the potential for sudden loss of a viable, well characterized culture, reduces the potential for loss of desired properties such as antibiotic sensitivity and resistance patterns, and reduces labor-intensive procedures for producing vegetative cell cultures.

Store the lyophilized pellets at 2−8°C.

34.6.1.1 Materials Required

a. LYFO-DISK lyophilized organisms (Micro-Bio-Logics, 217 Osseo Avenue North,St. Cloud, MN 56303-4452)K. rhizophila ATCC 9341a, K. rhizophila ATCC 15957and S. epidermidis ATCC 12228.]

b. Isotonic saline with 0.01% Tween

c. Sterile forceps

34.6.1.2 Rehydration of Assay Organisms from Pellets

a. Prepare isotonic saline with 0.01% Tween as described below. Quantities may bevaried if the proportions are retained. An appropriate measuring device (such as a positive displacement pipette) is needed to dispense the Tween, as the solution is veryviscous.

• Dissolve 8.5 g of sodium chloride in 1000 ml of distilled water.

• Add 0.1 ml of Tween 80.

•

Allow ample time for the solution to stir to insure thorough mixing.• Autoclave at 121°C for 15 minutes, @15 lbs. of pressure or filter sterilize.

b. Rehydrate the pellet. Aseptically transfer the lyophilized pellet(s) into a sterile tube

and add 2.0 ± 0.08 ml of room temperature saline-Tween solution for each pellet.Let this stand for approximately 90 minutes. It may be necessary to vortex the pelletmixture to aid in the dispersion of the pellet. Note: if the pellet does not dissolve it

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may be necessary to briefly warm the suspension to about 40°C. The pelletsuspension may be stored at 2-8°C for up to 5 days.

34.6.2 Spore Suspensions of Bacillus cereus ATCC 11778 and B. subtilis ATCC 6633

This protocol uses commercially available Bacillus cereus ATCC 11778 and B. subtilis ATCC6633 spore suspensions in ethanol from MedTox Diagnostics, Inc.

Laboratory-prepared spore suspensions may also be used. See the appendix to this chapter for instructions on preparing spore suspensions. Store all spore suspensions at 2−8°C.

34.6.3 Determination of Number of Colony Forming Units (cfu)

To determine the number of bacteria per ml of each culture suspension, prepare a tenfold dilutionseries (10

-2to 10

-8) in the following manner. If high counts are obtained (outside the countable

range on these plates) the procedure must be repeated using higher dilutions.

a. Pipette one ml of well mixed (use vortex mixer) stock suspension into 99 ml of Butterfield's phosphate buffer for the 10-2 dilution. Use a separate, sterile 10 ml pipette to prepare each

dilution. Pipette 10 ml of the 10

-2

dilution into a 90 ml dilution blank for the 10

-3

dilution.Continue to prepare additional dilutions by adding 10 ml from the newly prepared dilution tothe next 90 ml dilution blank. Make sure that each dilution is mixed thoroughly before usingit to make the next dilution. Repeat as needed to finish the dilution series.

b. Pipette one ml of each dilution into two 100 x 15 mm Petri dishes, and add 15 ml of molten

Plate Count Agar which has been cooled in a water bath to 48± 2°C. See the MicrobiologyLaboratory Guidebook Media Appendix for directions. Mix by swirling or tilting the plates

to disperse the inoculum evenly throughout the medium. Incubate for 24 to 48 h at 35± 1°Cor until colonies are easily visible. Some cultures grow more rapidly than others. Avoid incubating so long that colonies merge.

c. Count duplicate plates in a suitable range (30-300 colonies) on a colony counter. Record thedilution and number of colonies found. Average the counts obtained and record the counts per ml for each dilution of plates.

d. Using the counts obtained, calculate and record the stock concentration. The vegetative cellstocks should contain at least 1 x 108 cfu/ml. Preparations with lower counts of vegetativecells may be used, but will require greater quantities to prepare the plates. The minimum

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recommended count is 5 x 106 cfu/ml. The spore suspensions should contain at least 1 x 106 cfu/ml.

34.6.4 Optimization of Bioassay Plate Bacterial Lawn

Each antibiotic has a set of dilutions that are recommended for producing the standard curve.The standard curve is used for calculating the quantity of antibiotic residue in a sample. The

lowest concentration of each standard curve dilution should produce a clear zone of inhibition ona bacterial lawn of the proper organism with the appropriate cfu concentration. If necessary,adjust the bacterial lawn target level to optimize the bioassay results. To determine the amountof the rehydrated pellet or spore suspension necessary for making bioassay plates, use thefollowing formula:

X = (TV)/C

Where:X = amount of stock culture suspension to be added to the molten agar (ml)T = target (desired) number of bacterial cells in the lawn (cfu/ml)V = volume of molten agar (ml)

C = concentration of stock culture suspension (cfu/ml) EXAMPLE 1:

T = 4 x 104 cfu/ml lawnC = 8 x 10

6cfu/ml B. cereus stock culture suspension

V = 150 ml agar

X = (TV)/CX = (4 x 10

4cfu/ml)(150 ml)

8 x 106 cfu/mlX = 6 x 10

6cfu/8 x 10

6cfu/ml

X = 0.75 ml0.75 ml of B. cereus stock culture suspension should be added to the molten agar

EXAMPLE 2:

T = 1 x 105 cfu/ml lawnC = 2 x 108 cfu/ml culture from pellet, in isotonic Tween solutionV = 100 ml agar

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X = (TV)/CX = (1 x 105 cfu/ml)(100 ml)

2 x 108

cfu/mlX = 1 x 107 cfu/2 x 108 cfu/mlX = 0.05ml0.05 ml of rehydrated lyophilized culture should be added to the molten agar

34.7 Preparation of Bioassay Plates

34.7.1 General Instructions and Plate Quality Control

34.7.1.1 General Instructions

• To prepare each type of plate described below, prepare the required amount of the

specified agar and temper it to 48± 2° C in a water bath. If previously prepared agar is used, the agar should be heated gently to melt it. Do not melt prepared agar morethan once. Note: Do not use a microwave oven to melt the agar.

• Add the required volume of cell suspension to produce the desired concentration of

organisms. See the examples in section 34.6.4 above.• Add penicillinase if required.

• Mix the cell-agar solution by swirling and/or pipetting it several times. To make the plates, dispense 8.0 to 8.5 ml of the preparation into plastic petri plates (100 x 15mm).

• Tilt or swirl the plates gently so that the agar uniformly covers the surface of the plate. Let the agar harden on a flat, level surface.

• Properly identify each bioassay plate. Mark a vertical line on the side of the bottomof the plate for proper alignment of stainless steel spider. The date of preparation or expiration, and identity of the preparer should also be recorded.

• Store the prepared plates at 2-8°C.

• Prepared plates must not be allowed to freeze or dry out• Plates may be used for up to 7 days after preparation.

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Bioassay Plate Summary TablePlate No.

AntibioticAgar

OrganismCulture

PreparationWater BathIncubation*

Penicillinase

1 No. 8 Bacillus cereus

ATCC 11778 None 45 min 1ml/100ml agar

2 No. 2(with

dextrose)

Kocuria rhizophila

ATCC 9341aRehydrate

(90 minutes)none none

3 No. 2(with

dextrose)

Kocuria rhizophila ATCC 9341a

Rehydrate(90 minutes)

none 1ml/100ml agar

4 No. 5 Bacillus subtilis

ATCC 6633 None 75 min 1ml/100ml agar

5 No. 11Kocuria rhizophila

ATCC 9341aRehydrate

(90 minutes)none 1ml/100ml agar

6 No. 11Kocuria rhizophila

ATCC 15957Rehydrate


7 No. 11Staphylococcus

epidermidis ATCC 12228Rehydrate


* Using the 48± 2°C water bath, incubate spores in the molten agar before adding penicillinase

34.7.1.2 Critical Steps in Plate Preparation

• Assure thorough mixing of organisms and penicillinase throughout the agar before pouring the plates.

• Use no less than 8.0 ml and no more than 8.5 ml of seeded agar for each plate.

• Assure that the entire plate is covered with a uniform layer of seeded agar and that theagar layer is smooth with no holes or air bubbles.

• Work rapidly to assure that the agar does not prematurely harden while pouring the plates.

• Incubate the spore-inoculated agars for the required times before making plates.

• Pour the plates on a flat level surface to assure that when the agar hardens it has auniform thickness.

34.7.1.3 Bioassay Plate Quality Control

a. The inoculum concentrations specified for the preparation of each plate (Sectionsunder 34.7.2) have been standardized to achieve a confluent cell lawn growth. The

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lowest antibiotic standard concentration to which the organism is sensitive should produce a significant, readable zone of inhibition. If this is not achieved, prepare plateswith varying concentrations of the bacterial suspensions to determine the amount of inoculum that produces the desired zone of inhibition for each assay. If the concentrationof the organisms suspended in the agar is changed, prepare a new standard curve beforeusing the plates for the bioassay.

b. The prepared plates must not be used if stored for more than seven days after preparation. Each batch of prepared plates must be performance tested before use.

c. When plates are used for screening samples and identifying residues the followingquality control criteria must be met.

• Produce antibiotic sensitivity patterns as described in the table below. Sensi-Disksconcentrations other than those specified may be used to determine the pattern. Platesthat do not perform as expected must be discarded and new plates produced to meetthe requirements.

Plate QC: Antibiotic Sensi-Disk Patterns Table Antibiotic Sensi-DiscPlate No. & Organism Agar

Te30 P10 S10 E15 N5

1 - B. cereus ATCC 11778 # 8 S R S S S

2 - K. rhizophila ATCC 9341a# 2 without

penicillinaseS S R S R

3 - K. rhizophila ATCC 9341a# 2 with

penicillinaseS R R S R

4 - B. subtilis ATCC 6633 # 5 S R S S S

5 - K. rhizophila ATCC 9341a #11 S R R S S

6 - K. rhizophila ATCC 15957 # 11 S R S R S

7 - S. epidermidis ATCC 12228 # 11 R R R S S

S = Zone of inhibitionR = No zone of inhibition

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d. Plates that will be used for quantitative analysis, including plates for standard curves,should also meet the following QC procedures.

• A clear zone of inhibition (at least 8 mm) must be produced with the lowestconcentration of the plate-specific antibiotic standard-curve dilution. Note: Plates 3and 6 are confirmatory plates and will not produce a zone of inhibition with thespecific antibiotic.

• Plates 1, 2, 4, 5, and 7 must also be tested with the plate-specific standard referenceconcentration. Zones sizes must fall within 3 standard deviations of the historicalaverage. Any results greater than 2 standard deviations should be treated withcaution.

34.7.2 Preparation of Specific Plates

34.7.2.1 Preparation of Plates with B. cereus and Penicillinase (BC, Plate 1)

• Pipette the required quantity of B. cereus (ATCC 11778) spores into melted,

tempered Antibiotic Media No. 8 to make a final concentration of 5 x 10

3

cfu/ml of agar.

• Swirl the bottle gently to mix and incubate for 45 minutes in a 48° ± 2° C water

bath.

• After incubation add 1± 0.1 ml of penicillinase per 100 ml of seeded media (100,000units per ml of agar).

• Mix the agar suspension and prepare the plates as described in Section 34.7.1.

34.7.2.2 Preparation of Plates with K. rhizophila (KR, Plate 2)

• Pipette the required quantity of K. rhizophila cells (ATCC 9341a) into melted,

tempered Antibiotic Media No. 2 to make a final concentration of 8 x 105

cfu/ml of agar.

• Note: No penicillinase is added to this agar preparation.


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34.7.2.3 Preparation of Plates with K. rhizophila and Penicillinase (KR+P, Plate 3)

• Pipette the required quantity of K. rhizophila cells (ATCC 9341a) into melted,tempered Antibiotic Media No. 2 to make a final concentration of 8 x 105 cfu/ml of agar.

• Add 1 ± 0.1 ml of penicillinase per 100 ml of seeded media.


34.7.2.4 Preparation of Plates with B. subtilis and Penicillinase (BS, Plate 4)

• Pipette the required quantity of B. subtilis (ATCC 6633) spores into melted, tempered Antibiotic Media No. 5 to make a final concentration of 1 x 10

4cfu/ml of agar.

• Swirl the bottle gently and incubate for 75 minutes in a water bath at 48 ± 2°C.

• After incubation add 1± 0.1 ml of penicillinase per 100 ml of seeded media (100,000units per ml of agar).


34.7.2.5 Preparation of Plates with K. rhizophila ATCC 9341a and Penicillinase

(KR-11, Plate 5)• Pipette the required quantity of K. rhizophila cells (ATCC 9341a) into melted,

tempered Antibiotic Media No. 11 to make a final concentration of 8 x 105 cfu/ml of agar.



34.7.2.6 Preparation of Plates with K. rhizophila ATCC 15957 and Penicillinase

(KR-ER, Plate 6)

• Pipette the required quantity of K. rhizophila cells (ATCC 15957) into melted,

tempered Antibiotic Media No. 11 to make a final concentration of 1 x 105 cfu/ml of agar.

• Add 1 ± 0.1 ml of penicillinase per 100 ml of seeded media (100,000 units/ml).

• Mix the agar suspension and prepare plates as described in Section 34.7.1.

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34.7.2.7 Preparation of Plates with S. epidermidis and Penicillinase (SE, Plate 7)

• Pipette the required quantity of S. epidermidis (ATCC 1228) cells into melted,tempered Antibiotic Agar No. 11 to make a final concentration of 1 x 105 cfu/ml of agar.


• Mix the agar suspension and prepare plates as described in Section 34.7.1.

34.8 Standard Curves

Standard curves are generated for each antibiotic that is tested. The standard curve is calculated from thezones of inhibition produced by a series of antibiotic concentrations that have been tested on the platetype that is specific for that antibiotic. There are 5 antibiotic concentrations for each standard curve. Themiddle concentration is designated the antibiotic standard reference (SR) concentration. This standard reference concentration is also placed on plates along with unknown samples for quantitative analysisand is the point of comparison between the quantitative analysis and the standard curve.

34.8.1 Standard Curve QC Requirements

Antibiotic standard curves should be prepared at a minimum of once every three months, or when a new lot of culture, reference standard, or plating media is used. The standard curve isexpected to be relatively straight when plotted semi-logarithmically. Track the standard curveslopes and zone sizes. If the antibiotic SR zone size changes by more than three standard deviations of historic values the curve is considered unacceptable and a new curve must be produced. Trends, such as changes in slope, high and low points, and antibiotic zone sizes,should also be tracked. If significant changes are observed an investigation may be warranted. If the lowest concentration of the standard curve (as defined in this method) does not produce azone at least 8 mm in diameter, it may be necessary to change the antibiotic standard curveconcentrations in your lab so that a zone of at least 8 mm in diameter is produced at the lowest

concentration. If the lowest concentration of the standard curve (as defined in this method) produces too large a zone, it may be necessary to change the antibiotic standard curveconcentrations in your lab.

34.8.2 Antibiotic Working Standards

Authentic antimicrobial reference standards may be obtained from the United StatesPharmacopeia, 12601 Twinbrook Parkway, Rockville MD 20852, and the Committee on

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National Formulary, American Pharmaceutical Association, 2215 Constitution Avenue, NW,Washington DC 20037. It is recommended that USP quantitation reference standards be used. If USP quantitation reference standards are not available, equivalent standards should be used.

Follow the manufacturer's directions for storing and preparing the antibiotic ref erence standards.Use chemically clean glassware. Stock solutions, 1000 parts per million (ppm

1), should be

prepared in an atmosphere of 50% relative humidity or less. Carefully weigh a small amount of

the antibiotic into a chemically clean container. Dilute the weighed powder using the appropriateClass A volumetric glassware and/or volumetric dispensing device(s) and diluent to obtainsolutions of the required concentrations.

34.8.2.1 Tetracyclines

Tetracyclines are light sensitive. All tetracycline powders and solutions should be protected from light. Amber glassware may be used for preparing solutions. If amber glassware is not used, the containers should be wrapped in foil or otherwise protected from light.

Preparation of Tetracycline and Oxytetracycline Solutions

a. Calculate the quantity of tetracycline or oxytetracycline needed to produce 1000 ppmof free base in the desired volume. Use the free base content statement provided bythe manufacturer or calculate the free base content by using the molecular formulainformation and purity information provided by the supplier.

b. Accurately weigh the required amount of antibiotic. Add the required volume of 0.01 N hydrochloric acid to produce a 1000 ppm (ug/ml) stock solution. If the powder istransferred from the weighing container into another container for dilution, wash itinto the container with 0.01 N hydrochloric acid to ensure that the entire quantity istransferred. Oxytetracycline may be slow to go into solution. It may be placed on amagnetic stirrer until dissolved or held overnight at 2-8 °C.

c. Store the stock solution at 2-8°C and use it within seven (7) days.

1Parts per million (ppm) is used in this chapter to express a quantity of 1μg per ml.

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d. Preparation of Tetracycline and Oxytetracycline Dilutions. Dilute an aliquot of thetetracycline or oxytetracycline stock solution with enough 0.1 M, pH 4.5 phosphate buffer to obtain solutions with concentrations of 1.28, 0.64, 0.32, 0.16, and 0.08 ppm.The 0.08 ppm concentration is used to indicate test sensitivity. The 0.32 ppm dilutionis used as the SR concentration for the standard curve and for the quantitative bioassay.

The table below outlines a suggested dilution method for preparing the required concentrations of tetracycline and oxytetracycline.

From ppm Solution Take (ml) Dilute to (ml) Resulting concentration*

1000.0 5.0 50 100.0

100.0 4.0 50 8.0

8.0 4.0 25 1.28

8.0 2.0 25 0.64

8.0 1.0 25 0.32

8.0 1.0 50 0.16

8.0 1.0 100 0.08

* In ppm (μg/ml)

Preparation of Chlortetracycline Solutions

e. Calculate the quantity of chlortetracycline needed to produce 1000 ppm of free basein the desired volume. Use the free base content statement provided by the

manufacturer or calculate the free base content by using the percent purity and molecular formula information.

f. Accurately weigh the required amount of antibiotic. If the powder is transferred fromthe weighing container into another container for dilution, wash it into the container with 0.01 N hydrochloric acid to ensure that the entire quantity is transferred. Add the required volume of 0.01 N hydrochloric acid to produce a 1000 ppm (ug/ml)stock solution.

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g. Store the stock solution at 2-8°C and use it within seven (7) days.

h. Preparation of Chlortetracycline Dilutions. Dilute an aliquot of stock solution withenough 0.1 M, pH 4.5 buffer to obtain solution concentrations of 0.16, 0.08, 0.04,0.02, and 0.01 ppm. The 0.01 ppm concentration is used to indicate test sensitivity.The 0.04 ppm dilution is the SR concentration for the standard curve and for the

quantitative bioassay.

The table below outlines a suggested dilution method for preparing the required concentrations of chlortetracycline:

From Solution (ppm) Take (ml) Dilute to (ml) Resulting concentration*

1000.0 5.0 50 100.0

100.0 4.0 50 8.0

8.0 1.0 50 0.16

8.0 1.0 100 0.08

8.0 1.0 200 0.04

0.04 25.0 50 0.02

0.02 12.5 25 0.01

* In ppm (μg/ml)

34.8.2.2 Preparation of Penicillin and Ampicillin Solutions

a. Calculate the amount of Penicillin G potassium salt needed to produce 1000 ppm of penicillin G free base in the desired volume of buffer. Use the free base contentstatement provided by the manufacturer for these calculations (Ex. 1). If the free basecontent is not available but the units content is available, calculate the quantity byconverting units to micrograms (Ex. 2). See the formula and examples below. Pure penicillin G potassium salt contains 1595 penicillin G units per milligram and this isequivalent to 88.8% penicillin G free base.

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B = free base content (expressed decimally)V = target volume

C = stock solution concentration in milligrams: 1000μg/ml = 1mg/mlX = quantity of antibiotic in micrograms

Example 1

B (free base content of the powder) = 88.8%, (0.888)V = 25 mlC = 1 mg/mlX = (C*V)/B

X = 1(25)/0.888X = 28.15 mg.Weigh 28.2 mg into a 25 ml volumetric flask and dilute to volume.(Analytical balances are capable of measuring to 4 decimal places in grams, i.e. toone tenth of a milligram. Therefore 28.15 mg is rounded to 0.0282 g)

Example 2 - Converting units to free base content before calculating quantityThe antibiotic powder contains 1490 units per milligramCalculate the proportion: 1490/1595 = B/0.888B = (1490)(0.888)/1595B= 0.8295 (83.95%) penicillin G free base

B = 0.8295V = 25 ml,C = 1 mg/mlX = 1(25)/0.8295 = 30.14 mgWeigh 30.1 mg into a 25 ml volumetric flask and bring to volume.

b. Accurately weigh the required amount of antibiotic. If the powder is transferred fromthe weighing container into another container for dilution, wash it into the container with the suspension buffer to ensure that the entire quantity is transferred. Add sufficient phosphate buffer (0.1 M, pH 6.0) to make a stock solution with a finalconcentration of 1000 ppm (µg/ml).

c. Store the stock solution at 2-8°C and use it within seven days.

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d. Preparation of Penicillin Dilutions. Dilute an aliquot of the stock solution in 0.1 M, pH 6.0 buffer using volumetric flasks to prepare working solutions withconcentrations of 0.16, 0.08, 0.04, 0.02, and 0.01 ppm. The 0.01 ppm concentrationis used to indicate test sensitivity. The 0.04 ppm dilution is used as the SR concentration for the standard curve and for the quantitative bioassay.

The table below outlines a suggested dilution method for preparing the required concentrations of penicillin.

From Solution* Take (ml) Dilute to (ml) Resulting Concentration*

1000 5.0 50 100

100 2.0 100 2.0

2.0 4.0 50 0.16

2.0 2.0 50 0.08

2.0 1.0 50 0.04

2.0 1.0 100 0.02

2.0 0.25 50 0.01

* In ppm (μg/ml)

e. For ampicillin, calculate the free base value and prepare all dilutions using the penicillin dilution scheme.

34.8.2.3 Preparation of Streptomycin and Dihydrostreptomycin Solutions

Preparation of Streptomycin Solutions

a. Follow the manufacturer’s instructions for use, drying a portion if necessary or calculating out the water content. Calculate the quantity of powder needed to prepare1000 ppm of free base in the desired volume. Use the free base statement provided by the manufacturer. If the free base content is not available, calculate the quantity of powder needed for an equivalent content. Use the label potency statement (in units

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per mg) for the formula. Since 1 mg of pure streptomycin contains 785 units and theU.S.P. specifies not less than 650 µg and not more than 850 µg of streptomycin per mg, calculate potency of the standard by this formula:

([Potency units/mg] / [785 units/mg]) * (850 µg/mg) = Potency (µg/mg)

b. Accurately weigh the required amount of antibiotic. If the powder is transferred fromthe weighing container into another container for dilution, it may be washed into the

container with distilled water to ensure that the entire volume is transferred. Dilutethe weighed powder with distilled water to the required volume to achieve a 1000 ppm stock solution.

c. Store the stock solution at 2-8°C and use it within seven (7) days.

d. Preparation of Streptomycin Dilutions. Dilute an aliquot of stock solution in 0.1 M, pH 8.0 phosphate buffer using volumetric flasks to prepare working concentrations of 1.6, 0.8, 0.4, 0.2, and 0.1 ppm. The 0.1 ppm concentration is used to indicate testsensitivity. The 0.4 ppm concentration is used as the SR concentration for thestandard curve and in the quantitative bioassay.

The table below outlines a suggested dilution method for preparing the required concentrations of streptomycin.


1000 5.0 50 100.0

100 2.0 50 4.0

4.0 20.0 50 1.6

4.0 10.0 50 0.8

4.0 5.0 50 0.4

4.0 2.5 50 0.2

0.2 25.0 50 0.1

* In ppm (μg/ml)

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Preparation of Dihydrostreptomycin Solutions

e. Follow the procedures described above for streptomycin to prepare the 1000 ppmstock solution.

f. Preparation of dihydrostreptomycin dilutions. Dilute an aliquot of stock solution in0.1 M, pH 8.0 phosphate buffer using volumetric flasks to prepare workingconcentrations of 2.4, 1.2, 0.6, 0.3, and 0.15 ppm. The 0.15 ppm concentration is

used to indicate test sensitivity. The 0.6 ppm concentration is used as the SR concentration for the standard curve and in the quantitative bioassay.

The table below outlines a suggested dilution method for preparing the required concentrations of dihydrostreptomycin.

From Solution* Take (ml) Dilute to (ml) Resulting Concentration

1000 5.0 50 100.0

100 15 100 15

15 8 50 2.415 8 100 1.2

15 4 100 0.6

15 2 100 0.3

15 1 100 0.15

* In ppm (μg/ml)

34.8.2.4 Preparation of Erythromycin Solutions

a. Use either of the 2 methods below to prepare the 1000 ppm stock solution.

• Determine the quantity of erythromycin required to prepare a 1000 ppm solution.Use the free base content of the product to calculate the amount of powder needed to make the 1000 ppm solution. Weigh this amount into a suitable container (such as a clean disposable aluminum weigh boat). Add about 2.0 to 4.0 ml of pure methyl alcohol to the container and carefully swirl the container until theantibiotic powder is in solution. Add about 6 to 15 ml of buffer (pH 8.0, 0.2 M)

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to the methanol-antibiotic solution and mix again. Pour this solution into theappropriate volumetric flask. Rinse the weigh boat with additional buffer addingthe rinse to the volumetric flask. Add buffer to the volumetric flask to bring it tovolume.

or

• Weigh antibiotic powder into a container. Calculate the amount of diluent needed to prepare 1000 ppm solution of the free base. Add enough (about 2 to 3 ml)

30% methanol-buffer solution to dissolve the antibiotic powder. Subtract out thevolume of methanol solution used. Add the remaining buffer solution to bring tothe desired volume.

b. Store the stock solution at 2-8°C and use it within 14 days of preparation.

c. Preparation of Erythromycin Dilutions. Dilute an aliquot of the stock solution in 0.2M, pH 8.0 phosphate buffer using volumetric flasks to prepare workingconcentrations of 0.8, 0.4, 0.2, 0.1, and 0.05 ppm. The 0.05 ppm dilution is theconcentration used to indicate test sensitivity. The 0.2 ppm dilution is used as SR concentration on the standard curve and as the working standard in the quantitative bioassay.

The table below outlines a suggested dilution method for preparing the required concentrations of erythromycin.


1000 5.0 50 100.0

100 2.0 50 4.0

4.0 10.0 50 0.8

4.0 5.0 50 0.4

4.0 2.5 50 0.2

0.2 25.0 50 0.1

0.1 25.0 50 0.05

* In ppm (μg/ml)

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34.8.2.5 Preparation of Tylosin Solutions

a. Follow the manufacturer’s instructions for use, drying a portion if necessary or calculating out the water content. Use 0.2 M, pH 8.0 phosphate buffer for preparingall solutions.

b. Determine the quantity of tylosin required to prepare a 1000 ppm solution. Use thefree base content statement on the container if available. If the free base content is

not available, calculate the free base from the molecular formula and percent purity.Weigh the antibiotic powder into a suitable container. If the powder is transferred from the weighing container into another container for dilution, wash it into thecontainer with the suspension buffer to ensure that the entire quantity is transferred.Add sufficient 0.2 M, pH 8.0 phosphate buffer, to produce a concentration of 1000 ppm (µg/ml).

c. Store the stock solution at 2-8°C and use it within 7 days.

d. Preparation of Tylosin Dilutions. Dilute an aliquot of the stock solution in 0.2 M, pH8.0 phosphate buffer using volumetric flasks to give working concentrations of 3.2,1.6, 0.8, 0.4, and 0.2 ppm. The 0.2 ppm concentration is used to indicate test

sensitivity. The 0.8 ppm dilution is the SR concentration on the standard curve and the working standard in the quantitative bioassay.

The table below outlines a suggested dilution method for preparing the required concentrations of tylosin.


1000 5 50 100.00

100 5 50 10.0

10 8 25 3.2

10 8 50 1.6

10 8 100 0.8

10 2 50 0.4

10 1 50 0.2

* In ppm (μg/ml)

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34.8.2.6 Preparation of Neomycin Solutions


b. Determine the quantity of neomycin required to prepare a 1000 ppm solution. Usethe free base content statement on the container if available. If the free base content

is not available, calculate the free base from the molecular formula and percent purity. Weigh the antibiotic powder into a suitable container. If the powder istransferred from the weighing container into another container for dilution, wash itinto the container with the suspension buffer to ensure that the entire quantity istransferred. Add sufficient 0.1 M, pH 8.0 phosphate buffer, to produce aconcentration of 1000 ppm (µg/ml).


d. Preparation of Neomycin Dilutions. Dilute an aliquot of the stock solution in 0.1 M, pH 8.0 phosphate buffer using volumetric flasks to give working concentrations of 4.0, 2.0, 1.0, 0.5, and 0.25 ppm. The 0.25 ppm concentration is used to indicate test


The table below outlines a suggested dilution method for preparing the required concentrations of neomycin.


1000 10 100 100.00

100 10 100 10.0

10 4 10 4.0

10 2 10 2.0

10 1 10 1.0

10 0.5 10 0.5

10 0.25 10 0.25

* In ppm (μg/ml)

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34.8.2.7 Preparation of Gentamicin Solutions


b. Determine the quantity of gentamicin required to prepare a 1000 ppm solution. Usethe free base content statement on the container if available. If the free base content

is not available, calculate the free base from the molecular formula and percent purity. Weigh the antibiotic powder into a suitable container. If the powder istransferred from the weighing container into another container for dilution, wash itinto the container with the suspension buffer to ensure that the entire quantity istransferred. Add sufficient 0.1 M, pH 8.0 phosphate buffer, to produce aconcentration of 1000 ppm (µg/ml).


d. Preparation of Gentamicin Dilutions. Dilute an aliquot of the stock solution in 0.1 M, pH 8.0 phosphate buffer using volumetric flasks to give working concentrations of 1.6, 0.8, 0.4, 0.2, and 0.10 ppm. The 0.1 ppm concentration is used to indicate test


The table below outlines a suggested dilution method for preparing the required concentrations of gentamicin.


1000 10 100 100.00

100 10 100 10.0

10 8 50 1.6

10 4 50 0.8

10 4 100 0.4

10 2 100 0.2

10 1 100 0.1

* In ppm (μg/ml)

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34.8.3 Standard Curves

34.8.3.1 Procedure

a. Use prepared plates that have met all QC requirements. Label the platesappropriately with antibiotic concentrations. Use triplicate plates for each

concentration required for the standard curve, except for the standard reference (SR)concentration, for a total of 12 plates. Mark a vertical line on the side of the bottomof the plate for proper alignment of the stainless steel spider.

b. Place sterile spiders or 6 sterile cylinders (evenly spaced around the perimeter of the plate) on each plate. Align the spider orientation hole with the line previouslymarked on the plate. The well below the orientation hole designates the starting point. Use care with spider forceps when placing metal spiders on the agar. Spidersmay tear the agar if carelessly dropped or placed. Do not lift and re-place spiders ona plate.

c. Fill three alternate wells with the antibiotic SR concentration and the other three wells

with one of the four other concentrations of the antibiotic standard. Use 200 ± 4µl of solution for each well.

d. Incubate the plates 16 to 18 hours at the appropriate temperature(s) as described inSection 34.9.3.1.

e. After the plates have been incubated, remove the spiders or cylinders by inverting thetest plates over a bucket or tub. Cover the spiders or cylinders with distilled water.Spiders and cylinders must be decontaminated chemically or by autoclaving.Spiders must be carefully cleaned so that no organic, antibiotic, or chemicalresidues remain.

f. Read the diameters of the zones of inhibition to the nearest tenth of a millimeter usingthe zone reader or caliper. Start reading from the first well (marked line). Record thediameter of each zone of inhibition for both antibiotic concentrations on each plate.Also make the appropriate entry if no zone is produced. Record the data in anappropriate format such that the averages and corrected zones can be calculated.

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34.8.3.2 Calculations

Calculate the corrected standard curve and linear regression. This may be done manuallyor using an appropriate validated computer program. The manual method is described inthe Appendix 34A.5, Manual Calculation of a Standard Curve.

Apply a correction factor to compensate for between-plate variations. Each antibioticdilution value (except for the SR) will be calculated from a set of 3 plates.

Calculate cumulative average zones for all SR concentrations on the entire set of 12 plates.

For each set of three plates:

a. Average the readings of the nine SR zones and the nine readings of the other concentration used for the standard curve.

b. Subtract the nine-zone average of the SR from the cumulative SR average.

c. Algebraically add the resulting value to the value of the average zone reading for theantibiotic concentration used on that set of plates.

The standard curve is calculated on a semi-logarithmic graph (using the corrected values)with the zone diameters on the arithmetic scale and the antibiotic concentrations on thelogarithmic scale. Calculate the linear regression from the corrected standard curve. Usethe linear regression line for calculating values in quantitative analysis of test samples.

34.9 Bioassay Procedures for the Detection and Quantitation of Antibiotic Residues in Animal

Tissues

34.9.1 Sample Preparation and Storage

Tissues suspected of containing antimicrobials should be handled so that freezing and thawing

are kept to a minimum. Samples and sample extracts must be kept cold at all times withallowances for brief excursions at room temperature during processing and testing. Thelaboratory should take measures to ensure good housekeeping. Environmental conditions should be controlled so that they do not adversely affect the quality of the test results. Clean and disinfect the work area with an appropriate disinfectant. Take care to assure disinfectant residuedoes not contaminate testing materials (i.e. stainless steel spiders and cutting utensils). Use a

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clean set of equipment for cutting, weighing, or blending each tissue. Keep tissues isolated fromone another at all times to avoid commingling.

a. Use Tekmar ® filter bags, Whirl-Pak ® bags, or equivalent. Label a bag with the sampleidentification, tissue type, and buffer pH. Four bags will be required for each tissue.

b. Dice tissue into 0.5 cm pieces or homogenize a large enough portion of the sample tocomplete all anticipated tests. Use a blender (or similar apparatus) to homogenize the

samples. The frozen, homogenized tissue portion may be used for up to 14 days after preparation. If possible, retain an intact portion of the frozen tissue as a sample reserve.

c. Weigh sample portions into the four, labeled bags. If a large amount of sample (i.e. >

100 g) is available, weigh 10± 0.2 g of tissue (avoid fat) into each bag. If a small amount

of sample (i.e. < 100g) is available, use 5± 0.1 g per bag and make a note so that a proportional amount of diluent is added. Keep the sample to buffer ratio at 1 part sampleto 4 parts buffer.

It is suggested that the sample bags be set up in a manner to prevent confusion and facilitate sample preparation. An example of how to separate the bags into racks by pHof the buffer is shown below.

Rack No. 1(Buffer pH4.5)

Rack No. 2(Buffer pH6.0)

Rack No. 3(Buffer pH 8.0,0.1M)

Rack No. 4(Buffer pH 8.0,0.2M)

Muscle Muscle Muscle Muscle

Kidney Kidney Kidney Kidney

Liver Liver Liver Liver

d. For 10 gram samples, dispense 40± 1.0 ml of the appropriate buffer into each bag. For 5

gram samples, use 20 ± 0.5 ml of buffer. Stomach the diced muscle tissue samples for 60seconds. Stomach the diced kidney and liver tissues for 30 seconds. Thoroughly mixsamples into the buffer if the tissues have already been homogenized. After stomachingor mixing, allow the tissue to extract/settle for a minimum of 45 minutes before use.Follow the assay procedure as described below. The sample extracts should berefrigerated if they will be held for more than 2 hours before use.

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e. The extracts may be stored refrigerated for 24 hours, or frozen for 14 days for additionaltesting. Reserve tissues should be held for the time prescribed by the laboratory.

34.9.2 Preparation of Spiked Tissues

In order to validate a new assay or changes to an assay, to detect the presence of a new antibiotic

in tissue, or to prepare a daily process control sample, it is necessary to analyze tissues whichhave been artificially inoculated (spiked) with the antibiotic to be assayed. Follow the proceduredescribed below:

a. Select only those tissues from food animals that are known to be free of antimicrobials.

b. Prepare individual tissue samples and inoculate each with a known quantity of antibiotic.Individual samples may be inoculated with different types and quantities of antibiotic(s).Select concentration(s) that give a response that is expected to fall within the range of concentrations used for preparing the standard curve.

c. Follow the extraction procedures (Section 34.9.1) for these samples in parallel with non-inoculated (non-spiked) samples.

d. Determine the recovery of the antibiotic from the spiked tissues following the proceduresdescribed for sample quantitative analysis (Section 34.9.3.4). This information may beused in the laboratory’s control charts.

34.9.3 Performing the Seven Plate Bioassay

The bioassay can be performed in several combinations. A screening test may be done todetermine the type of antimicrobial residue present, followed by the appropriate quantitativeanalysis. Alternatively, the full seven-plate bioassay may be done on the sample(s),accomplishing both identification and quantitation at the same time. If the antimicrobial residue present is already known, the quantitative step may be performed to determine the concentrationof the residue. Each of these 3 approaches is described below. The individual assays aredescribed in Section 34.10. Instructions on reading the plates are given in Section 34.11.Interpretation of results is found in Section 34.12.

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Note: it is important to decontaminate spiders or cylinders after use. Chemical disinfection or sterilization by autoclaving may be used. In either case, it is essential that the spiders becarefully cleaned so that no organic, antibiotic, or chemical residues remain.

34.9.3.1 General Instructions

a. Obtain a sufficient number of plates and spiders for the assays that are to be performed.

Label the plate lids according to the extracts under analysis. Mark a vertical line onthe side of the bottom of the plate for proper alignment of the stainless steel spider.

The following plates are used with the extracts listed to test for each of the standard antibiotic residues. For the full assay, place the plates in the sequence listed.

Plate 1 BC (tetracycline detection – pH 4.5 buffer extract)Plate 2 KR (beta-lactam detection – pH 6.0 buffer extract)Plate 3 KR+P (penicillin confirmation – pH 6.0 buffer extract)Plate 4 BS (streptomycin detection – pH 8.0, 0.1M buffer extract)Plate 5 KR-11 (macrolide detection – pH 8.0, 0.2M buffer extract)Plate 6 KRER (erythromycin confirmation – pH 8.0, 0.2 M buffer extract)Plate 7 SE (aminoglycoside detection – pH 8.0, 0.1M buffer extract)

b. To perform the assay:

• Using a pair of spider forceps, GENTLY place one sterile spider on each plate.Align the spider orientation hole with the line previously marked on the plate. Thewell below the orientation hole designates the starting point.

• Fill the wells with 200 ± 4 μl of the appropriate test sample extract or SR.

• Place the lids on the plates and incubate plates 1 through 6 at 29± 1°C for 16 to

18 h. Incubate plate 7 at 37± 1°C for 16-18 h.

• After the plates have been incubated, remove the spiders or cylinders by invertingthe test plates over a bucket or tub. Cover the spiders or cylinders with distilled water. Decontaminate the spiders and cylinders chemically or by autoclaving.Carefully clean the spiders so that no organic, antibiotic, detergent, or chemicalresidues remain.

NOTE: If an error is made in dispensing an extract, repeat the procedure with a fresh plate.

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34.9.3.2 Screening for Identification of Antimicrobial Residues.

a. Arrange the full set of seven plates in the order described above (Section (34.9.3.1),and label them appropriately. Place a spider or a set of cylinders on each plate.

b. Use the extracts prepared as described in Section 34.9.1. Fill one well on each of the

seven plates with 200± 4 μl of the appropriate buffered sample extract. Repeat for additional samples. Make sure that a record is kept of the sample placement in thewells.

c. Use the following antibiotic SRs in one well each day the screen test is run: Plate 1,tetracycline; Plates 2 and 3, penicillin; Plate 4, streptomycin; Plates 5 and 6,

erythromycin; Plate 7, neomycin or gentamicin. Pipette 200± 4 μl of the SR concentration into the test well. When screen-testing samples, Sensi-Discs may beused on a separate plate in place of using reference dilutions on each plate.

d. Incubate as described in Section 34.9.3.1.

e. After incubation, remove the spiders or cylinders. Read and record the zones (or absence of zones) on each of the seven plates as described in 34.11 “Reading theBioassay.”

34.9.3.3 Identification and Quantitative Analysis Using the Full 7-plate Bioassay

a. For each tissue to be analyzed arrange the full set of seven plates in the order described above (Section 34.9.3.1), and label them appropriately.

b. Place a spider or a set of 6 cylinders on each plate, aligning the spider orientation

grove with the mark on the bottom edge of the plate.

c. Moving clockwise from the starting well, fill three alternate wells with 200± 4 μl of the appropriate buffered sample extract. Fill the other three with the appropriate SR antibiotic dilution. The entire plate will be used for each tissue. Refer to the listunder Section 34.9.3.1 (General Instructions) to match the plates, extracts, and SR antibiotics. Include a plate with a blank tissue (negative) and a positive (spiked)

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tissue to indicate the degree of precision and accuracy of analysis. This is in additionto the SR controls that are used on each plate.


e. After incubation, remove the spider or cylinders and read and record the zones (or absence of zones) on each of the seven plates as described in Section 34.11 “Reading

the Bioassay.”

f. Use the appropriate standard curve to calculate the sample test results for quantitativedetermination.

Note: Some plates may identify multiple antibiotics. Further testing may be necessary todetermine the specific antibiotic present. If the antibiotic identified differs from thestandard reference antibiotic that was used in this assay proceed to Section 34.9.3.4 toquantitate the antibiotic.

34.9.3.4 Quantitative Analysis of Known Antibiotic Residues in Tissue

a. To determine the amount of known antibiotic residue in the tissue extract use the plate and buffered extract specific for that residue.

b. Use at least one plate for each individual muscle, kidney, and liver sample extract.

c. Moving clockwise from the starting well, fill three alternate wells with 200± 4 μl of the buffered sample extract. Fill the other three with the reference concentration of the known antibiotic standard solution. Include a plate with a blank tissue (negative)and a positive (spiked) tissue to indicate the degree of precision and accuracy of analysis. This is in addition to the SR controls that are used on each plate.


e. After incubation, read and record the zones (or absence of zones) on each of theseven plates as described in 34.11 “Reading the Bioassay.”

f. Use the appropriate standard curve to calculate the sample test results for quantitativedetermination.

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34.10 Individual Antibiotic Assays

The following sections describe the use of each of the individual bioassay plates. The 7-plate bioassaywas designed to identify a limited number of antibiotics that were in use at a particular point in time.The antibiotic industry and chemical methods have both evolved since the bioassay was developed. Now many of the antibiotics must be identified, confirmed, or quantitated using chemical methods. The

role of the bioassay in some instances is simply to indicate the presence of a class of antibiotic.

34.10.1 Tetracyclines – Plate 1

This method is used in detecting the presence of all tetracyclines. This is followed by anidentification procedure to determine which tetracycline compound is present.

34.10.1.1 Performing the Assay

• Use Plate 1 (prepared with antibiotic medium No 8, Bacillus cereus ATCC 11778and penicillinase as described in Section 34.7.2.1).

•

Add 200 ± 4 μl of the sample extracted in 0.1M, pH 4.5 buffer to the test wells.• Tetracycline is the standard reference antibiotic for this plate.

• Incubate the plates at 29 ± 1°C for 16 to 18 hours.

34.10.1.2 Tentative Identification of Tetracycline in Tissue from the Seven Plate

Bioassay System

The tetracyclines are tentatively identified by zones of inhibition on BC Plate 1. Whenthe concentration of tetracycline is low, there may be no zones of inhibition (ZI) on anyother plate. Various concentrations of tetracyclines may produce zones on any or all of the plates except the Plate 7. The specific tetracycline compound may be identified using

HPLC following the method in the USDA, FSIS Chemistry Laboratory Guidebook,Method # CLG-TET2 or other appropriately validated method.

34.10.1.3 Quantitation of Tetracycline in Tissue

After the residue is identified, perform a quantitative analysis on Plate 1 using theidentified antibiotic as the standard reference (SR). Use the appropriate standard curve tocalculate the sample test results as described in Section 34.13.

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34.10.2 Penicillin and Antibiotics Giving the Penicillin Pattern – Plates 2 and 3

34.10.2.1 Performing the Assay

• Use Plate 2 (prepared with K. rhizophila ATCC 9341a, antibiotic medium No. 2without penicillinase) and Plate 3 with penicillinase (prepared as described in Section

34.7.2.2. and 34.7.2.3).• Add 200 ± 4 μl of the sample extracted in 0.1M, pH 6.0 buffer to the test wells.

• Penicillin G potassium salt is the reference antibiotic for these plates.


34.10.2.2 Tentative Identification of Penicillin and/or Penicillin-Like Residues in

Tissue from the Seven Plate Bioassay System

The presence of penicillin in a sample is indicated by zones of inhibition on Plate 2 and no zone on Plate 3. The identity of residues matching this plate pattern may beconfirmed by additional testing.

34.10.2.3 Quantitation of Penicillin or Penicillin-Like Residues in Tissue

Upon identification of the penicillin-like residue, perform quantitative analysis. Use thesample zone size, the Standard Reference (SR) zone size, and the standard curve of theidentified antibiotic to calculate the sample test results for quantitative determination.Examples of other penicillin-like antibiotics include ampicillin and amoxicillin. See theStandard Curve Summary Table in Section 34.14 for additional antibiotics.

34.10.3 Streptomycin and Dihydrostreptomycin - Plate 4

34.10.3.1 Performing the assay

• Use plate 4 prepared with B. subtilis ATCC 6633 and antibiotic medium No. 5 (SeeSection 34.7.2.4).

• Add 200 ± 4 μl of the sample extracted in 0.1 M, pH 8.0 buffer to the test wells.

• Streptomycin is the reference antibiotic for this plate.


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34.10.3.2 Tentative Identification of Streptomycin or Dihydrostreptomycin in Tissue

from the Seven Plate Bioassay System

The presence of streptomycin or dihydrostreptomycin is tentatively identified by zones of inhibition on BS Plate 4. There will be no zones on any other plates if the residue is present at a low concentration. If it is in high concentration, there may be zones on Plates1 and Plate 6. There should be no zones of inhibition on Plates 2, 3, 5, and 7.

Identification must be confirmed by other methods. Spectinomycin and lowconcentrations of fluoroquinolones may produce similar patterns. The specific antibioticmust be confirmed by chemical tests.

34.10.3.3 Optional: Use of an Additional Plate with a Resistant Organism for

Streptomycin Confirmation

Use plates prepared with Antibiotic Medium No. 5, K. rhizophila ATCC 9341a (which isresistant to streptomycin), and penicillinase to test the sample extracted in 0.1 M, pH 8.0 buffer. This plate is prepared in the same manner as the other K. rhizophila plates. Add sample and standard reference antibiotic as described in Section 34.10.3.1. There should be no zone of inhibition produced by the sample or the streptomycin SR.

34.10.3.4 Quantitation of Streptomycin or Dihydrostreptomycin in Tissue

Upon confirmation of streptomycin or dihydrostreptomycin, perform quantitativeanalysis using Plate 4. Use the sample zone size, the SR antibiotic zone size, and theappropriate standard curve to calculate the quantity of streptomycin in the sample asdescribed in Section 34.13.

NOTE: At times, both streptomycin and penicillin may be present in the same tissue. Inthat case, there will be zones of inhibition on Plates 2 and 4. Measure the zones and calculate the antibiotic contents of the sample for penicillin from Plate 2 and for confirmed streptomycin or dihydrostreptomycin from Plate 4.

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34.10.4 Erythromycin and Tylosin – Plates 5 and 6


• Use Plate 5 (prepared with K. rhizophila ATCC 9341a, and Antibiotic Agar No. 11),and Plate 6 (prepared with K. rhizophila ATCC 15957 and Antibiotic Agar No. 11)(Sections 34.7.2.5 and 34.7.2.6).

• Add 200 ± 4 μl of the sample extracted in 0.2 M, pH 8.0 buffer to the test wells.• Erythromycin is the reference antibiotic for this assay.


34.10.4.2 Identification and Confirmation of Erythromycin in Tissue from the Seven

Plate Bioassay System

The presence of erythromycin in tissue is indicated by zones of inhibition on KR-11 Plate5 and no zones of inhibition on KRER Plate 6. Use the zone size on Plate 5 and theappropriate standard curve to calculate the quantity of erythromycin residue present in thesample as described in Section 34.13.

34.10.4.3 Quantitation of Erythromycin in Tissue

Upon confirmation of erythromycin, use the sample zone size on Plate 5, theerythromycin SR zone size, and the appropriate standard curve to calculate the quantityof erythromycin in the sample. This plate is also used to calculate the quantity of tylosinresidue in tissue using the tylosin SR dilution as described in the Standard CurveSummary Table in Section 34.14.

34.10.5 Neomycin and Gentamicin – Plate 7


• Use Plate 7 (prepared with antibiotic agar No. 11,S. epidermidis ATCC 12228 and penicillinase as described in Section 34.7.2.7).

• Add 200 ± 4 μl of the sample extracted in 0.1 M, pH 8.0 buffer to the test wells.

• Neomycin is the reference antibiotic for this assay.


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34.10.5.2 Identification of Neomycin in Tissue from the Seven Plate Bioassay System

Neomycin residue present in tissue at low concentrations (<1.0μg/g) produces zones of inhibition only on Plate 4 and 7. The largest zones of inhibition are usually observed onPlate 7. At higher concentrations, neomycin may also produce zones of inhibition onadditional plates, even all 7 plates. Identification of neomycin must be confirmed usingadditional tests. Other macrolides and aminoglycosides, such as tylosin and gentamicin,

may produce similar patterns. Chemical tests must be used to establish the identity of theantibiotic.

34.10.5.3 Quantitation of Neomycin in Tissue

Use the sample zone size on Plate 7, the neomycin SR antibiotic zone size, and theappropriate standard curve to calculate the quantity of neomycin residue present in thesample as described in Section 34.13. This plate is also used to calculate the quantity of gentamicin residue in tissue using the gentamicin SR dilution as described in theSummary Table in 34.14.

34.11 Reading the Bioassay

a. After the plates have been incubated, remove spiders or cylinders by inverting test plates over a bucket or similar receptacle. Cover the spiders or cylinders with distilled water. Spiders and cylinders must be decontaminated chemically or by autoclaving. Spiders must be carefullycleaned so that no organic, antibiotic, or chemical residues remain.

b. Arrange test plates according to numerical sequence of the plates.

c. Align the zone reader at ‘0’ and, starting clockwise from the first well (marked line), read the zone of inhibition using the dial on the zone reader. Alternatively, calipers may be used. Record thediameter to the nearest tenth of a millimeter of each zone of inhibition for both the unknown and thestandard reference antibiotic. Also, make the appropriate entry if no zone is produced.

34.12 Additional Information on Test Interpretation

To identify an antibiotic, the pattern of the zones of inhibition produced by a sample should be compared with the patterns produced by known antibiotics. The presence and/or absence of zones of inhibition on

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each of the 7 plates and the relative sizes of the zones are used in interpreting the results. The patternobtained may not match known patterns. In that case, alternative approaches may be taken. Further testing may be necessary using more dilute sample preparations, or using other methods of analysis.Some concentrations of antibiotic residues may produce patterns that differ from typical patterns for thatantibiotic. Antibiotics, other than the ones described here, may produce similar or different patterns tothe ones described under the individual antibiotic tests. Further discussion is found in Section 34.15“Additional Testing Procedures.”

34.13 Calculating the Concentration of Antibiotic Residue in Tissue

The zone sizes on the test plate are compared to the standard curve to calculate the residue concentrationin the test sample. To compensate for day-to-day and plate-to-plate variations, the sample zone sizemust be adjusted so that it is comparable to the standard curve. To calculate the antibiotic concentrationin a test sample, use the steps described below. Calculations may be done manually or with a validated computer program.

a. Adjust the test sample zone size

• For each plate, average the zone readings of the SR concentration and of the sample.

•

Subtract the zone average for the SR on the test plate from the average standard curve SR concentration.

• Algebraically add the resulting value to the value of the average zone reading for the testsample.

Example 1:The average zone size of the SR concentration on the standard curve = 20.5The average zone size of the SR concentration on the test plate = 19.1The difference = 1.4

The average zone size of the test sample = 17.3.

Add 1.4 to 17.3 = adjusted zone size of 18.7. This is used to determine the concentration of theresidue in the sample from the standard curve.

Example 2:The average zone size of the SR concentration on the standard curve = 20.5The average zone size of the SR concentration on the test plate = 21.1

The difference = −0.6

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The average zone size of the test sample = 17.3.

Add ‘−0.6’ to 17.3 = adjusted zone size of 16.7. This is used to determine the concentration of the residue in the sample from the standard curve.

b. Calculate the residue concentration using the adjusted sample zone size.

• With the adjusted value of the sample on the arithmetic scale for the zone diameters of the

standard curve determine the concentration of antibiotic from the log scale for the antibioticconcentration. This may be done using a validated computer program.

• In calculating the final concentration in a tissue sample, take into consideration the dilutionfactor of the sample extract. This is usually five (one part of sample to 4 parts of buffer).

• In those instances where the sample zone size exceeds the highest concentration on thestandard curve, dilutions of the extract may be made so that the test concentration falls withinthe linear range of the standard curve.

• Use the appropriate current standard curve in your calculations. Round off the calculated concentration to the nearest hundredth microgram.

34.14 Summary of Antibiotic Reference Standards

In order to assist in the antibiotic bioassay, a summary table is presented, describing appropriateantibiotic plates, microorganisms, buffers, and antibiotic standard concentrations necessary to preparestandard curves for many antimicrobials with or without an established antibiogram pattern. A standard curve prepared by using these specifics may be used to quantitate the amount of an antimicrobial residuein a tissue. For some antibiotics, the FDA NADA (New Animal Drug Application) method must be used for quantitation for regulatory purposes.

In the table below, the first concentration shown for a standard curve (column four) is used for demonstrating cell lawn sensitivity. The concentration shown in bold (middle dilution) is the SR concentration. A key to the bioassay plate composition is found below the table. Note that Plates 3

and 6 are confirmatory-type plates and are not used for standard curves.

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Standard Curve Summary Table

Antimicrobial Bioassay Plate*Antibiotic Buffer Diluent

Concentrations (μg/ml) used for theStandard Curve

Ampicillin Plate 2, KR 0.1 M, pH 6.0 0.01, 0.02, 0.04, 0.08, 0.16

Bacitracin Plate 2, KR 0.1 M, pH 6.0 0.2, 0.4, 0.8, 1.6, 3.2

Chloramphenicol Plate 2, KR 0.1 M, pH 6.0 4.0, 8.0, 16.0, 32.0, 64.0

Chlortetracycline iii Plate 1, BC 0.1 M, pH 4.5 0.01, 0.02, 0.04, 0.08, 0.16

Cloxacillin Plate 2, KR 0.1 M, pH 6.0 0.32, 0.64, 1.28, 2.56, 5.12

Dihydrostreptomycin iv Plate 4, BS 0.1 M pH 8.0 0.15, 3.0, 6.0, 1.2, 2.4

Erythromycin i Plate 5, KR-11 0.2 M, pH 8.0 0.05, 0.1, 0.2, 0.4, 0.8

Flavomycin ii Plate 1, BC 0.1 M, pH 4.5 0.05, 0.1, 0.2, 0.4, 0.8

Gentamicin Plate 7, SE 0.1 M, pH 8.0 0.1, 0.2, 0.4, 0.8, 1.6

Hygromycin Plate 4, BS 0.2 M, pH 8.0 5.0, 10.0, 20.0, 40.0, 80.0

Lincomycin Plate 5, KR-11 0.2 M, pH 8.0 0.3, 0.6, 1.2, 2.4, 4.8

Neomycin Plate 7, SE 0.1 M, pH 8.0 0.25, 0.5, 1.0, 2.0, 4.0

Novobiocin Plate 7, SE 0.2 M, pH 8.0 0.25, 0.5, 1.0, 2.0, 4.0

Oleandomycin Plate 5, KR-11 0.2 M, pH 8.0 0.25, 0.5, 1.0, 2.0, 4.0

Oxytetracycline iii Plate 1, BC 0.1 M, pH 4.5 0.08, 0.16, 0.32, 0.64, 1.28

Penicillin Plate 2, KR 0.1 M, pH 6.0 0.01, 0.02, 0.04, 0.08, 0.16

Spectinomycin Plate 4, BS 0.2 M, pH 8.0 10, 20, 40, 80, 160

Streptomycin iv Plate 4, BS 0.1 M, pH 8.0 0.1, 0.2, 0.4, 0.8, 1.6

Sulfamethazine Plate 5, KR-11 0.2 M, pH 8.0 30, 60, 120, 240, 480Tetracycline iii Plate 1, BC 0.1 M, pH 4.5 0.08, 0.16, 0.32, 0.64, 1.28

Tilmicosin i Plate 5, KR-11 0.2 M, pH 8.0 0.1, 0.2, 0.4, 0.8, 1.6

Tylosin Plate 5, KR-11 0.2 M, pH 8.0 0.2, 0.4, 0.8, 1.6, 3.2

Virginiamycin Plate 5, KR-11 0.2 M, pH 8.0 1.28, 2.56, 5.12, 10.24, 20.48

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i. Dissolve the antibiotic powder in a small volume of methanol or 30% methanol before diluting it inthe phosphate buffer solution.ii. Dissolve the antibiotic powder in a small volume of methanol or 50% methanol before diluting itin the phosphate buffer solution.iii. Dissolve the tetracycline powder in 0.01 N hydrochloric acid, to make the 1000 ppm solution, before preparing the dilutions.iv. Dissolve streptomycin and dihydrostreptomycin powder in distilled water to make the 1000 ppm

solution before preparing dilutions.

*Bioassay Plate Identity Plate Composition

Plate 1, BC B. cereus ATCC 11778 in Antibiotic Medium 8Plate 2, KR K. rhizophila ATCC 9341a in Antibiotic Medium 2Plate 4, BS B. subtilis ATCC 6633 in Antibiotic Medium 5Plate 5, KR-11 K. rhizophila ATCC 9341a in Antibiotic Medium 11Plate 7, SE S. epidermidis ATCC 12228 in Antibiotic Medium 11

34.15 Additional Testing Procedures

34.15.1 ELISA Tests

Enzyme-Linked Immunosorbent Assay (ELISA) tests may be used as screen tests or to identifythe antibiotic residues present in a sample. ELISA tests for sulfonamides are used for screeningsuspect tissues. ELISA tests for gentamicin, neomycin, and tylosin are currently used to identifyresidues that give bioassay patterns indicative of these antibiotics. Commercially available testsmay be used. If the test is not designed for use with tissue extracts, it must be validated for thetissues with which it will be used. Non-commercial tests may be used with appropriatevalidation or equivalency studies as prescribed by the laboratory’s operating procedures. AllELISA tests are validated for the intended application before they are used. In addition, each lotmust pass lot acceptance criteria. Lot acceptance criteria may be found in the laboratory SOPs.Positive and negative tissue controls must always be used when running ELISA tests.

34.15.2 Chemical Methods of Antibiotic Residue Confirmation and/or Quantitation

The FSIS Chemistry Laboratory Guidebook describes chemical methods that may be used toidentify and/or quantitate some residues. Tissues that give a pattern indicative of the presence of aminoglycosides can be tested to identify the antibiotic present using the LC/MS/MS method CLG-AMG1. Amikacin, apramycin, dihydrostreptomycin, gentamicin, hygromycin, kanamycin,

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neomycin, spectinomycin, streptomycin, and tobramycin are among the antibiotics that can beidentified by this method. Macrolides and lincosamides can be identified using the LC/MS/MSmethod CLG-MAL, including clindamycin, erythromycin, lincomycin, pirlimycin, tilmicosin,and tylosin. Fluoroquinolones, including ciprofloxacin, difloxacin, enrofloxacin, norfloxacin,danofloxacin and sarafloxacin can be identified using the CLG-FLQ2. Tilmicosin can beidentified using method CLG-TIL2, and quantified using method CLG-TIL1. Florfenicol can beidentified using CLG-FLOR2 and quantitated using CLG-FLOR1. Chloramphenicol can be

identified and quantitated using CLG-CAM. Many sulfonamide residues can be identified and quantified using the method CLG-SUL4.

Additional chemical methods of identifying and quantitating antibiotic residues may also beavailable.

34.16 References

Brun, Y., J. Fleurette, and F. Forey. 1978. Micromethod for biochemical identification of coagulase-negative Staphylococci. J. Clin. Microbiol.8(5):503-508.

Food and Drug Administration. 1968. Information for Assay and Reporting of Data Pertaining toAntibiotic Residues in Milk, Dairy Products, and Animal Tissue. Revised. Food and DrugAdministration, Washington, DC.

Fugate, H. G. 1974. Determination of antibiotic residues in animal tissues. In MicrobiologicalLaboratory Guidebook, USDA, Food Safety and Inspection Service, Washington, DC.

Gemmel, C. G., and J. E. Dawson. 1982. Identification of coagulase-negative Staphylococci with theAPI system. J. Clin. Microbiol. 16(5):874-877.

Helrich, K. (ed.). 1990. Official Methods of Analysis of the Association of Official Analytical Chemists.15th Edition. Published by the Association of Official Analytical Chemists, Inc., Gaithersburg, MD20877.

Jonas, M., J. B. Comer, and B. A. Cunha. 1984. Tetracyclines, p. 219-234. In A. M. Ristuccia and B. A.Cunha (ed.), Antibiotic Therapy. Raven Press, New York, NY.

Kotrina, A. et. al. 2000. Epidemiology and pathogenesis of Bacillus cereus infection. Microbes Infect.2:89-198.

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Kramer, J., G. G. Carter, B. Arret, J. Wilner, W. W. Wright, and A. Krishbaum. 1968. AntibioticResidues in Milk, Dairy products, and Animal Tissues, p. 344-837. U. S. Government Printing Office,Washington, DC.

Official Methods of Analysis of AOAC International (2000) 17th

Ed., AOAC International,Gaithersburg, MD, USA, Official Method 957.23, Part F.

Oka, H.; Suzuki, M.; Martz, V.; MacNeil, J. D.; Harada, K. I.; Ikai, Y.; Hayakawa, J.; Masuda, K. 1993Journal of Agricultural and Food Chemistry41: 410-415 Improvement of chemical analysis of antibiotics. 18. Identification of residual tetracyclines in bovine tissues by TLC/FABMS with a samplecondensation technique.

Reamer, R., B. P. Dey, and N. Thaker. 1995. Cryopreservation of bacterial vegetative cells used inantibiotic assays. J. Assoc. Off. Anal. Chem.78(4):997-1001.

Reynolds, J. E. F. (ed.). 1989. Martindale The Extra Pharmacopoeia, pgs. 132 and 297. 29th Edition.Published by the Pharmaceutical Press, London, England.

Ristuccia, A. M. 1984. Aminoglycosides, p. 305-328. In A. M. Ristuccia and B. A. Cunha (ed.),Antimicrobial Therapy. Raven Press, New York, NY.

Tang, Jane S. and Gillevet, Patrick M. Reclassification of ATCC 9341 from Micrococcus luteus toKocuria rhizophila. Int. J. Syst. Evol. Microbiol. 53 (2003) 995-997 International Union of Microbiological Societies.

USDA Food Safety and Inspection Service, Chemistry Laboratory Guidebook, available online at:http://www.fsis.usda.gov/OPHS/clg/index.htm

Yamai, S., O. Yasushi, T. Nikkawa, Y. Shimada, and Y. Miyamoto. 1981. Preservation and transportation of bacteria by a simple disk method. J. Clin. Microbiol.14:61-66.

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34.A Appendix

34.A.1 General Information

The material in this appendix is included primarily for informational purposes. FSIS laboratories do notroutinely use these procedures. Some of the materials and techniques may be outdated. The informationis included for reference. However, these procedures (or similar procedures) may be used when needed.

34.A.2 Safety Precautions

The microorganisms used in the bioassay are Biosafety Level 1 and 2 organisms. Safe laboratory practices should be followed in working with all organisms. Exercise caution in working with hazardouschemicals by wearing appropriate protective safety clothing, gloves and eyeglasses or face shields.Consult a Material Safety Data Sheet (MSDS) for each hazardous chemical before working with it.Collect hazardous chemical wastes in separate containers. Dispose of them in accordance with thestandard chemical waste management procedures for your laboratory. FSIS personnel should follow theFSIS Laboratory Environmental, Health, and Safety Handbook.

34.A.3 Preparation, Storage, and Use of Vegetative Cell Cultures for Assay

34.A.3.1 Equipment, Materials, Reagents, and Media (for 34.A.3 and 34.A.4)

Equipment and Materialsa. Bausch & Lomb Spectronic 20 or comparable spectrophotometer

b. Refrigerator, 2 to 8 °C

c. Freezer capable of maintaining –15°C

d. Incubators capable of maintaining temperatures of 29± 1°C, 35 ± 1°C, and 37 ± 1°C

e. Water bath, 40 to 48°C range with ± 2°C of set temperaturef. Colony counter

g. Analytical balanceh. General laboratory balance 0.1 to 200 g capacityi. Microscope, at least 400X magnification j. Centrifuge, super speed k. Centrifuge, refrigerated, 1500 to 5000X gravity capacityl. Magnetic Stirrer – Heated m. Vortex mixer n. Roux Bottles (optional)

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o. Sterile glass beads p. Homogenizer or blender q. Virtis jars or homogenizer jarsr. Centrifuge tubes, 50 mls. Tubes with caps, 12 x 75 mm plastic storage typet. Assorted tubes, graduated cylinders volumetric flasks, volumetric pipettesu. Petri dishes 100 x 15 mm

v. Erlenmeyer flasks, 250 mlw. Semi-logarithmic graph paper x. Autoclave

Reagents and Mediaa. Hydrogen Peroxide 3% b. Glycerolc. Methylcellulose (See Section 34A.3.5.3b)d. Dehydrated Alcohol, Ethyl Alcohol, 200 Proof, or 50% ethyl alcohol in purified water e. 3 M, pH 7.1 phosphate buffer (recipe in Section 34.A. 4.2 e.) or density gradient forming

agents, such as Percollf. Polyethylene glycolg. A-K Agar # 2 (Sporulating Agar) with extra 0.5% purified agar h. Blood agar plates (Sheep blood in Tryptose Soy Agar)i. Plate count agar (PCA) j. Antibiotic Media No’s 1, 8, and 11k. Standard reference antibiotics: penicillin, erythromycin, neomycin, and streptomycinl. Brain Heart Infusion Broth regular strength and 2X (double strength)m. Tryptose soy agar (TSA)n. Gram stain reagentso. Sterile physiological saline (0.85% NaCl) p. Biochemical identification systems such as API Staph Trac, VITEK, MicroId, or biochemical

reagents as needed to identify the microorganisms per SOP LW-0015:q. Phenol Red Dextrose Broth (optional)r. Phenol Red Mannitol Broth (optional)s. Phenol Red Lactose Broth (optional)t. Trypticase Soy Agar slants or plates (TSA)u. Butterfield’s phosphate buffer v. Glycerol- saline (See Section 34.A.3.5.3)w. Sodium chloride and magnesium sulfate for preparation of reagents listed above

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x. O/F glucose brothy. Methyl Red/Voges-Proskauer (MR/VP) test brothz. Mannitol brothaa. Malachite green stain bb. Safranin staincc. Mannitol Egg Yolk Polymyxin (MYP) Agar (Difco agar base with 50% egg yolk

enrichment, and antimicrobic vial P) (Optional)

dd. Motility Medium

34.A.3.2 Test Organisms

The following test organisms are used in the preparation of agar plates used for the standard curves and the seven-plate bioassay system. Use aseptic technique when working with all bacterial cultures.

a. Kocuria rhizophila, ATCC 9341a (KR) b. Kocuria rhizophila, ATCC 15957 (KRER)c. Staphylococcus epidermidis, ATCC 12228 (SE)d. Bacillus cereus var. mycoides spores, ATCC 11778 (BC)e. Bacillus subtilis spores, ATCC 6633 (BS)

34.A.3.3 Determination of Purity and Properties of Vegetative Test Organisms

34A.3.3.1 Purity and Identity

All cultures must be confirmed to be pure and their identity biochemically confirmed before they are acceptable for use. Manufacturer’s certificates are acceptable for commercial preparations. Laboratory tests for purity and biochemical confirmation may be done as described below. Appropriate commercial biochemical test kits may be used in place of the traditional biochemical tests described. Before preparing cell suspensionsfrom fresh or lyophilized cultures for assay, use a portion of each test organism todetermine its purity and other important characteristics as follows:

a. Streak two plates of tryptose soy agar each with K. rhizophila ATCC 9341a (KR),K.

rhizophila ATCC 15957 (KRER), and S. epidermidis ATCC 12228 (SE), streaking

for isolation. Incubate the plates overnight at 35°C to 37°C.

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b. Examine the plates for purity. Check the color of colonies. Colonies of both strainsof Micrococcus are yellow, and colonies of Staphylococcus are white. Prepare aGram stain of each test organism. All cultures are Gram positive. Micrococcus spp.usually appear as cocci in packets of eight. S. epidermidis appear as cocci in irregular clusters.

c. Check biochemical reactions of the cultures. Use one TSA plate of each organism to

test for presence of catalase. Place a drop of hydrogen peroxide on the plate and observe for bubble formation. Generation of bubbles indicates a positive catalasetest. All organisms are catalase positive. Select isolated colonies from the second

TSA plate and inoculate dextrose, lactose, and mannitol broths. Incubate at 35± 1°Covernight. The biochemical patterns should be as follows:

Organism Dextrose Lactose Mannitol Catalase

SE + - - +

KR - - - +

KRER - - - +

34.A.3.3.2 Determination of Antibiotic Sensitivity

All organisms must be tested to confirm antibiotic sensitivity. Prepare bioassay plateslisted in the table below as described in Section 34.7with the exception that

penicillinase should not be added to the agar. Place a ‘spider’ or 5 cylinders on each plate. Add 200 microliters of the antibiotic concentrations specified in the table below toindividual wells. Each prepared plate will be tested with all five of the antibiotics listed inthe table. Antibiotics with a ‘>’ should not produce a zone of inhibition greater than 8mm at the indicated concentration on that plate. For the other antibiotic-plate

combinations, a measurable zone of inhibition of 8 to 15 mm should be produced at theconcentrations listed. Replace any test organism that is not consistently capable of meeting these criteria with a new ATCC culture.

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Antibiotic concentration in µg per ml Test Organism

Medium No. Erythromycin Neomycin Penicillin Streptomycin Tetracycline

K. rhizophila9341a

2 0.05 5.0 0.0125 > 200 0.5

K. rhizophila9341a

11 0.025 1.5 0.0125 250 0.4

K. rhizophila15957

11 > 200 1.5 0.0125 1.0 0.2

S. epidermidis12228

11 0.075 0.25 0.8 1.5 250

B. cereus11778

8 0.25 2.00 20 1.00 0.08

B. subtilis6633

5 0.04 0.25 0.0125 0.2 0.08

34.A.3.4 Propagation of Test Organisms (Non-spore Formers)

This procedure is used when cultures are transferred on a weekly basis instead of using preserved cultures.

a. Open a vial of each lyophilized culture:Kocuria rhizophila ATCC 9341a (KR), K. rhizophila ATCC 15957 (KRER), and S. epidermidis ATCC 12228 (SE). Suspend the organisms inseparate tubes of Brain Heart Infusion broth.

b. Incubate K. rhizophila ATCC 9341a (KR) and K. rhizophila ATCC 15957 (KRER) at 29

±1°C for 18 h and S. epidermidis ATCC 12228 (SE-TR/SE-SR) at 37 ± 1°C for 24 h.

c. After incubation, inoculate duplicate slants of Antibiotic medium # 1 withK. rhizophila ATCC 9341a, K. rhizophila ATCC 15957, and S. epidermidis ATCC 12228. Incubate allK.

rhizophila slants at 29 ± 1°C for 18-24 h and S. epidermidis slants at 37 ± 1 °C for 18-24 h.

d. Store the slants at 4-8°C and transfer cultures to fresh slants every other week. Use the same procedures described in step ‘c.’ above for culture transfers.

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34.A.3.5 Preparation and Preservation of Stock Cultures

Cultures may be preserved for long term storage instead of transferring them on a weekly basis,or using commercial preparations as described in 34.6.1

34.A.3.5.1 Preparation of Batch Cultures for Preservation

If results agree with criteria in Section 34.A. 3.3 at the next culture transfer:

a. Inoculate each culture onto two agar slants of the appropriate media described in ‘b’

below. Incubate 18-24 h at 35 to 37°C.

b. Wash the growth from each slant with 2 ml of sterile normal saline and transfer thesuspension to the surface of each of two Roux bottles containing 300 ml of the proper media.

i. Use Antibiotic Medium No.1 for K. rhizophila ATCC 9341a and S. epidermidis ATCC 12228.

ii. Use Antibiotic Medium No. 11 for K. rhizophila ATCC 15957.

c. Spread the suspension evenly over the entire agar surface with the aid of sterile glass beads.

d. Incubate for 18-24 h at 35 to 37°C.

e. Wash growth from the agar surface of each Roux bottle with 40 ml of sterile saline.(There are 2 Roux bottles for each culture. Keep each Roux bottle suspensionseparate at this point.) Place each suspension in a 50 ml centrifuge tube, and

centrifuge at 5000 x G for 30 minutes at 10± 2°C.

f. Pour off the supernatant, resuspend each pellet in 40 ml sterile saline, and centrifugeas above. Repeat one more time.

g. After the final centrifugation, pour off the supernatant and use 10 ml of sterile salineto resuspend the pellet from each tube of the three bacterial cultures. Pool together

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only the identical bacterium from the duplicate Roux bottles for a total of 20 ml of stock culture each. Do not mix the bacteria. Pool bacteria of the same strain only.

34.A.3.5.2 Determining Number of Colony Forming Units (cfu)

Follow the procedures described in Section 34.6.3 to determine the number of bacteria per milliliter in each fresh culture suspension.

34.A.3.5.3 Preservation of Vegetative Cultures

A procedure for preserving vegetative culturesin situ for readily preparing assay plateshas been developed. The procedure described here is simple and reliable for storingstandardized, non-spore-forming organisms for up to one year. The method is as follows:

a. Preparation of Glycerol-Saline Solution

• Add 8.5 g NaCl to 850 ml of distilled water.

• Mix on a magnetic stirrer until the solution is clear.

•

Add 150 ml of glycerol and mix until clear.• Autoclave for 15 minutes at 15 lbs. pressure (121°C). The glycerol-saline

mixture is now ready for use.

b. Preparation of Methylcellulose Solution

• Weigh out 12 g MgSO4 and dissolve in 1 liter of distilled water.

• Place the solution on a heated magnetic stirrer.

• Add, with stirring, 10 g of methylcellulose and slowly bring to a boil.

• Boil 5-10 minutes until small amorphous aggregates of methylcellulose areformed.

• Immediately autoclave for 15 minutes at 15 lbs. pressure and 121°C. On cooling,the methylcellulose disperses and stays in solution and is ready for use.

c. Aseptically pipette 15 ml of each stock culture containing at least 5 x 108 cfu/ml intothe sterile 250 ml Erlenmeyer flask and add 135 ml of either the glycerol-saline or themethylcellulose solution.

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d. Prepare racks containing 140, properly identified and labeled, 12 x 75 mm plasticstorage tubes with caps.

e. Mix the flask contents on a magnetic stirrer and then aseptically pipette 1 ml from theflask into each tube.

f. Close the tube caps securely.

g. Store properly labeled cultures according to conditions described in 34.A.3.5.4.

34.A.3.5.4 Shelf Life of Preserved Cultures

The prepared tubes may be handled according to the following chart:

OrganismStorageTime

Preserving Fluid Storage

Temperature

KR, KRER 1-5 months Glycerol-Saline 4°C

KR, KRER & SE 1-5 months Glycerol-Saline -14°C

KR, KRER & SE 1 year Methyl-Cellulose -14°C

34.A.3.5.5 Reconstituting Preserved Vegetative Organisms for Use

a. Thaw preserved methylcellulose or glycerol-saline preserved cultures slowly at roomtemperature.

b. Vortex until the suspension appears smooth.

c. Warm the suspension in a 40°C water bath, vortex again or aspirate eight times upand down using a 1 ml pipette. Inoculate the appropriate melted agar medium withthe required volume of the suspension based on stock cfu and desired cfu per plate.

d. If the culture is used for other tests, then pipette the remaining portion into a 3 ml

tube of 2x Brain Heart Infusion Broth. Incubate the tube at 29°C for 18-24 h.

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34A.3.6 Use of Fresh Vegetative Cultures in Assay

If preserved cultures are not available, fresh organisms from slants may be prepared and used for bioassay plates. The method described here is an AOAC recommended procedure for antibioticassays. However, the procedure is subjective and thus does not allow finer adjustment inorganism concentration to achieve desired assay sensitivity, which can be better achieved by

making dilutions of cell suspensions of known cfu/ml.

a. Maintain cultures by transferring onto Antibiotic Agar No. 1 slants as described in Section34.A.3.4.

b. Inoculate the surface of duplicate slants of Antibiotic medium # 2 withK. rhizophila ATCC9341a, and Antibiotic medium # 11 slants with S. epidermidis ATCC 12228 and K.

rhizophila ATCC 15957. Incubate all K. rhizophila slants at 29°C for 18-24 h and S.

epidermidis slants at 37°C for 18-24 h.

c. Turn on the spectrophotometer to warm it up. Set it to 580 nm. Transfer 2 ml of sterile

0.85% NaCl solution (saline) onto each fresh agar slant culture. Emulsify the cell suspensionthoroughly, taking care not to scratch the agar.

d. Pipette 1 ml of cell the suspension into 15 ml of saline. Vortex the new mixture. Blank thespectrophotometer with saline to 100% transmittance. Adjust the percent transmittance of the K. rhizophila suspensions to 14% and the transmittance of the S. epidermidis suspensionto 40% by adding saline or additional culture suspension as needed.

e. Add 0.5 ml of each cell suspension to the appropriate antibiotic medium in 100 x 15 mmPetri dishes to make assay plates. Do not use these cell suspensions more than 1 week.

f. If the bioassay plates appear contaminated, discard the cell suspension. Check the stock cultures, or purchase a new culture from ATCC.

34.A.4 Preparation of Suspensions of Spore Forming Test Organisms

B. cereus ATCC 11778 and B. subtilis ATCC 6633 may be maintained on agar by weekly transfers.Alternatively spore suspensions may be prepared for long term storage preserved in 50% ethanol. To

maintain cultures, grow B. cereus on slants of Antibiotic Medium No. 8. Incubate at 29 to 30°C

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overnight. Grow B. subtilis on slants of Antibiotic Medium No. 1. Incubate at 35 to 37°C overnight.Store the cultures between transfers at 2-8°C.

34.A.4.1 Determination of Purity and Properties of Spores

Before preparing spore suspensions for long-term storage, the purity and properties of thecultures must be confirmed. If the cultures are carried by regular subculture on agar slants, the

purity and properties must also be confirmed at regular intervals as described in the laboratoryquality system. To perform biochemical confirmation of either organism, use appropriate,recognized commercial test kits or follow the biochemical procedures described below.

a. If a lyophilized culture is used, reconstitute it in Brain Heart Infusion (BHI) broth. Streak thesuspension onto two plates of Tryptose Soy agar with 5% defibrinated sheep blood for isolation using a loopful of culture from the BHI broth or from the regular agar slant culture.Inoculate a Brain Heart Infusion agar slant at the same time. The optional MYP agar plateand motility medium may also be inoculated at the same time. Incubate all culturesovernight at the appropriate temperatures.

Examine the Blood Agar plates for purity. Note the size and type of hemolytic zone. B.cereus should produce a zone of beta hemolysis around the colonies. B. subtilis should not produce a zone of hemolysis.

b. Prepare a Gram stain and Malachite Green stain from three well-isolated colonies. Counter-stain the Malachite green slide with Safranin. The cells should be Gram positive. On theslide stained with Malachite green and Safranin spores will appear green and the vegetativecells will be red or pink. Endospores should be oval and centrally located. The spore should not distend the vegetative cell.

c. Use the BHI slant to test for catalase activity. Both organisms should be catalase positive.(A positive test is production of bubbles when a drop of hydrogen peroxide is added to thecell growth.)

d. Inoculate O/F glucose, Methyl Red/Voges-Proskauer (VP), and mannitol broths with isolated colonies from the blood agar plate. Incubate overnight and record results. The mannitol broth is not required if the MYP agar plate is used. They should agree with the followingchart:

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Organism β hemolysis O/F glucose Mannitol MR/VP

B. cereus Positive Oxidative & Fementative Negative Negative/Positive

B. subtilis Negative Oxidative Positive Positive/Positive

MYP and Motility Agar Results

Organism MYP lecithinase MYP mannitol Motility

B. cereus zone of precipitation Negative (pink) PositiveB. subtilis No zone Positive (yellow) Positive

Any organism that is not pure or does not meet the biochemical identification listed should bereplaced. NOTE: Any organism that is non-motile, lecithinase positive, mannitol negative,and non-hemolytic is possibly Bacillus anthracis. This organism is potentially lethal tohumans. Stop work with this culture and thoroughly disinfect all work surfaces with 0.5%sodium hypochlorite for at least 15 minutes.

34.A.4.2 Preparation of Spore Suspensions of Bacillus cereus ATCC 11778

a. Reconstitute a lyophilized culture of B. cereus ATCC 11778 in Brain Heart Infusion brothand incubate overnight at 35 to 37°C.

b. Transfer several well isolated colonies from the blood agar plate onto A-K Sporulating Agar

# 2 slants with extra 0.5% Purified Agar (Difco or equivalent) and incubate at 37°C.

c. Add 4 to 6 sterile glass beads and 2 to 3 ml of sterile distilled water to the agar slants. Shakethe slants gently for 2 minutes to dislodge the bacterial growth. Aseptically pipette the bacterial suspension into a Roux bottle containing 300 ml of A-K Sporulating Agar # 2 withextra 0.5% Purified Agar (Difco or equivalent) and spread over the surface. Multiple culturesmay be prepared and pooled for transfer to Roux bottles. Incubate the Roux bottles

horizontally at 35-37°C for 18 to 24 h, and then incubate at room temperature for theremainder of 1 week (6 days).

d. Harvest the growth from the Roux bottles by adding 20-30 sterile glass beads and approximately 25 ml of sterile distilled water per bottle. Gently agitate the bottle to dislodgethe bacterial growth. Care must be taken not to break the agar during harvesting. Asepticallytransfer this suspension to sterile centrifuge tubes (40 ml volume) and heat for 10 minutes in

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boiling water at 100°C. Wash the spore suspension three times with sterile distilled water bycentrifuging and decanting in the following manner:

i. Centrifuge at 5°C for 20 minutes at 27,000 x G.ii. Pour off supernatant.iii. Resuspend the pellet in 20 ml sterile distilled water.iv. Repeat Steps i, ii, and iii two more times.

e. The spores may be further purified using density gradient centrifugation. The method described here or other appropriate method may be used.

Prepare 3 M, pH 7.1 phosphate buffer.K 2HPO4 306.9 gKH2PO4 168.6 g0.1 N HCl or 0.1 N NaCl

Dissolve the potassium salts in distilled water. Adjust the pH with hydrochloric acid or sodium hydroxide if necessary to a final pH of 7.1. Bring the volume to 1.0 liter. Filter-

sterilize the solution.

f. Using 3 M, pH 7.1 phosphate buffer, wash and coat a sterile Virtis jar in the followingmanner:

Combine 34.1 ml of sterile 3 M, pH 7.1 phosphate buffer and 11.8 g of sterile polyethyleneglycol in a sterile 100 ml glass stoppered graduated cylinder or volumetric flask. Shakevigorously. Bring to volume with sterile distilled water. Pour the mixture into a sterile Virtis jar and place the jar on the homogenizer. Blend for 5 minutes at 5,000 RPM. Discard themixture. Repeat.

g. Combine 34.1 ml of sterile 3 M, pH 7.1 phosphate buffer and 11.8 g of sterile polyethyleneglycol in a sterile 100 ml glass stoppered graduated cylinder or volumetric flask. Add 25 mlof the washed spore mixture and bring to volume with sterile distilled water. Shakevigorously. Pour the mixture into the coated Virtis jar and homogenize for 5 minutes at5,000 RPM.

h. Dispense the resulting mixture equally into four sterile centrifuge tubes and centrifuge in aswinging bucket rotor at 1,500 x G for 2 minutes at room temperature.

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i. A two-phase system with an interface will be formed in the centrifuge tube. The spores arein the upper phase, sub-cellular debris collects at the interface, and vegetative cells collect inthe bottom phase. Being careful not to disturb or disperse the interface layer, use a sterile 10ml pipette to transfer the upper phase to a second sterile centrifuge tube. Discard theinterface and bottom layers according to acceptable procedures for disposal of microbialwaste.

j. Centrifuge this upper phase layer at 27,000 x G for 20 minutes at 5°C. Pour off thesupernatant. Resuspend the spores with 20 ml sterile distilled water and pool in a sterilevessel. Pipette 25 ml aliquots of spores into sterile centrifuge tubes and wash five times with

20 ml of sterile distilled water by centrifuging (at 27,000 x G for 20 minutes at 5°C),decanting the supernatant, and re-suspending the pellet with sterile distilled water.

k. After the last wash step, resuspend each spore pellet in 20 ml filter-sterilized 50% dehydrated alcohol, USP, ethyl alcohol, 200 Proof. Pool all spore suspensions in a sterile bottle

containing 15-20 sterile glass beads. Store this stock suspension at 35-40°F (2-4.4°C). Thestock spore suspension may be used indefinitely if protected from evaporation and

contamination.

34.A.4.3 Preparation of Spore Suspension of Bacillus subtilis ATCC 6633

Use the same procedure as described for B. cereus ATCC 11778 (Section 34.A.4.2)

34.A.4.4 Preparation of Working Spore Suspensions

To determine the number of spores/ml in each new spore stock suspension, perform plate counts

following the procedure described in Section 34.6.3. Incubate at 35± 1°C for 46 to 50 hours.

To determine the dilution required to obtain 1 x 106 spores/ml in 50% ethyl alcohol use the

following formula:

D = W/S

Where:S = Stock concentrationW = Working concentrationD = dilution

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Example:Stock concentration = 5 x 10

8

Working concentration desired = 1 x 106

D = 1 x 10

6/5 x 10

8

D= 1/500

34A.5 Manual Calculation of a Standard Curve

Use the method below or the AOAC procedure Section 957.23, F for calculation.

Average all 36 readings of the standard reference (SR) concentration from the 12 plates. Thiscumulative average is the correction point for the standard curve.

For each set of three plates:

a. Average the readings of the nine SR zones and the nine readings of the other concentrationused for the standard curve.

b. Subtract the nine-zone average of the SR from the cumulative SR average.

c. Algebraically add the resulting value to the value of the average zone reading for theantibiotic concentration used on that set of plates.

Example 1. If, in correcting the second concentration of the standard curve, the cumulativeaverage of the 36 readings of the reference concentration is 20.0 mm and the average of thenine readings of the SR on this set of three plates is 19.8 mm, then the correction is + 0.2mm. (20.0 minus 19.8 = 0.2). If the average of the second concentration on triplicate plates is17.0 mm, then corrected value is 17.2 mm.

Example 2. If the grand average of the reference concentration is 20.0 but the average of thenine readings of the reference concentration on the set of plates is 20.3, the correction factor would be ‘-0.3’. If the average of the second concentration on triplicate plates is 17.0 mm,then corrected value is 16.7. (The sum of 17.0 and ‘-0.3’ is 16.7.)

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d. Use either the method described below or the AOAC 957.23, F procedure to construct thecurve.

• Plot the corrected values, including the correction point, on semi-logarithmic graph paper, using the log scale for the concentration (the concentration of antibiotic for the ̀ y'axis) and the arithmetic scale for the zone diameters (the adjusted value of a sample onthe ̀ x' axis).

• Draw a line of best fit between the high and the low end points of a curve derived fromthe equations below.

L = (3 a + 2 b + c -e) / 5H = (3 e + 2 d + c -a) / 5,

Where:L and H = calculated zone diameters for the low and high concentrations, respectively, onthe standard response line.a, b, c, d, and e = the corrected average zone diameters for each concentration on the

response line, where “a” equals the lowest concentration of antibiotic used and “e” equalsthe highest concentration used.

i Food and Drug Administration. 1968. “Information for assay and reporting of data pertaining toantibiotic residues in milk, dairy products, and animal tissue”. Revised. Food and DrugAdministration, Washington, D.C.

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CHAPTER 35. DETECTION OF ANTIBIOTIC AND SULFONAMIDE RESIDUES INMEAT TISSUE BY COMMERCIALLY AVAILABLE IMMUNOASSAYKITS

Anne M. Dulin, Clarence A. White and Nitin H. Thaker

35.1 Introduction

In recent years, the application of immunochemical methods fordetecting veterinary drug residues in animal tissue has increased.These methods are based on highly specific antigen-antibodyreactions on a solid phase matrix involving conjugation of anenzyme to the drug analyte and specific antibody directed againstthe analyte. Advantages of enzyme immunoassays include sensitivity(usually in the ng/ml range), simplicity of test performance,stability of reagents, lack of radioisotope use and associated hazards, potential for automation, and relatively inexpensiveequipment.

In direct competitive enzyme immunoassays, enzyme labeled drugantigen and unlabeled drug antigen (sample analyte) compete forlimited antibody binding sites. Specific antibody is generally bound to a solid phase support. The amount of enzyme labeled drugthat binds to antibody is inversely proportional to the amount ofunlabeled drug antigen (analyte) present in the tissue sample, which also competitively binds to the same antibody. Upon additionof substrate to the reaction mixture, to provide a visibleindication of the test reaction, the amount of colored end product produced is inversely proportional to the amount of unlabeled druganalyte bound to the antibody. Thus, positive reactions indicating

the drug's presence in the sample are generally indicated by nocolor change, while negative reactions indicating absence of thedrug analyte are usually colored products. The exact color of theend product depends upon the specific substrate - chromogen system used in the particular assay. The applications of directcompetitive enzyme-linked immunosorbent assays (ELISA) have provided additional support to the FSIS regulatory programs byenabling the detection of drug residues in food animal tissues atappropriate levels.

Presently, there are a number of screen test kits commerciallyavailable for detecting the presence of antibiotic and sulfonamideresidues. However, regulatory action cannot be based on screen

test results alone, since they are not quantitative, do not relateto biological activity of the detected drug and they are notconsidered to be absolutely definitive and confirmatory in nature.The presence of antibiotic residues, therefore, must be confirmed by bioassay and/or chemical methods, when a chemical method exists.

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35-2

35.2 Commercially Available Test Kits

Currently there are commercially available, immunoassay, screentest kits, in several different formats, for such antimicrobialagents as:

penicillin (ββ-lactams)ceftiofur (cephalosporin)chloramphenicolgentamicinsulfonamidestetracyclinesneomycin

Many of these test kits, however, were originally developed forspecific application in bulk milk tank testing for antimicrobialresidues. Before any of these kits can be used in an FSISLaboratory for testing meat tissue extracts for antimicrobialresidues, they must first be thoroughly evaluated to determinetheir suitability and applicability with regard to appropriate performance characteristics. They must perform in a manner to meet minimum sensitivity detection levels for the drug in questionrelative to that drug's established tolerance level, be specific,show excellent lot-to-lot reproducibility, have stability over thereported shelf life of the kit, and also have very low (0-5%) false positive and false negative reaction rates. Non-government usersof this Guidebook must assume individual responsibility forevaluating commercial test kits for their applicable suitability with regards to the above performance parameters. 35.3 Equipment

This generic list applies to all test kits. Depending on theexact kit used, other supplies might be required.

a. Tekmar stomacher®, Model 400 (Tekmar Company,Cincinnati, OH)

b. Eppendorf centrifuge, Model 5412 (Thomas Scientific Co.,Swedesboro, NJ)

c. Timerd. Tekmar strainer bags, 18 oz capacity. (Tekmar Company,

Cincinnati, OH)e. Micro centrifuge tubes, 1.5 ml volume. (Thomas

Scientific Co., Swedesboro, NJ)

35.4 Reagents

a. 0.1 M phosphate buffer, pH 8.0 (+ 0.1). Dissolve16.73 g dibasic potassium phosphate and 0.523 g monobasic potassium phosphate in distilled water and

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35-3

dilute to 1 liter with distilled water. Check pH of thenonsterile buffer before autoclaving. If necessary,adjust by the dropwise addition of 0.1 N HCl or NaOH.

Autoclave for 15 minutes at 121°°C and 15 lbs pressure.

b. 0.1 M phosphate buffer, pH 4.5 (+ 0.1). Dissolve 13.6 g

monobasic potassium phosphate in distilled water and dilute to l liter with distilled water. For pHadjustment, proceed as in (a) above.

c. 0.1 M phosphate buffer, pH 6.0 (+ 0.1). Dissolve 11.2 g monobasic potassium phosphate and 2.8 g dibasic potassium phosphate in distilled water and dilute to lliter with distilled water. For pH adjustment, proceed as in (a) above.

d. U. S. Pharmacopeia (USP) antibiotic and sulfonamidestandard reference materials

35.5 Tissue Extraction Procedure

This procedure generally applies to all test kits:

a. Weigh out 10 g of sample (muscle, kidney, or livertissue) into a sterile container.

b. Place the sample into a labeled Tekmar strainer bag.

c. Add 40 ml of appropriate phosphate buffer for theantibiotic or sulfonamide residue under evaluation.

d. Place strainer bag in a Tekmar stomacher® and stomachfor 30 seconds for kidney or liver and 60 seconds for muscle tissue.

e. Allow the extract to settle for 45 minutes.

f. Place 1.5 ml of the settled extract into a labeled microcentrifuge tube.

g. Centrifuge for 10 minutes in an Eppendorf microcentrifuge at maximum speed.

h. Pipette supernatant fluid into another labeled test tube

avoiding any fat and debris.

i. The 1:5 extracts prepared for bioassay analysis (Chapter34) can be used instead of performing steps a through e.

35.6 Performing the Assay

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35-4

Perform the assay procedure according to the specific test kit manufacturer's directions if no modifications were found to benecessary, or according to any specific instructions provided bythe Microbiology Division, OPHS, for a particular test kit, whenextensive protocol modifications were necessary.

35.7 Reporting and Confirmation of Screen Test Results

All positive screen test results should initially be reported as a presumptive positive finding. All samples presenting a positivescreen test result must be subjected to confirmatory testing by the bioassay procedure or an appropriate chemical analysis procedure,if available for that particular drug, to confirm the drug'sidentity and determine it's quantitation. All sulfonamides must beconfirmed by appropriate chemical methods. Final violative resultreports must be based on confirmed drug quantitative levels presentabove that of established tolerance levels for that drug in aspecific animal slaughter class.

35.8 Quality Assurance Procedures

a. Maintain a written log of all kits purchased, used and appropriate dates.

b. Test kits must be stored under refrigeration (4-8oC).

Do not freeze.

c. Upon receipt of new test kits, perform positive and negative control testing at appropriate drugconcentration levels.

d. Do not mix reagents and test components from kits withdifferent serial numbers or from different manufacturerskits that detect the same analyte.

e. Do not use kits past their expiration date.

f. Use a separate pipet and test device for each sample.

g. Before performing the test, allow all reagents to reachroom temperature. If the room temperature is not within

the range of 18-29°°C (65-85°°F), perform test in anotherarea within the proper temperature range.

h. Observe all test time intervals accurately by using atimer.

i. Two weeks before test kit expiration, perform positive

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35-5

and negative control tests at appropriate drug levels toassure proper kit performance as expiration approaches.

j. U.S. Pharmacopeia (USP) standards of antibiotics and sulfonamides at appropriate quantitative levels should be used.

k. Record the results of all positive and negative controltests in a log book.

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Agarwal, V. K. 1992. Analysis of Antibiotic/Drug Residues inFood Products of Animal Origin. Plenum Press, New York, NY10013.

Boison, J. O., and J. D. MacNeil. 1995. New test kittechnology, p. 77-119. In H. Oka, H. Nakazawa, K. Harada and J. D. MacNeil (ed.), Chemical Analysis for Antibiotics Used in Agriculture. AOAC International, Gaithersburg, MD 20877.

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products may be available for laboratory use.

_________________________________________________________________________________




_______________________________________________________________________________________________


Notice of Change


Title: Avian Influenza Detection in the Chicken Heart Using Real-Time Reverse

Transcriptase PCR



This new method utilizes a Real-Time Reverse Transcriptase (RT) PCR to identify Avian Influenza,

and specifically, H5 subtype in the chicken heart. Viral RNA is extracted and purified from raw

chicken heart. The Real-Time RT PCR assay amplifies the AIV Matrix gene, which is common to all

Type A avian influenzas. A presumptive positive result requires a second Real-Time RT PCR assay toidentify subtype H5 in the same sample extract.



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Title: Avian Influenza Detection in the Chicken Heart Using Real-Time Reverse Transcriptase PCR

Revision: 00 Replaces: N/A Effective: 10/8/07

Approved by: Laboratory Quality Assurance Division (LQAD)

Procedure Outline

40.1 Introduction

40.2 Limit of Detection (LOD)40.3 Safety


40.4.1 General40.4.2 Required Extraction Controls

40.4.3 Required Real-Time RT PCR Controls40.4.4 Preparation of Extraction Controls

40.5 Equipment, Materials, Media, Reagents and Test Kits40.5.1 Equipment


40.5.3 Test Kits40.6 Viral RNA Isolation Procedure: Preparation of Homogenate and Trizol Extraction of RNA

40.7 Viral RNA Purification Procedure: Ambion Magnetic Bead Purification of Viral RNA

40.8 Viral RNA Amplification and Detection: Real-Time RT PCR using Lyophilized Beads40.8.1 Interpretation of Sample Results

40.8.2 Interpreting and Troubleshooting Control Results

40.9 Confirmation by NVSL40.10 Selected References

40.1 Introduction

Avian Influenza (AI) is an economically and epidemiologically important disease of

chicken, turkey and other fowl. The agent responsible for AI is a single-stranded RNA

virus, avian influenza (AIV), which is a member of the Orthomyxoviridae family. As of 2005, there were at least 16 antigenically different hemagglutinin (H) subtypes and 9

different neuraminidase (N) subtypes. Avian Influenza Virus exists with one H subtype and

one N subtype in any combination. The H5 and H7 subtypes have been identified to causehigh pathogenicity avian influenza (HPAI) in birds with clinical signs ranging from sudden

death to respiratory signs. Although most AI strains are classified as low-pathogenicity

(LPAI), H5 and H7 subtypes have been designated as reportable. Due to the concern of

animal to human transmission, AI, particularly H5 subtype, is important to both human andanimal health.

The method described utilizes a Real-Time Reverse Transcriptase (RT) PCR to identifyAvian Influenza, and specifically, H5 subtype in the chicken heart. Viral RNA is extracted

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and purified from raw chicken heart. The Real-Time RT PCR assay amplifies the AIV

Matrix gene, which is common to all Type A avian influenzas. A presumptive positive

result requires a second Real-Time RT PCR assay to identify subtype H5 in the samesample extract. If either PCR assay provides positive results, confirmation will be

determined from the reserve sample by Animal and Plant Health Inspection Services(APHIS) / National Veterinary Service Laboratory (NVSL) in Ames, Iowa.

40.2 Limit of Detection (LOD)

SEPRL-ARS-USDA (Das et al., unpublished manuscript) determined the live virus LODin experimentally-infected tissue to be approximately 2.2 Log10 EID50/gm tissue (viral

titer).

40.3 Safety

When working with diagnostic samples, use protective attire such as gloves and lab coats.

Samples originating from an area (defined by APHIS or FSIS) with heightened concern for

HPAI transmission should be handled with enhanced biosecurity, preferably with limitedaccess to the laboratory area. Even though HPAI can be processed in BSL-2 laboratories,

these laboratories must use the following BSL-3 work practices:

1. Any procedure generating aerosols or droplets should be performed in a Class II,HEPA filtered biological safety cabinet.

2. Laboratory workers should wear protective equipment, including disposable

gloves, lab coats, head coverings and, where applicable, close-toed shoes and

eye protection (safety glasses, surgical masks, or face shields) due to risk of aerosol/droplet exposure.

3. Centrifugation of specimens should be performed using sealed centrifuge rotors.

After centrifuging, specimens should be unloaded from the rotors in a Class II,HEPA filtered biological safety cabinet.

4. Work surfaces and equipment should be decontaminated after specimens are

processed using standard decontamination agents according to the

manufacturer’s recommendations or freshly prepared bleach solutions that areappropriate for biohazardous spillage. Several types of disinfectants including

70% alcohol, 10% sodium hypochlorite and peroxygen compounds will

inactivate AIV by destroying the lipid envelope of the virus. Sodiumhypochlorite and peroxygen compounds also degrades nucleic acids (Suarez et

al., 2003).

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5. Biological waste contaminated with suspect or confirmed HPAI should be

treated as outlined in the WHO laboratory biosafety manual

(http://www.who.int/csr/resources/publications/biosafety/Labbiosafety.pdf).

NOTE: Once samples have been treated with lysis buffer, the samples can be handled under normal BSL-2 conditions.


40.4.1 General

Inactivated antigen preparation from NVSL (Ames, Iowa) is used as a positiveextraction control. The inactivated antigen is produced in specific pathogen-free

(SPF) embryonating chicken eggs inoculated by the allantoic sac route. The viral

antigen is inactivated with beta-propriolactone, but contains viral RNA detectable by PCR. The crude antigens, 192-ADV H9/N2 and 190 ADV low path H5/N9,

were prepared by NVSL.

40.4.2 Required Extraction Controls

a. H5N1 or H5N9 antigen, used in NVSL’s Agar Gel Immunodiffusion testing, shall be used as a positive extraction control for the AIV matrix and H5 assays. The

antigen contains inactivated virus that lacks the ability to replicate but contains RNA

detectable by Real-Time RT PCR.

b. New Castle Disease Virus (NDV) available as inactivated antigen, or if unavailable, RNase-free water shall be used as an extraction negative control.

40.4.3 Required Real-Time RT PCR Controls

a. AIV matrix transcript (203 ADV 0601 is provided by NVSL) should be used as

a PCR positive control for the AIV matrix screen.

b. H5 gene transcript (202 ADV 0704 provided by NVSL) should be used as aPCR positive for the H5 Real-Time RT PCR assay.

c. RNase-free water should be used as a No-Template control in the Real-Time RT

PCR reactions.

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40.4.5 Preparation of Extraction Controls

a. Prepare the control in lysing matrix tubes with 0.5 ± 0.05 g of heart tissue spikedwith 40 µl of H5N1 or H5N9 antigen.

b. Prepare the negative control tissue the same way as the positive control tissue, but add NDV or RNase-free water instead of H5 antigen.

40.5 Equipment, Materials, Media, Reagents and Test Kits

40.5.1 Equipment

a. Cepheid®

Smart Cycler II b. Cepeid

®Smart Cycler Tubes (25 µl volume)

c. Lysing Matrix A bead tubes for sample homogenization (Q-Biogene

Cat#6910-500), or equivalent.d. Vortex

e. Refrigerated microcentrifuge

f. Sterile RNase-free Pipet Tips with Filtersg. 1.5 ml Microcentrifuge Tubes

h. Gloves

i. Ambion®

Magnetic Stand-96 (Ambion, Cat#10027) j. Orbital Shaker for 96 well plates

k. Pipettors dedicated to RNA work

l. MoBIO®

Vortex Adapter (MoBIO Cat#13000-V1-24), or equivalent

m. Sterile Scalpelsn. Sterile Forceps


a. Chloroform, 99% pure (Sigma Cat#366927-100ml)

b. 2-Propanol, 99% pure (Sigma Cat#I9516-500)

c. Molecular Biology Grade Water (RNase-free water)d. Transcribed AIV (Cat.# 203 ADV) and H5 RNA (Cat.# 202 ADV) as

supplied by NVSL Ames, Iowa

e. Trizol®

LS reagent (Invitrogen, Cat#10296-010)f. RNase Inhibitor (Promega, Cat#N2511)

g. Cepheid®

PCR Beads containing probes and primers

h. RNA/DNA decontaminant solution

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40.5.3 Test Kits

a. Qiagen®

OneStep RT-PCR Kit (Qiagen®, Cat#210210)

b. Ambion®

MagMax™

-96 AI/ND Viral RNA Isolation Kit (Ambion®

,

Cat#1835-plate kit)

40.6 Viral RNA Isolation Procedure: Preparation of Homogenate and Trizol Extraction

of RNA

NOTE: Before beginning analysis, frozen heart sample should be removed from ≤ -70

0C and allowed to thaw. All reagent preparation must be done in a “clean area”

Biosafety cabinet. Work with RNA or amplified product should not be done in this

area. Keep all samples, reagents and enzymes on ice during all steps.

A. Add 0.5 ± 0.05 gram of heart tissue to a sterile tube containing lysing matrix(glass beads) and 250 ± 2.5 µl of BHI broth. Add 750 ± 7.5 µl of Trizol

®to the

tubes.

B. Agitate the meat/beads mixture on MoBio®

vortex adaptor and shake at maximum

speed for approximately 10 minutes.

C. Hold the samples at room temperature for approximately 15 min. Add 200 ± 2.0

μl of chloroform and vortex the samples for approximately 15 seconds. Let the

samples stand for at least 15 min at room temperature.

D. Centrifuge the samples for 15 - 20 min @ 12,000 rpm set at 4oC. Remove the

aqueous layers from all tubes and place into clean RNase-free microcentrifuge

tubes.

40.7 Viral RNA Purification Procedure: Ambion®

Magnetic Bead Purification of Viral

RNA

This procedure is designed for high-throughput loads. Paramagnetic beads with a nucleic

acid binding surface bind nucleic acids and are detained with use of a magnet.

Contaminants and cellular debris are washed away during wash steps. Make up solutionsfor day of use.

A. Follow kit instructions to prepare Wash Solutions I and II. Prepare ViralLysis/Binding Solution as outlined in TABLE 1.

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TABLE 1: Viral Lysis/Binding Solution Preparation

Ingredients Volume Per Sample

Lysis Binding Solution 50.0 µl

Carrier RNA 1.0 µl

Mix briefly, then add

2-Propanol 50.0 µl

Total Volume per Sample 101.0 µl

B. Prepare the Bead Re-suspension Mix as outlined in TABLE 2.

TABLE 2: RNA Binding Bead Re-Suspension

Ingredients Volume Per Sample

Bead Re-suspension Solution 6.0 µlRNase-Free Water 4.0 µl

Mix briefly, then addRNA Binding Beads 4.0 µl

Mix briefly, then add2-Propanol 6.0 µl

Total Volume per sample 20.0 µl

C. Transfer 50 ± 0.5 µl of sample into the corresponding well on a 96 well

processing plate supplied in the kit. Add 101 ±1.0 µl of prepared virallysis/binding solution (TABLE 1) to each well containing sample. Shake the

plate on a plate shaker at 550- 600 rpm for approximately 30 seconds.

D. Add 20 ± 0.2 µl of RNA Binding Beads solution (TABLE 2) to each well withsample. Shake the plate for approximately 4 minutes at 550-600 rpm.

E. Capture/pellet the RNA Binding Beads on a magnetic stand for approximately 2 minutes. Remove and discard the clear supernatant from the

beads.

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F. Remove the plate from the magnetic stand and add 100 ± 1.0 µl Wash Solution I

mix to each well. Shake for approximately 30 seconds at 550 - 600 rpm.

G. Pellet the beads again on the magnetic stand for approximately 2 minutes and

remove/discard the supernatant. Remove the plate from the magnetic stand. Add100 ± 1.0 µl Wash Solution II Mix to each well. Shake for approximately 30

seconds at 550 -600 rpm.

H. Pellet the beads on the magnetic stand for approximately 2 minutes and

remove/discard the supernatant. Remove the plate from the magnetic stand andrepeat the wash with Wash Solution II. Shake for approximately 30 seconds at

550 - 600 rpm.

I. Pellet the beads again for approximately 2 minutes on the magnetic stand and

discard the supernatant. Shake the plate vigorously for 2 - 5 minutes to dry the beads and remove ethanol.

J. Add 50 ± 0.5 µl of Elution Solution from the kit and shake for approximately 4minutes at 1000 rpm.

K. Pellet the beads for approximately 2 minutes on the magnetic stand and transfer the RNA into an RNase-free sample tube for Real-Time RT PCR. Keep the

sample, also referred to as extraction template at this point, on ice or in the

refrigerator for immediate use. Store the sample at ≤ -70 C if not testing

immediately.

40.8 Viral RNA Amplification and Detection: Real-Time RT PCR using Lyophilized

Beads

PCR is performed using the SmartCycler system made by Cepheid®. The lyophilized

beads contain primers, probes and an internal positive control (IPC).

• Each bead makes 4 reactions.

• Count the number of samples and controls and divide by 4 to determinethe number of beads to reconstitute.

• All PCR runs must include a RNA gene transcript positive control and a

no template control (NTC), usually RNase-free water.

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• Do not combine the contents of One-step PCR kits. Record lotinformation for each sample if different.

• Dilute RNase Inhibitor (40U/µl) 1:3 with RNase-free water to get 13 U/µlExample: 2µl of RNase Inhibitor to 4µl of RNase-free water for finalconcentration of 13 U/µl.

A. Reconstitute each bead as outlined in TABLE 3.

NOTE: Keep reagents on ice.

TABLE 3: PCR Master Mix for four reactions.

Ingredients of Master Mix Volume per 4 reactions

# of Lyophilized beads 1RNase-free water 58.8 µl

dNTPs 3.2 µl

Enzyme mix (RT + Taq) 4.0 µlRNase inhibitor (diluted) 2.0 µl

Total 68.0 µl

B. Add 17 μl of master mix to each SmartCycler tube in the cold block rack..

C. Add 8 μl of extraction template to each tube (total volume of 25 μl in each tube).

D. Pulse spin all tubes before loading the machine and then place samples in

appropriate locations on the SmartCycler machine.

E. Create a run by adding sites for detection and choosing the program for AIV

Matrix or the H5 assay. Select the viewable graphs including “FAM threshold”,“logFAM threshold”, “Texas Red threshold”, “logTexas Red threshold”, and

“Temperature”. Name the run in the “Run Name ID” field. Select the “Start

Run” button.

The run time for the assay is approximately 1.5 hours. Program the SmartCycler with the

parameters shown in TABLE 4. Amplification of viral amplicon should be monitored in

the FAM channel and the IPC should be monitored in the Texas Red Channel.

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TABLE 4: Thermocycling Conditions for the two PCR tests.

Target Gene Cycle Time Temp Step

1 cycle 30 min 50°C

15 min 95°C

Reverse Transcription

40 cycles 20 sec 94°C DenaturationAIV Matrix

20 sec 60°C Annealing/Extension

1 cycle 30 min 50°C

15 min 95°C

Reverse Transcription

40 cycles 1 sec 94°C Denaturation20 sec 54°C Annealing

H5

15 sec 72°C Extension

NOTE: Thermocycling conditions for the AIV Matrix contain only 2 steps. Turn

Optics “On” for the annealing step of AIV Matrix and extension step of H5 assay.

The Optics remain “Off” for all other steps. In the protocol setup, accept the

default settings of all parameters except the following: The background maximum

cycle should be “28” instead of the default “40”. The Manual Threshold

Fluorescence units should be set to “25” instead of the default “30”. The lower

threshold will decrease the chance of a false negative result.

40.8.1 Interpretation of Sample Results

Both AIV Matrix and H5 gene targets, which are monitored in the FAM channel, areconsidered positive when the fluorescence crosses the cycle threshold (CT) and negative

if the fluorescence does not cross the CT . However, for samples which have CT values

≥ 35.0 on the matrix PCR assay, repeat the matrix PCR assay. If the second matrix PCR

assay yields a positive result for that sample, then the sample is considered a suspect positive. Samples testing negative by the matrix PCR assay on the initial test or on the

repeated matrix PCR assay (when initial test CT values are ≥ 35.0) are considered screen

negative so the H5 PCR assay is not performed. Samples testing positive by the matrixPCR assay, either on the initial test or the repeated test, are considered suspect positive so

the H5 PCR assay is performed.

40.8.2 Interpreting and Troubleshooting Control Results

The performance criteria for the positive controls for a valid assay shall include a positiveextraction control and a positive PCR control that are both positive with C T < 35.0.

Every valid assay shall also include a negative extraction control and a NTC in which the

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MLG 40.00 Page 10 of 11




fluorescence does not cross the cycle threshold. If any of the controls do not perform

within the listed specifications, troubleshoot using the following criteria:

A. If a positive extraction control tests PCR negative, repeat the PCR assay. When a

false negative occurs with a control that should be positive, this usually indicateseither a deficient viral RNA extraction or PCR error. If the positive extraction

control is negative on the repeated PCR assay, repeat the assay from the

extraction step.

B. If a negative extraction control tests PCR positive, repeat the PCR assay. If afalse positive occurs with a control that should be negative, this usually indicates

either cross-contamination during extraction, pipetting error during PCR setup or high background fluorescence noise. If a suspected extraction error occurred,

repeat the assay starting with the extraction step. If a suspected pipetting error

occurred during PCR setup, repeat the assay from the PCR step.

C. If the positive PCR control is negative or is < 35.0 CT, repeat the PCR assay.

D. If the no template control (NTC) tests positive, repeat the PCR assay.

40.9 Confirmation by NVSL

FSIS reserve samples that are Real-Time RT PCR positive for AIV Matrix and/or H5

subtype assay are considered presumptive and forwarded to NVSL for viral isolation and

further characterization. The number of samples sent to NVSL shall be determined by FSISand NVSL. They are forwarded to NVSL following the “Transfer of Select Agents”

guideline which states:

FSIS may transfer select agent from other government or private laboratories. Boththe shipper and receiver shall follow the transport requirements for the select agents

listed in their APHIS permit. Prior to shipment, the APHIS transfer form shall be

completed by the recipient and sender, and faxed to APHIS for shipment approval.

APHIS will fax the form back to the sender and /or recipient with an approvalauthorization number after verification of the information on the form. This form is

completed for each transfer of a select agent and must be kept as documentation for

at least three years. Copies of this form, instructions for completing the form, andthe Health and Human Services and USDA list of select agents are located on the

APHIS website. The requirements for transfer of a select agent are stated in 42 CFR

Part 73.

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MLG 40.00 Page 11 of 11





Das, A., Spackman, E., Senne, D., Pederson, J., and Suarez, D. 2006. Development of an Internal Positive Control for Rapid Diagnosis of Avian Influenza Virus Infections by

Real-Time Reverse Transcription-PCR with Lyophilized Reagents. J. Clin. Microbiol.(44): 3065-3073.

Das, A., Spackman, E., Thomas, C., Swayne, D.E., and Suarez, D.L. (Unpublishedmanuscript, submitted for publication). Detection of H5N1 High Pathogenecity Avian

Influenza Virus in Meat and Tracheal Samples from Experimentally Infected Chickens.

Department of Health and Human Services: Office of Inspector General. 2005. 42 CFR Parts 72 and 73: Possession, Use, and Transfer of Select Agents and Toxins, Final Rule

(Part III).

National Veterinary Services Laboratories Protocol AVPRO1505.02. 2005. Real-Time

PCR Test for the Detection of Avian Influenza Virus.

Spackman, E., Senne, D.A., Myers, T.J., Bulaga, L.L., Garber, L.P. Perdue, M.L.,

Lohman, K., Daum, L.T., and Suarez, D.L. 2002. Development of a Real-Time Reverse

Transcriptase PCR Assay for Type A Influenza Virus and the Avian H5 and H7Hemagglutinin Subtypes. J. Clin. Microbiol. (40): 3256-3260.

Suarez, D.L., Spackman, E., Senne, D.A., Bulaga, L., Welsch, A.C., and Froberg, K.

2003. The Effects of Various Disinfectants on Detection of Avian Influenza Virus byReal Time RT-PCR. Avian Diseases. 47 (3 Suppl.): 1091-1095.

Swayne, D and Beck, J.R. 2005. Experimental Study to Determine if Low-PathogenicityAvian Influenza Viruses Can Be Present in Chicken Breast and Thigh Meat Following

Intranasal Virus Inoculation. Avian Diseases. (49): 81-85.

World Health Organization. Laboratory Biosafety Manual: Epidemic Alert and Response,2

ndedition (revised), 2003.

(http://www.who.int/csr/resources/publications/biosafety/Labbiosafety.pdf).

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United StatesDepartment of

Agriculture

Food Safetyand Inspection

Service

Office of Public Health

Science

Laboratory QA/QC Division950 College Station Road

Athens, GA 30605

___________________________________________________________________________________________


Notice of Change

Chapter new, revised, or archived: MLG Appendix 1.03

Title: Media and Reagents



This MLG chapter has been revised to include:• Addition of modified Tryptone Soya Broth with novobiocin plus casaminoacids (mTSB+n),

Neutralizing Buffer, and Dey-Engley (DE) Neutralizing Broth preparation instructions.

• Removal of modified EC Broth with novobiocin (mEC+n) preparation instructions.

• A correction to a parameter for weekly testing of microbiologically suitable water from <2.0

microSiemens-cm conductivity at 25°C to <1.0.

• Additional recommendation in introduction for checking pH of media made from individual

components before autoclaving.

• Revised names for Antibiotic Medium #2 with Dextrose and SOB + Ampicillin Medium.

• Addition of Antibiotic Medium #4 formula and an option to use Antibiotic Medium #4 in place of

Antibiotic Medium #2 with Dextrose.

• Addition of a note in BHI Broth and BHI Agar that states different manufacturers may usedifferent formulations which can be used for non-critical applications.

• Addition or revision of the final pH requirements for Brilliant Green Sulfa Agar, Brucella-FBP Broth

and Agar, Double Modified Lysine Agar, EY-Free Tryptose Sulfite Cycloserine (TSC) Agar,

Mannitol Yolk Polymyxin (MYP) Agar, Modified Campylobacter Charcoal Differential Agar

(MCCDA), MOX, Motility-Nitrate Medium, Motility Test Medium, and MR-VP.

• Addition of the formula for the FBP supplement under Brucella-FBP Broth and Brucella-FBP Agar

and a change in storage temperature of FBP supplement in the Fraser Broth formulation from -20 ºC

to -10ºC or colder.

• New catalogue number for E Buffer.

• Revised formulation for Fraser Broth including formulations for acriflavin stock and ferric

ammonium citrate.

• Minor editing to the instructions for BHI Agar, Baird Parker, HBO, Modified Cooked Meat Medium,DMLIA, MOX, MYP, Modified UVM Broth, MOPS-BLEB, Mueller Hinton, Rainbow Agar,

Trypticase Soy Agar (with and without blood), and TT Broth.

• A note regarding use of the Oxford Supplement or any other supplement with the MOX formula.

• Addition of BC Motility Medium preparation instructions.



products may be available for laboratory use.________________________________________________________________________________________QD-F-Micro-0004.03 Issuing Authority: Laboratory Quality Assurance Division (LQAD)


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SOP No: MLG Appendix 1.03 Page 1 of 42


Revision: 03 Replaces: MLG Appendix 1.02 Effective: 1/28/08


APP 1 Specific Procedure(s)

APP 1.1 Introduction

• All media and reagents necessary for each analysis are listed in each chapter. Theformulations and procedures for preparing the special media and reagents used

throughout this Guidebook are presented in alphabetical order in this appendix.

• Formulations and preparations for basic media that may be used for general

microbiological procedures, which are not listed in this appendix, may be obtained byconsulting readily available reference materials such as general microbiology textbooks,commercially available media formulation handbooks, FDA's Bacteriological Analytical

Manual, and APHA's Compendium of Methods for the Microbiological Examination of

Foods.

• The carbohydrates (sugars) should be chemically pure and suitable for biological use;inorganic chemicals used as reagents should be American Chemical Society (ACS)

grade; dyes must be certified by the "Biological Stain Commission" for use in media.

• The ingredients and the chemicals used for preparing media and reagents may be the product of any manufacturer if comparative tests show satisfactory results. For

convenience, dehydrated media of any brand equivalent to the formulation may be used

unless instructions indicate otherwise. Pre-mixed, dehydrated media should beexamined before use for indications of separation or deterioration. Each batch of

medium should be tested for sterility and growth promotion/inhibition characteristics, as

appropriate following the QC procedures described by the manufacturer.

• Hydrogen ion concentration (pH) of media should be determined using an electronic pHmeter which is standardized against known buffers, prepared according to the Official

Methods of Analysis of the Association of Official Analytical Chemists (16th Edition).If necessary the pH of a medium should be adjusted by adding sufficient 1 N sodium

hydroxide or 1 N hydrochloric acid. For testing the pH of agar media, the use of an

automatic temperature adjusting pH meter/probe and/or a surface-testing probe are

recommended. If a recipe is made from individual components instead of acommercially available dehydrated media, it is recommended that the pH be checked

prior to sterilization.

• Pre-cautions: All manufacturers’ precautions should be followed. The personnel whohandle the material should read the product’s Material Safety Data Sheets. Chemicals

with ‘†’ are of particular concern.

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• Unless otherwise indicated, up to 1 liter of a medium should be sterilized by steam under

pressure at 121°C (15-16 psi) for 15 minutes. Alternatively, media may be filter-sterilized.

• Any departures from standard media preparation practices/techniques (i.e. preparation

volumes, sterilization/heating requirements, formulations, etc.) will require equivalency

data to support the change(s). The laboratory will retain all records. Where theinstructions say to dissolve by gently heating, the media shall be checked visually to

determine that it is well dissolved. Do not over heat. This can be an essential step inobtaining the correct pH for the final medium.

• Depending on the type and quantity of media needed, tubed media may be either

dispensed directly into tubes and sterilized by autoclaving or may be autoclaved in

bulk and then aseptically dispensed into pre-sterilized tubes. Dilution tubes, or any

tubes where the exact volume is critical, should only be dispensed after autoclaving.

• If commercial dehydrated medium is used, follow the manufacturer’s instructions for specified pH, time and temperature of sterilization, etc.

• Microbiology Suitable (MS) water requirements.

Only water that has been treated to be free from traces of dissolved metal,

bactericidal, and inhibitory compounds shall be used to prepare culture media,reagents, and dilution blanks. Inhibitor free water is referred to as microbiologically

suitable (MS) water. The following tests are performed on the water source to ensure

that the water is inhibitor free. Records of the following parameters shall be kept.

Weekly testing (or prior to use):

• >1.0 megohms-cm resistance at 25ºC or

• <1.0 microSiemens-cm conductivity at 25°C.

Monthly testing:

• Total Residual Chlorine shall be < 0.1 mg/l

• Aerobic Plate Count shall be < 1,000 colony forming unit (cfu) mlAnnual testing:

• Heavy Metals (Cd, Cr, Cu, Ni, Pb, and Zn-single) shall be < 0.05 mg/L

• Heavy Metals (total) shall be < 1.0 mg/L

The suitability of water for microbiological analyses shall pass the test for toxicity

annually.

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APP 1.2 Preparation Instructions

A-K AGAR #2 (SPORULATING AGAR)

Pancreatic Digest of Gelatin

Pancreatic Digest of CaseinYeast Extract

Beef Extract

DextroseAgar

Manganous Sulfate (MnSO4.7H2O)MS water

6.0 g

4.0 g3.0 g

1.5 g

1.0 g15.0 g

0.3 g1.0 L

Suspend above ingredients. Heat and check visually to ensure that it is well dissolved.Dispense and autoclave at 121ºC for 15 minutes. Final pH 6.6 ± 0.2 at 25

oC.

ANTIBIOTIC MEDIUM #2 with Dextrose.

Antibiotic Medium #4 may be used instead of adding dextrose to Medium #2.

Bacto Peptone 6.0 g

Beef Extract 1.5 gYeast Extract 3.0 g

Agar 15.0 g

Dextrose solution, sterile (10g/100 ml) 10.0 ml

MS water 1.0 L

Combine all ingredients except the dextrose solution. Heat the mixture until it is well

dissolved. . Dispense and autoclave at 121°C for 15 minutes. When cooled but still

liquid (60-65°C), add sterile dextrose solution to a final concentration of 1 g/L.Commercially available powdered media may be used with the addition of the dextrose

solution after autoclaving. Final pH 6.5 to 6.6 at 25oC.

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ANTIBIOTIC MEDIUM #4

Bacto Peptone 6.0 g

Beef Extract 1.5 g

Yeast Extract 3.0 g

Dextrose 1.0 g

Agar 15.0 g

MS water 1.0 L

Heat the mixture until visual examination shows that it is well dissolved. Dispense

and autoclave at 121°C for 15 minutes.

Final pH 8.0 ± 0.1 at 25oC


Bacto Peptone 6.0 g

Beef Extract 1.5 g

Yeast Extract 3.0 g

Agar 15.0 g

MS water 1.0 L

Heat the mixture until visual examination shows that it is well dissolved. Dispense

and autoclave at 121°C for 15 minutes.

Final pH 8.0 ± 0.1 at 25oC or as specified by the manufacturer if using commercial

dehydrated medium.


Bacto Peptone 6.0 g

Beef Extract 1.5 gYeast Extract 3.0 g

Agar 15.0g

MS water 1.0 L

Heat the mixture until visual examination shows that it is well dissolved. Dispense and

autoclave at 121°C for 15 minutes.

Final pH 5.8 ± 0.1 at 25oC or as specified by the manufacturer if using commercial

dehydrated medium.

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ANTIBIOTIC MEDIUM #11 (NEOMYCIN ASSAY AGAR)

Gelsate™ Peptone or Bacto Peptone 6.0 g

Trypticase Peptone or Bacto Casitone* 4.0 g

Yeast Extract 3.0 g

Beef Extract 1.5 g

Dextrose 1.0 g

Agar 15.0 g

MS water 1.0 L

*Pancreatic digest of casein

Heat the mixture until visual examination shows that it is well dissolved. Dispense and

autoclave at 121°C for 15 minutes. Refrigerate.

Final pH 7.95 ± 0.05 at 25ºC or as specified by the manufacturer if using commercialdehydrated medium. Adjust pH if necessary.

APT AGAR

Pancreatic digest of casein 12.5 g

Dextrose 10.0 gYeast Extract 7.5 g

Sodium Chloride 5.0 g

K 2HPO4 5.0 g

Sodium Citrate 5.0 g

Na2CO3 1.25 g

MnCl2.4H2O 0.14 g

MgSO4.7H2O 0.8 g

Polysorbate 80 0.2 g

FeSO4.7H2O 0.04 g

Thiamine Hydrochloride 1.0 mg

Agar 15.0 gMS water 1.0 L

Add components to MS water, bring volume to 1.0 L, and mix thoroughly. Heat the

mixture until visual examination shows that it is well dissolved. Distribute into tubes

or flasks and sterilize by autoclaving at 118ºC - 121°C at 13 psi for 15 minutes. Avoid

excessive heating. Pour into sterile Petri dishes or leave in tubes.Final pH 6.7 ± 0.2 at 25ºC.

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BC (BACILLUS CEREUS) MOTILITY MEDIUM

Trypticase 10.0 g

Yeast Extract 2.5 g

Glucose 5.0 g

Disodium Hydrogen Phosphate 2.5 g

Agar 3.0 g

Distilled water 1.0 L

Dissolve the ingredients in distilled water and heat to boiling to completely dissolve the

agar. Mix thoroughly and dispense 2.0 ml into 13X100 mm tubes. Autoclave at 121°C

for 15 minutes. Allow medium to solidify and store at room temperature for up to 2 or 3

days for best results.

Final pH 7.4 ± 0.2 at 25°C.

BAIRD-PARKER MEDIUM

Basal Medium

Tryptone 10.0 g

Beef Extract 5.0 g

Yeast Extract 1.0 g

Sodium Pyruvate 10.0 g

Glycine 12.0 g

Lithium Chloride 6H20 5.0 g

Agar 20.0 g

MS water 950.0 ml

Suspend ingredients in water. Heat the mixture until visual examination shows that it

is well dissolved. Autoclave at 121°C for 15 minutes.

Final pH 7.0 ± 0.2 at 25°C.

Complete medium.

a. Add 50 ml prewarmed (to at least room temperature) Bacto EY telluriteenrichment to 950 ml molten basal medium, which has been tempered to

45-50°C.

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b. Mix well (avoiding bubbles) and pour 15-18 ml into sterile 100 x 15 mm Petri

dishes.

c. Plates of complete medium should be stored in refrigerator for no longer than 4

weeks before use.d. Ensure that the surface of the plate is dry before use.

BRAIN HEART INFUSION (BHI) AGAR

Calf Brain (infusion from 200 g) 7.7 g

Beef Heart (infusion from 250 g) 9.8 g

Proteose peptone or gelysate 10.0 g NaCl 5.0 g

Na2HP04 2.5 g

Dextrose 2.0 g

Agar 15.0 g

MS water 1.0 L

Dissolve ingredients in MS water. Heat the mixture until visual examination shows

that it is well dissolved. Dispense as desired and autoclave at 121ºC for 15 minutes.

This may also be prepared by adding 15 g of agar to each liter of BHI broth,

Final pH 7.4 ± 0.2 at 25oC. .

Note: Different manufacturers may use different formulations. For non-critical

applications any of these formulations may be used.

BRAIN HEART INFUSION (BHI) BROTH

Pancreatic digest of gelatin 14.5 g

Brain heart, solids from 6.0 g

Peptic digest of animal tissue 6.0 g

NaCl 5.0 g

Glucose 3.0 g

Na2HP04 2.5 g

MS water 1.0 L

Add components to MS water. Mix thoroughly. Dispense and autoclave at 121ºC for

15 minutes.

Final pH 7.4 ± 0.2 at 25ºC.

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Note: Different manufacturers may use different formulations. For non-critical

applications any of these formulations may be used.

BRILLIANT GREEN SULFA AGAR (OSBORN AND STOKES)

Yeast Extract 3.0g

Polypeptone 10.0 g


Lactose 10.0 g

Sucrose 10.0 gPhenol Red 0.08 g

Agar 20.0 g

Sulfapyridine 1.0 g

Brilliant Green 0.0125 g

MS water 1.0 L

Mix thoroughly and heat with frequent agitation to dissolve. Autoclave at 121°C for 15

minutes. Cool to approximately 50ºC and pour approximately 20 ml into sterile 100 x

15 mm Petri dishes.

Final pH 6.9 ± 0.2 at 25 C

BROMCRESOL PURPLE (BCP) DEXTROSE BROTH

Peptone 10.0 g

Beef Extract (optional) 3.0 g


Bromcresol Purple (0.16 g/ 10.0 ml of 95%

ethanol).

2.0 ml

MS water 1.0 L

Combine the above ingredients with 5 g dextrose per liter. (Other carbohydrates such asadonitol, arabinose, mannitol, maltose, sucrose, lactose, sorbitol, cellobiose, salicin or

trehalose may also be used individually at a quantity of 5 g per liter to prepare these

individual BCP carbohydrate fermentation broths). Adjust to pH 7.0. Dispense 8.0 ml

aliquots into 16 x 150 mm tubes containing inverted 12 x 75 mm fermentation tubes.Autoclave for 10 minutes at 121ºC.

Final pH 6.9 ± 0.1 at 25ºC. NOTE: Dehydrated prepared medium not available commercially.

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BRUCELLA-FBP (BFBP) AGAR

Bacto Peptamin 20.0 g

Bacto Dextrose 1.0 g

Bacto Yeast Extract 2.0 g


Sodium Bisulfite 0.1 g

Bacto Agar 15.0 g

MS water 1.0 L

Brucella agar (dehydrated; Difco), 43.0 g, may be substituted for the first six ingredients

above. Suspend the dehydrated ingredients in MS water. Heat the mixture until visual

examination shows that it is well dissolved. Autoclave at 121ºC for 15 minutes. Coolto approximately 50ºC and add 4 ml filter-sterilized ferrous sulfate-sodium

metabisulfite-sodium pyruvate (FBP) solution or 2 vials of Oxoid FBP supplement.

FBP Solution

Ferrous Sulfate 0.25 g

Sodium Metabisulfite 0.25 g


MS Water 30.0 mL

Mix components thoroughly to dissolve. Filter sterilize.

Mix combined media thoroughly and pour into sterile petri dishes (approximately 20

ml/100 x 15 mm plate). Dry the agar surfaces prior to inoculating by placing the plates

on a bench top (protected from light) overnight.


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BRUCELLA-FBP (BFBP) BROTH

Bacto Tryptone 10.0 g









MS water 1.0 L

Brucella broth (dehydrated; Difco), 28.0 g, may be substituted for the first six

ingredients above. Dissolve the dehydrated ingredients in MS water and autoclave at

121ºC for 15 minutes. Cool the medium to room temperature and add filter-sterilized

FBP solution. (Use Oxoid FBP supplements SR84 or the FBP solution, prepared asdescribed for Brucella FBP agar.) Aseptically dispense into tubes.

Final pH 7.0 ± 0.2 at 25ºC

BUFFERED PEPTONE WATER

Peptone 10.0 g


Sodium Phosphate, dibasic 3.5 g

Potassium Phosphate, monobasic 1.5 g

MS water 1.0 L

Dissolve dry ingredients in MS water, dispense into appropriate containers, and sterilize

in the autoclave at 121ºC

for 15 minutes.


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CARBOHYDRATE FERMENTATION BROTH (EWING)

Fermentation Broth Base

Peptone 10.0 g

Meat Extract 3.0 g


Andrade's indicator 10.0 ml

MS water 1.0 L

Adjust pH to 7.1-7.2. Dispense in tubes with inverted insert tubes and sterilize at 121ºCfor 15 minutes. (See exceptions)

Dextrose, lactose, sucrose, and mannitol are employed in a final concentration of 1%.Other carbohydrates such as galactitol, salicin, etc., may be used in a final concentration

of 0.5%. Dextrose, mannitol, galactitol, salicin, adonitol, and inositol may be added to

the basal medium prior to sterilization. Medium containing neutral glycerol should be

sterilized at 121°C for 10 minutes. Disaccharides such as lactose, sucrose, and

cellobiose (10% solution in MS water, neutral pH) should be sterilized by filtration or at121ºC for 10 minutes and added to previously sterilized basal medium. Arabinose,

xylose, and rhamnose also should be sterilized separately. If basal medium is tubed in

3.0-ml amounts, add 0.3 ml of sterile aqueous carbohydrate solution, i.e., one-tenth the

volume. The natural occurring forms of the carbohydrates are used.

DEY-ENGLEY (DE) NEUTRALIZING BROTH

Tryptone 5.0 g

Yeast Extract 2.5 g

Glucose 10.0 g

Sodium thioglycollate 1.0 g

Sodium thiosulfate 6.0 g

Sodium bisulfite 2.5 g

Polysorbate 80 5.0 g

Lecithin (soy bean) 7.0 g

Brom cresol purple .02 g

MS water 1.0 L

Dissolve dry ingredients in MS water, dispense into appropriate containers, and sterilize

in the autoclave at 121ºC for 15 minutes. Final pH 7.6 ± 0.2 at 25

oC.

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DOUBLE MODIFIED LYSINE IRON AGAR (DMLIA)

Lysine Iron Agar 34.0 g

Bile Salts No. 3 1.5 g

Lactose 10.0 g

Sucrose 10.0 g

Sodium Thiosulfate 6.76 g

Ferric Ammonium Citrate 0.3 g

MS water 1.0 L

Sodium Novobiocin 0.015 g

Suspend all ingredients except Sodium Novobiocin in 1.0 L MS water and heat to

boiling using a hotplate or equivalent (or heat to 100ºC for 10 minutes). DO NOT

HEAT ABOVE 100ºC.. Cool to approximately 50ºC and add Sodium Novobiocin froma filter-sterilized stock solution. Pour 15-20 ml/ plate. Stored refrigerated for up to 3

weeks.

Final pH 6.7 ± 0.2 at 25oC.

This medium is also commercially available as a dehydrated powder with a separate

novobiocin supplement.

E BUFFER

Bovine Albumin(Sigma # A7906-500G or equivalent)

0.5 g

Tween-20 50 µl

Buffered Peptone Water (BPW). 100 ml

Prepare by mixing Bovine Albumin and Tween-20 into Buffered Peptone Water (BPW). Filter sterilize (0.2 µm) and store at 2-8°C.


ENRICHED SEMISOLID BRUCELLA MEDIUM

Bacto Tryptone 10.0 g





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Agar 5.0 g

MS water 1.0 L

Sterile defibrinated sheep blood 100.0 ml

Brucella broth (dehydrated; Difco), 28.0 g, may be substituted for the first six

ingredients above. Heat the mixture until visual examination shows that it is well

dissolved. Autoclave at 121ºC for 15 minutes. Cool to approximately 50ºC and add thesheep blood.


EY-FREE TRYPTOSE SULFITE CYCLOSERINE (TSC) AGAR

The above medium is made exactly as that shown for Tryptose Sulfite Cycloserine

(TSC) Agar except, omit the 50 ml addition of sterile egg yolk emulsion. Add 50 mlMS water instead of the egg yolk emulsion. Final pH 7.6 ±0.2 at 25ºC.

FRASER BROTH

Proteose Peptone 5.0 g

Tryptone 5.0 g

Beef Extract (Oxoid LabLemco) 5.0 g

Yeast Extract 5.0 g

NaCl 20.0 g

KH2PO4 1.35 g

Na2HPO4 12.0 g

Esculin 1.0 g

Naladixic Acid † (2% in 0.1 M NaOH) 1.0 ml

Acriflavin 25mg

Lithium Chloride 3.0 g

MS water 1.0 L

Acriflavin Stock

Acriflavin Hydrochloride (Sigma) 13 mg

MS water 10 ml

Dissolve and add to 1 L of Fraser Broth.

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Ammonium iron (III) citrate (Ferric Ammonium Citrate)

Ammonium iron (III) citrate (Sigma) 5 g

MS water 100 ml

In a 100 ml volumetric flask, dissolve 5g of ammonium iron (III) citrate (Sigma) in MS

water. Bring to volume and filter sterilize. Store at 2-8°C.

Frazier Broth may be prepared from commercially available Modified UVM by adding

the appropriate amounts of lithium chloride and acriflavin before sterilizing and

ammonium iron (III) citrate after sterilization.

Mix well to resuspend the media and dispense into test tubes. Sterilize at 121ºC for 15

minutes. Store in the refrigerator.

Just before use, 0.1 ml of ammonium iron (III) citrate in MS water to each 10 ml tube.


HORSE BLOOD OVERLAY MEDIUM (HL)

a. Base Layer

Columbia Blood Agar Base 1.0 L

Prepare according to manufacturer's specifications and sterilize at 121ºC for 15 minutes.

Pour 10 ml per 100 mm diameter Petri dish. Allow to solidify, overlay with blood agar as described below.

b. Top Layer

Add 4 ml of sterile horse blood to each 100 ml of melted/tempered Columbia Blood

Agar Base which has been cooled to 46ºC . Stir or swirl to mix evenly. Quickly place 5to 6 ml on top of the base layer and tilt the plates to spread top layer evenly. Store platesrefrigerated up to 2 weeks. Discard any plates which become discolored.


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HUNT ENRICHMENT BROTH (Hunt, 1992)

a. Basal Broth

Nutrient broth #2 (Oxoid CM 67) 25.0 g

Yeast Extract (Oxoid L 21) 6.0 g

MS water 950.0 ml

Dissolve the nutrient broth #2 and yeast extract in MS water. Autoclave at 121oC for 15

minutes.

Final pH 7.5 ± 0.2 at 25oC

Cool media and add supplements (FBP, filter-sterilized antibiotics, and horse blood) just

before use and mix thoroughly.

FBP Supplement




FBP Stock Solution




Dissolve ingredients in MS water in a 100 ml volumetric flask, bring to volume and filter sterilize. Dispense and store at -10ºC or colder. Use 4 ml for each liter of

enrichment broth. Discard frozen FBP stock solution after 2 months.

Alternatively, use Oxoid FBP (Campylobacter Growth Supplement; SR84). Rehydratethe supplement with 2 ml sterile MS water and add to the cooled medium. Add 2 vials

for each liter of broth.

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c. Antibiotics

Vancomycin Stock Solution

Vancomycin Hydrochloride (Sigma) 10.0 mg

In a 100 ml volumetric flask, dissolve 0.25 g vancomycin in MS water, bring to volume,mix well, and filter sterilize. Store at 2–8ºC. Use 4 ml for each liter of enrichment

broth. Discard the vancomycin solution after 2 months.

Trimethoprim Lactate Stock Solution

Trimethoprim Lactate † (Sigma) 12.5 mg

In a 100 ml volumetric flask, dissolve 0.3125 g trimethoprim lactate in MS water, bring

to volume, mix well, and filter sterilize. Store at 2–8ºC. Use 4 ml for each liter of enrichment broth. Discard the trimethoprim lactate solution after 12 months.

Cefoperazone Stock Solution

Cefoperazone Sodium (Sigma) 15.0 mg

In a 100 ml volumetric flask, dissolve 0.375 g cefoperazone in MS water, bring to

volume, mix well, and filter sterilize. Store at -70ºC in 4 ml aliquots. Initially, use 4 ml

for each liter of enrichment broth (for the first four hours, incubation is at 37ºC). After

four hours, add an additional 4 ml/liter, to bring the final concentration to 30 mg/liter,and increase the incubation temperature to approximately 42ºC. Discard the frozen

cefoperazone solution after 5 months.

Cycloheximide Stock Solution

Cycloheximide † (Sigma) 100.0 mg

Prepare as a 10% solution in 50% ethanol. In a 50 ml volumetric flask, dissolve 5 gcycloheximide in 50 ml 50% ethanol, mix, and bring to volume. Filter sterilize and store

at 2º to 8ºC up to one year. Use 1 ml for each liter of broth.

d. Sterile lysed horse blood 50.0 ml

Lyse horse blood by subjecting it to two freeze/thaw cycles. Store frozen and discard

blood after 12 months.

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KF BROTH



Yeast Extract 10.0 g


Sodium Glycerophosphate 10.0 g

Maltose 20.0 g

Lactose 1.0 g

Na2CO3 0.636 g

Sodium Azide† 0.4 g

Phenol Red 0.018 g

MS water 990.0 ml

Stock 2,3,5-triphenyltetrazolium chloride solution

Place 0.1 g 2,3,5-triphenyltetrazolinum chloride in MS water to make a total volume of

10 ml. Filter sterilize through a 0.2 µm filter.

Place the above components, except for the 2,3,5-triphenyltetrazolium chloride solution,

in MS water, bring volume to 990.0 ml, and mix thoroughly. Gently heat and bring to boiling. Autoclave for 15 minutes at 121oC. Cool to 45º - 50ºC and aseptically add the

10 ml sterile, stock 2,3,5-triphenyltetrazolium chloride solution to the base medium.Mix thoroughly. Aseptically distribute in 5 - 8 ml volumes in sterile tubes.


LYSINE IRON AGAR (EDWARDS AND FIFE)

Peptone 5.0 g

Yeast Extract 3.0 g

Dextrose 1.0 gL-lysine HCl 10.0 g



Bromcresol Purple 0.02 g

Agar 15.0 g

MS water 1.0 L

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Dispense into tubes and autoclave for 12 minutes at 121°C. Slant with deep butt and

short slant. Final pH 6.7 ± 0.2 at 25ºC

MANNITOL YOLK POLYMYXIN (MYP) AGAR

Preparation A

Beef Extract 1.0 g

Peptone 10.0 g

D-Mannitol 10.0 g

NaCl 10.0 g

Phenol Red 0.025 g

Agar 15.0 g

MS water 900.0 ml

Preparation B

Egg yolk Enrichment 50%

Preparation C

Polymyxin B Sulfate - Dissolve 500,000 units of sterile polymyxin B sulfate (Burroughs

Welcome Co., Research Triangle Park, NC or equivalent) in 50.0 ml of sterile MS

water. Filter sterilize the solution and store in the dark at 4°C. If the solution is

prepared under sterile conditions, the filter sterilizing step may be omitted.

Mix the ingredients (Preparation A) in MS water. Heat the mixture until visual

examination shows that it is well dissolved. Adjust the pH to 7.2 ± 0.1, and dispense.Autoclave at 121ºC for 20 minutes, cool to 50ºC in a waterbath, and add 50 ml of

Preparation B and 10 ml of Preparation C . Mix well, pour into Petri dishes, allow to

solidify, and dry for 24 h at room temperature. Plates may be stored at 2º to 8ºC for 30

days. Final pH 7.1 ± 0.1 at 25ºC.

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M BROTH

M Broth is commercially available. The formula per liter is:

tryptone 12.5 g

yeast extract 5.0 g

D-mannose 2.0 g

sodium citrate 5.0 g

sodium chloride 5.0 g

dipotassium phosphate 5.0 g

manganese chloride 0.14 g

magnesium sulfate 0.8 g

ferrous sulfate 0.04 g

Tween 80® 0.75 g

Dissolve ingredients in 1 liter distilled or deionized water. Heat the mixture untilvisual examination shows that it is well dissolved. Dispense into appropriate

containers and autoclave at 121°C for 15 minutes.

Final pH 7.0±

0.2 at 25°C.

MODIFIED CAMPYLOBACTER CHARCOAL DIFFERENTIAL AGAR (MCCDA),

(Hutchinson and Bolton, 1984)

Nutrient broth No. 2 (Oxoid)* 25.0 g

Bacteriological charcoal 4.0 g

Casein Hydrolysate 3.0 g

Sodium Deoxycholate 1.0 g



Agar 12.0 gSodium Cefoperazone 0.032 g

MS water 1.0 L

*Formula: Lab Lemco Powder (Oxoid: powdered meat extract), 10.0 g;Peptone, 10.0 g: sodium chloride, 5.0 g

Preparation of cefoperozone solution: Add 0.032 g sodium cefoperazone to MS water

and bring volume to 10.0 ml. Mix well. Filter sterilize.

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Preparation of medium: Add components, except cefoperazone solution to MS water

and bring volume to 990 ml. Mix thoroughly. Heat the mixture until visual

examination shows that it is well dissolved. Autoclave at 121 °C for 15 minutes.Cool to 45º– 50 ºC. Aseptically add 10.0 ml sterile cefoperazone solution. Mix

thoroughly. Dispense into Petri dishes or tubes.

CCDA (Campylobacter Blood-free Selective Agar) available from Oxoid (CM739) or

Remel (Campylobacter Blood Free Selective Agar, 452722) may be used with the

addition of the cefoperazone solution.

Dry the agar surfaces prior to streaking by placing the plates on a bench top (protected from light) overnight.. Final pH 7.4 ± 0.2 at 25°C.

MODIFIED COOKED MEAT MEDIUM

a. Cooked Meat Medium (dehydrated prepared medium available commercially)

Beef Heart 454.0 g


Dextrose 2.0 g


b. Diluent (not available commercially)

Trypticase or Tryptone 10.0 g

Sodium Thioglycollate 1.0 g

Soluble Starch 1.0 g

Dextrose 2.0 g

Neutral Red (1% aqueous) 5.0 ml

MS water 1.0 L

Adjust to pH 6.8 ± 0.2. Add about 1 gram of (a) and 16 ml of (b) to screw-capped tubes

no smaller than 20 x 150 mm. Tighten caps, vortex tubes to disperse meat, loosen caps,

and autoclave at 121°C for 15 minutes. Note: b. may be heated to dissolve starch if

necessary.

Final pH 6.8 ± 0.2 at 25oC.

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MODIFIED OXFORD MEDIUM (MOX)

MOX Agar Base

Columbia Blood Agar Base (depending on brand)

38-44.0 g

Esculin 1.0 g


Lithium Chloride (Sigma L0505) 15.0 g

Colistin 0.01

MS water 1.0 L

Rehydrate commercial Modified Oxford Agar Base with constant stirring using a

magnetic mixer. Autoclave this base at 121oC for 15 minutes, mix again, and cool to

45º to 50ºC in a water bath. Add 2 ml of 1% filter sterilized Moxalactam Solution tomake the complete MOX medium, mix well, and pour 12 ml per plate.

Final pH 7.0 ± 0.2 at 25 ºC.

1% Moxalactam Solution or use commercially available supplement at same level

Sodium (or Ammonium) Moxalactam (Sigma) 1.0 g

0.1 M Potassium Phosphate Buffer, pH 6.0 100.0 ml

Dissolve, sterilize by filtration, dispense in small quantities for use and store in freezer at

-10ºC or below. Refreezing may decrease potency.

CAUTION: DO NOT use the Oxford Supplement or any other supplement with this

formula.

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MODIFIED TRYPTONE SOYA BROTH WITH NOVOBIOCIN (mTSB+n)

modified Tryptone Soya Broth (Oxoid Product #

CM0989B or equivalent)

33.0 g

Casaminoacids (casein acid hydrolysate) 10.0 g

MS water 1.0 L

The use of other manufacturer’s modified Tryptone Soya broth or Trypticase (Tryptic) Soy Broth base (other than Oxoid) is permitted if the formula is equivalent.

Rehydrate mTSB by stirring then autoclave for 20 minutes @ 121ºC. Let media coolto approximately 50ºC. Add 5ml of filter sterilized, aqueous solution of 4mg/ml

sodium novobiocin (adjusted for potency; Sigma N1628) for each liter of medium (20

mg/L). If refrigerated, media must be pre-warmed to 18-35ºC prior to use.


MODIFIED UVM BROTH


Tryptone 5.0 g

Lab Lemco Powder (Oxoid) 5.0 g

Yeast Extract 5.0 g

NaCl 20.0 g

KH2PO4 1.35 g

Na2HPO4 12.0 g

Esculin 1.0 g

Naladixic Acid (2% in 0.1 M NaOH) 1.0 ml

Acriflavin 12.0 mg

MS water 1.0 L

(Remel catalog # 455254-2/4/6, Difco catalog # 222330 or BBL catalog # 212348, or equivalent, may be used in lieu of the above formulation.)

Sterilize at 121ºC for 15 minutes. Store in the refrigerator.


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MORPHOLINEPROPANESULFONIC ACID-BUFFERED LISTERIA ENRICHMENT

BROTH (MOPS-BLEB)

Powdered Listeria Enrichment Broth 36.1 g

MOPS free acid

(3-[N-Morpholino]propanesulfonic acid)

6.7 g

MOPS sodium salt

(3-[N-Morpholino]propanesulfonic acid sodium salt)

10.5 g

MS water 1.0 L g

Listeria Enrichment Broth (LEB)Use commercial powdered media or the following ingredients:

Pancreatic Digest of Casein 17.0g

Soytone 3.0 g

Dextrose 2.5 g


Dipotassium Phosphate 2.5 g

Yeast Extract 6.0 g

Cycloheximide † 0.05 g

Acriflavine HCL 0.015 g Nalidixic Acid 0.04 g

Weigh out ingredients as listed above for MOPS-BLEB and mix well to dissolve.

Dispense and sterilize for 15 minutes at 121°C.


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MOTILITY-NITRATE MEDIUM (BUFFERED)

Beef Extract 3.0 g

Peptone 5.0 g

Potassium Nitrate 1.0 g

Disodium Phosphate 2.5 g

Agar 3.0 g

Galactose 5.0 g

Glycerol 5.0 g

MS water 1.0 L

Dissolve the ingredients, except agar, in MS water, and adjust the pH to 7.4. Add the

agar, and heat the mixture until visual examination shows that it is well dissolved.

Dispense and sterilize by autoclaving for 15 minutes at 121oC, and cool quickly in cold

water. If the medium is not used within 4 h after preparation, heat for 10 minutes in

boiling water or flowing steam and chill in cold water before use. Final pH 7.4 ± 0.2 at

25oC

MOTILITY TEST MEDIUM (EWING)

Meat Extract 3.0 g

Peptone 10.0 g


Agar 4.0 g

MS water 1.0 L

Adjust to pH 7.4. Add agar. Heat the mixture until visual examination shows that it is

well dissolved. Dispense and sterilize at 121ºC for 15 minutes.

Final pH 7.3 ± 0.2 at 25 ºC.

MR-VP MEDIUM (EWING)

Buffered Peptone 7.0 g

Dextrose 5.0 g

K 2HPO4 5.0 g

MS water 1.0 L

Dissolve, dispense and sterilize at 121oC for 15 minutes.


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MUELLER HINTON AGAR

Beef Extract 2.0 g

Acid hydrolysate of casein 17.5 g

Starch 1.5 g

Agar 17.0 g

MS water 1.0 L

Suspend ingredients and heat the mixture until visual examination shows that it is well

dissolved. Dispense and autoclave at 121ºC for 15 minutes.Final pH 7.3 ± 0.1 at 25ºC.

NEUTRALIZING BUFFER

Monopotassium Phosphate 42.5 mg

Peptone 0.16 g

Agar 5 g

MS water 1.0 L

Dissolve all ingredients in MS water. Dispense and autoclave for 15 minutes at 121ºC.


NITRATE BROTH

Beef Extract 3.0 g

Peptone 5.0 g

Potassium Nitrate 1.0 g

MS water 1.0 L

Suspend above ingredients in MS water and heat the mixture until visual examinationshows that it is well dissolved. Dispense and autoclave for 15 minutes at 121oC.


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NUTRIENT AGAR

Beef Extract 3.0 g

Peptone 5.0 g

Agar 15.0 g

MS water 1.0 L

Heat the mixture until visual examination shows that it is well dissolved. . Dispenseinto tubes or flasks. Autoclave 15 minutes at 121ºC.

Final pH, 6.8 ± 0.2 at 25ºC.

NUTRIENT BROTH, SEMI-SOLID (Holding Media)

Beef Extract 3.0 g

Peptone 5.0 g

Agar 7.5 g

MS water 1.0 L

Heat the mixture until visual examination shows that it is well dissolved. . Dispense

and autoclave 15 minutes at 121ºC.

Final pH, 6.8 ± 0.2 at 25ºC

PLATE COUNT AGAR (STANDARD METHODS AGAR)

Pancreatic digest of casein USP 5.0 g

Yeast Extract 2.5 g

Dextrose 1.0 g

Agar 15.0 g

MS water 1.0 L

Suspend ingredients in MS water. Heat the mixture until visual examination showsthat it is well dissolved. Sterilize at 121°C for 15 minutes.


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RAINBOW AGAR O157

Rainbow agar base 60.0 g

Potassium Tellurite solution 0.8 ml

Sodium Novobiocin solution 2.5 ml

MS water 1.0 L

Potassium Tellurite Solution

Potassium tellurite 0.010 g

MS water 10.0 ml

Dissolve the potassium tellurite in the MS water. Filter sterilize. Store in the dark at

2-8°C for up to 8 days.

Sodium Novobiocin Solution

Sodium novobiocin 0.40 g

MS water 100 ml

Dissolve the sodium novobiocin in the MS water. Filter sterilize. Store in the dark

up to one year at 2 to 8°C.

Add 60g of Rainbow agar base (Biolog Inc., Hayward California, 94545) to 1 liter of

MS water. Boil gently until dissolved. Autoclave for 10 minutes at 121°C. Cool to

50°C. Add 2.5 ml of sodium novobiocin solution and 0.8 ml of potassium telluritesolution and mix well. Dispense approximately 20 ml per plate into petri plates. Store

in a closed container in the dark. Shelf life of the prepared medium is two weeks if stored under refrigeration in sealed container such as sealed plastic bags.


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RAPPAPORT-VASSILIADIS R10 BROTH (Available from Difco)

Bacto Tryptone* 4.54 g


Potassium Dihydrogen Phosphate 1.45 g

Magnesium Chloride, Anhydrous 13.4 g

Malachite Green Oxalate 0.036 g

MS water 1.0 L

* Papaic digest of soybean meal.

Suspend the ingredients in MS water. Heat the mixture until visual examination showsthat it is well dissolved. Dispense and sterilize at 115-116ºC for 15 minutes.


RVS BROTH (Available from Oxoid Unipath or EM Science)

EM Science Oxoid

Magnesium Chloride 29 g (hexahydrate) 13.58g (anhydrous)

Sodium Chloride 8.0 g 7.2 g

Peptone from soymeal* 4.5 g 4.5 g

Potassium Dihydrogen Phosphate 0.6 g 1.26 g

Dipotassium Hydrogen Phosphate 0.4 g 0.18 g

Malachite Green 0.036 g 0.036 g

MS water 1.0 L 1.0 L

*Papaic digest of soybean meal

Add ingredients to MS water. Mix thoroughly. Dispense and autoclave for 15 minutesat 10 psi – 115ºC.

Final pH 5.2±

0.2 at 25ºC.

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SEMISOLID BRUCELLA GLUCOSE MEDIUM (Holdeman et al., 1977)



Dextrose 1.0 g

Yeast Extract 2.0 g



Agar 1.6 g

Dextrose 10.0 g

Phenol Red 0.02 g

MS water 1.0 L

Brucella broth (Albimi; dehydrated; BBL), 28.0 g, may be substituted for the first sixingredients above. Suspend all ingredients except phenol red and agar in MS water, and

adjust pH to 7.4 with NaOH solution. Add the agar. Heat the mixture until visual

examination shows that it is well dissolved. Cool to 55°C and add 2.5 ml of phenol red stock solution (0.08 g/10 ml of 0.1 N NaOH). Readjust pH to 7.4 if necessary, dispense

and autoclave at 121ºC for 10 minutes.


SOB + Ampicillin MEDIUM

Bacto-tryptone 20.0 g

Bacto-yeast extract 5.0 g

NaCl 0.5 g

Bacto-agar (For SOB agar only) 15.0 g

MS water 950.0 ml

Shake and mix until all solutes have dissolved. Add 10 ml of a 250 mM solution of

KCl. Adjust the pH to 7.0 with 1 N NaOH (less than two ml). Adjust the volume of thesolution to 1 liter with MS water. Sterilize by autoclaving for 20 minutes at 15 psi onliquid cycle.

To the autoclaved and tempered medium, add 5 ml of a sterile solution of 2 M MgCl2,

10 ml of a sterile solution of 2M MgSO4, and a filter sterilized solution of ampicillin(sodium salt) to give a final concentration of 100 µg/ml.


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250 mM KCL

KCL 1.86 g

MS Water 100.0 ml

2M MgCL2

MgCL2 19.0 g

MS Water 100.0 ml

Sterilize by autoclaving for 20 minutes at 15 psi on liquid cycle.

2M MgSO4

MgSO4 24.1 g

MS Water 90.0 ml

Adjust the volume of the solution to 100 ml with MS water. Sterilize by autoclaving for

20 minutes at 15 psi on liquid cycle.

TRIPLE SUGAR IRON (TSI) AGAR

Beef Extract 3.0 g

Yeast Extract 3.0 g

Pancreatic Digest of Casein 15.0 g

Proteose Peptone No. 3 5.0 g

Lactose 10.0 g

Sucrose 10.0 g

Dextrose 1.0 g


Sodium Chloride 5.0 gSodium Thiosulfate 0.3 g

Agar 12.0 g

Phenol Red 0.024 g

MS water 1.0 L

Heat the mixture until visual examination shows that it is well dissolved. Dispenseand autoclave at 121ºC

for 15 minutes. Slant tubes for generous butt.


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TRYPTICASE™ SOY AGAR

(TRYPTIC SOY AGAR)

Trypticase (Tryptic-pancreatic digest of

casein)

15.0 g

Phytone (papaic digest of soybean meal) 5.0 g


Agar 15.0 g

MS water 1.0 L

Add components to MS water and bring volume to 1.0 liter. Mix thoroughly. Heat the

mixture until visual examination shows that it is well dissolved. Autoclave 121°C for

15 minutes. Do not overheat. Pour into sterile petri dishes or leave in tubes.


TRYPTICASE™ SOY AGAR (TS BLOOD AGAR)

Trypticase™ (Tryptic) 15.0 g

Phytone 5.0 gSodium Chloride 5.0 g

Agar 15.0 g

MS water 1.0 L

Suspend ingredients in water. Heat the mixture until visual examination shows that it

is well dissolved. Sterilize at 121°C for 15 minutes. If desired, cool to approximately50ºC, add 5% sterile, defibrinated, sheep blood and swirl. Avoid bubble formation.

Pour 15 ml quantities into sterile 100 x 15 mm Petri dishes and allow to harden.

Final pH 7.3 ± 0.2 at 25°C.

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TRYPTICASE™ SOY AGAR-YEAST EXTRACT (TSA-YE)

Trypticase (Tryptic) 15.0 g

Phytone 5.0 g


Yeast Extract 6.0 g

Agar 15.0 g

MS water 1.0 L

Suspend the above ingredients in MS water. Heat the mixture until visual examination

shows that it is well dissolved. Autoclave for 15 minutes at 121ºC. Temper themedium to 45 - 50ºC and pour into sterile Petri dishes.

Final pH 7.3 ± 0.2 at 25°C.

TRYPTICASE™ SOY BROTH

Trypticase (Triptic) 17.0 g

Phytone 3.0 g


Dipotassium Phosphate 2.5 g

Dextrose 2.5 gMS water 1.0 L

Dispense into tubes and sterilize at 121°C for 15 minutes.

Final pH 7.3 ± 0.2 at 25°C.

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TRYPTICASE™ SOY BROTH (TSB) WITH 10% SODIUM CHLORIDE AND 1%

SODIUM PYRUVATE (PTSBS)


Trypticase (Tryptic) Pancreatic Digest of Casein)

17.0 g

Phytone (Papaic Digest of Soya Meal) 3.0 g

K 2HPO4 2.5 g

Dextrose 2.5 g


MS water 1.0 L

To make from commercial TSB, add 95 g of NaCl to 30 g of dry ingredients, and

dissolve in 1.0 L MS water. Dispense and sterilize at 121°C for 15 minutes.


NOTE: Dehydrated complete medium is not available commercially

TRYPTOSE SULFITE CYCLOSERINE (TSC) AGAR

Tryptose 15.0 g

Agar 14.0 g

Beef Extract 5.0 g

Pancreatic digest of soybean meal 5.0 g

Yeast Extract 5.0 g


Na2S2O5 1.0 g

Egg Yolk Enrichment (50%) 50.0 ml

Cycloserine † Solution 10.0 ml

MS water 940.0 ml

Note: First 7 ingredients available commercially as SFB Base

Cycloserine Solution

D-Cycloserine † 0.4 g

MS water 10.0 ml

Add cycloserine to MS water, bring volume up to 10.0 ml, mix thoroughly and filter

sterilize through a 0.2 µm filter.

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To prepare this medium, add the above components, except for the egg yolk emulsion

and the cycloserine solution, to MS water and bring volume up to 940.0 ml. Mix

thoroughly. Gently heat and bring to boiling. Autoclave for 15 minutes at 121ºC. Cool

to 45 - 50ºC and aseptically add 50 ml of the prepared egg yolk emulsion and the sterile10 ml cycloserine solution. Mix thoroughly and pour into sterile Petri dishes.

Final pH 7.6 ± 0.2 at 25ºC.See preparation of Egg Yolk Free Tryptose Sulfite Cycloserine Agar (EY-free TSC).

TT BROTH (HAJNA AND DAMON, 1956)

Yeast Extract 2.0 g

Tryptose 18.0 g

Dextrose 0.5 g

d-Mannitol 2.5 g

Sodium Desoxycholate 0.5 g



Calcium Carbonate 25.0 g

Brilliant Green 0.01 g

MS water 1.0 L

Dissolve and heat to boiling using a hotplate or equivalent. DO NOT AUTOCLAVE.

Cool below 50°C. Add 40 ml iodine solution. Do not heat after the addition of iodine.

Dispense into sterile containers while keeping the solution well mixed and use the day it

is prepared.

Final pH 7.6 ± 0.2 at 25ºC after addition of iodine.

Iodine Solution

Potassium Iodide 8 g

Iodine † crystals 5 g

MS water 20 ml

Dissolve potassium iodide in 20 ml MS water. Add iodine crystals and stir until

completely dissolved. Add MS water to volume of 40 ml. Mix thoroughly. Store in the

dark at 2-8ºC.

Final pH 7.6 ± 0.2 at 25ºC after addition of iodine.

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XLT4 AGAR

XL Agar Base (Difco #0555-01-8) 47.0 g

Bacto Agar (Difco #0140-01-0) 3.0 g


Sodium Thiosulfate (Anhydrous) 6.8 g

Proteose Peptone #3 (Difco #0122-01-2) 1.2 g

Niaproof 4 (Sigma Chemical Co, formally Tergitol 4) 4.6 ml

MS water 1.0 L

a. Dissolve Niaproof 4 in distilled or deionized water in a 2 L or larger Erlenmeyer

flask and mix with a magnetic stir-bar.

b. Add other ingredients, mix well using stir-bar and Heat the mixture until visualexamination shows that it is well dissolved.

c. Cool to 45 - 50ºC in a water bath and mix again gently.

d. Pour plates fairly thick (about 5 mm deep). The plates may appear dark at first

but should lighten up after cooling overnight. Allow plates to remain at roomtemperature overnight to dry, then refrigerate (in plastic bags or containers) at 3-

8ºC.

e. Remove plates from the refrigerator 24 h prior to use for further drying.f. pH of XLT4 plates = 7.5 + 0.2 (usually no adjustment is necessary).

NOTE: Poured XLT4 plates have a shelf life of at least 3 months when stored refrigerated in closed plastic bag or other container.

Neither XLD agar nor Tergitol 7 can be used in place of plain XL agar base or Tergitol4, respectively.

CAUTION: Consult a Material Safety Data Sheet (MSDS) before working with KCN.

Do not dispose of hazardous fluids such as sodium azide by pouring down sink drains.

Accumulation of sodium azide in lead drains may result in an explosion.

Collect sodium azide wastes and dispose of in accordance with the standard chemical

waste management procedures for your laboratory.

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APP 1.3 REAGENTS

ANDRADE'S INDICATOR (EWING)

Acid fuchsin 0.2 g

MS water 100.0 ml

Sodium hydroxide (1.0 N) 16.0 ml

The fuchsin is dissolved in the MS water, and the sodium hydroxide is added. If, after

several hours, the fuchsin is not sufficiently decolorized to a golden color, add anadditional 1 or 2 ml of alkali. Sterilize by filtration. The dye content of different

samples of acid fuchsin varies quite widely, and the amount of alkali that should be used with any particular sample usually is specified on the label. The reagent improvessomewhat on aging and should be prepared in sufficiently large amounts to last for

several years (up to ten years). The indicator is used in the amount of 10 ml per liter of

medium.

BUFFERED GLYCEROL SALT SOLUTION

Glycerol (glycerin) 100.0 ml

Dipotassium Phosphate (anhydrous) 12.4 g

Monopotassium Phosphate (anhydrous) 4.0 g

Sodium Chloride 4.2 gMS water 900.0 ml

Dissolve the sodium chloride in part of the water, and make up to 900.0 ml. Add the

glycerol and phosphates, and adjust the pH to 7.2. Autoclave for 15 minutes at 121oC.

For double strength (20%) glycerol solution, use 200 ml of glycerol and 800.0 ml of MSwater.

BUTTERFIELD'S PHOSPHATE DILUENT

a. Stock solution

Dissolve 34 g KH2P04 in 500 ml MS water, adjust to pH 7.2 with ca. 175 ml 1 N NaOH, and dilute to 1 liter. Store under refrigeration.

b. Diluent

Dilute 1.25 ml stock solution (a) to 1 liter with MS water. Readjust the pH to

7.2, if necessary, by the drop-wise addition of 0.1 N HCl or 0.1 N NaOH.

Autoclave at 121oC for 15 minutes.

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ENDOSPORE STAIN

a. Solution A

Dissolve 5.0 g of Malachite green in 100 ml MS water. Filter to remove

undissolved dyes.

b. Solution B

Dissolve 0.5 g Safranin O in 100 ml of MS water.

GRAM STAIN (HUCKER MODIFICATION)

a. Crystal violet solution

Crystal Violet (90% dye) 2.0 g

Ethanol (95%) 20.0 ml

b. Oxalate solution

Ammonium Oxalate 0.8 g

MS water 80.0 ml

Working crystal violet solution

Mix the above two solutions together and store in a glass-stoppered bottle.

c. Gram's iodine solution

Iodine crystals 1.0 g

Potassium Iodide 2.0 g

MS water 300.0 ml

Dissolve potassium iodide completely in 5 ml MS water, dissolve the iodine crystals,and then bring to volume with MS water. Mix well and store in an amber glass bottle.

d. Decolorizer

Ethanol, 95% 500.0 ml

Store in glass-stoppered bottle.

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e. Stock safranin (Counterstain)

Safranin O (2.5% solution in 95% ethanol) 10.0 ml

MS water 100.0 ml

Mix well and store in a glass-stoppered bottle.

OXIDASE REAGENT

Tetramethyl-p-phenylenediamine dihydrochloride 1.0 g

MS water 100.0 ml

Prepare fresh daily or refrigerate for not longer than 1 week. Alternatively, usecommercial oxidase reagents.

KOVAC'S REAGENT (EWING)

Pure Amyl or Isoamyl Alcohol 150.0 ml

Paradimethylaminobenzaldehyde 10.0 g

Concentrated HCl 50.0 ml

Dissolve aldehyde in alcohol and slowly add acid. The dry aldehyde should be light incolor. Prepare reagent in small quantities. Store in refrigerator.

METHYL RED REAGENT (EWING)

Methyl Red 0.1 g

Ethyl Alcohol (95-96%) 300.0 ml

Dissolve dye in the alcohol and then add MS water to make 500 ml. Use 5 or 6 drops

per 5.0 ml of culture.

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NITRATE REDUCTION REAGENTS

Solution A

Sulfanilic Acid 0.5 g

Glacial Acetic Acid 30.0 ml

MS water 120.0 ml

Solution B

N(1-naphthyl)ethylenediamine

dihydrochloride (*Marshal's Reagent)

0.2 g

Glacial Acetic Acid 30.0 ml

MS water 120.0 ml

Cleve's acid (5-amino-2 naphthalene sulfonic acid) may be substituted for Marshal's

Reagent.

PEPTONE WATER DILUENT (0.1%)

Peptone 1.0 g

MS water 1.0 L

Dissolve peptone in MS water and adjust pH to 7.0 ± 0.1. Prepare dilution blanks with

this solution, dispensing a sufficient quantity to allow for loss during autoclaving.

Autoclave at 121oC for 15 minutes.

PHOSPHATE BUFFERED SALINE (PBS)

Anhydrous Na2HPO4 12.0 g

NaH2PO4.H2O 2.2 g NaCl 85.0 g

Dissolve dry ingredients in MS water and bring volume to 1 L (10X PBS). Adjust pH to7.4 with 0.1 N HCl or 0.1 N NaOH. To make 1X PBS, dilute 100 ml 10X PBS in 900

ml MS water. Check and adjust pH (7.4) if necessary. Sterilize at 121oC for 15 minutes.

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0.15 M PHOSPHATE BUFFERED SALINE at pH 7.2 (PBS)

“Acid” solution

Anhydrous Na2HPO4 10.36 g

NaCl 4.38

RO water 1.0 L

“Base” solution

NaH2PO4.H2O 10.65 g

NaCl 4.38 g

RO water 1.0 L

Prepare ‘acid’ and ‘base’ solutions by added ingredients to RO water. Dissolve

completely. While mixing with a magnetic stirrer and monitoring the pH on a pH meter,

add a sufficient quantity of the ‘acid’ solution to the ‘base’ solution to achieve a final,

stabilized pH of 7.2. Dispense into glass containers. Autoclave at 121oC for 15 minutes.

Store at room temperature.

PHOSPHATE BUFFERS

0.1 M phosphate buffer, pH 4.5 (+ 0.1)

Dissolve 13.6 g of potassium dihydrogen phosphate (KH2PO4) in about 800 ml of

laboratory grade water. Check the pH of the solution. Adjust, if necessary, by the

dropwise addition 0.1 N HCl or NaOH. Dilute to l liter. Autoclave for 15 minutes at

121°C.


Potassium dihydrogen phosphate (KH2PO4) 11.2 g

Dipotassium hydrogen phosphate (K 2HPO4) 2.8 g

Dissolve in laboratory grade water. Check the pH of the solution. Adjust, if necessary,

by the dropwise addition of 0.1 N HCl or NaOH. Dilute to l liter. Autoclave for 15

minutes at 121°C.

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Dissolve in about 800 ml of laboratory grade water. Check the pH of the solution.Adjust if necessary by the dropwise addition of 0.1 N HCl or NaOH. Dilute to 1 liter.

Autoclave for 15 minutes at 121°C.




Dissolve in about 800 ml of laboratory grade water. Check the pH of the solution.Adjust if necessary by the dropwise addition of 0.1 N HCl or NaOH. Dilute to 1 liter.

Autoclave for 15 minutes at 121°C.

PHYSIOLOGICAL SALINE SOLUTION 0.85% (STERILE)


MS water 1.0 L

Dissolve salt completely in MS water and autoclave at 121oC for 15 minutes.

TRIS BUFFER (0.02 M, pH 7.75)

Trishydroxymethylaminomethane 7.5 g

MS water 3.0 L

Dissolve tris completely in MS water and adjust pH to 8.5 with 20% HCl. Dispense into150 ml portions and autoclave at 115oC for 15 minutes.

V-P REAGENT OF O'MEARA, MODIFIED (EWING)

Potassium Hydroxide 40.0 g

Creatine 0.3 g

MS water 100.0 ml

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Dissolve alkali in water. Add creatine. Keep refrigerated. Make new reagent every 3

weeks. Use equal parts of reagent and culture. Aerate by shaking. Place test tube at

37°C. Read in 4 hours.

APP 1.4 Reference(s):

Atlas, Ronald M. 2004. Handbook of Microbiological Media, 3nd

Ed. CRC Press, Boca

Raton, FL.

Difco & BBL Manual: Manual of Microbiological Culture Media. 2003. Published by Becton,

Dickinson and Company.

Downes, F. P. and K. Ito (Editors), 2001. Compendium of Methods for the Microbiological

Examination of Foods. Fourth Edition. Published by American Public Health Association,

Washington, D.C.

Ewing, W. H. 1986. Edwards and Ewing's Identification of Enterobacteriaceae, 4th Edition.

Elsevier Science Publishing Co., Inc., New York.

Hajna, A., and S. R. Damon. 1956. New enrichment and plating media for the isolation of Salmonella and Shigella organisms. Appl. Microbiol. 4:341-345.

Holdeman, L. V., E. P. Cato, and W. E. C. Moore. 1977. Campylobacter , p.114-115. In Anaerobe Laboratory Manual. 4th Edition. Published by Virginia Polytechnic Institute and

State University, Blacksburg, Va.

Hunt, J. M, C. Abeyta and T. Tran. 2001. Chapter 7: “Campylobacter ” In FDA Bacteriological

Analytical Manual Online.

Hutchinson, D. N., and F. J. Bolton. 1984. Improved blood free selective medium for the

isolation of Campylobacter jejuni from faecal specimens. J. Clin. Pathol. 37: 956-957.

Palumbo, S. A., F. Maxino, A. C. Williams, R. L. Buchanan, and D. W. Thayer. 1985. Starch-ampicillin agar for the quantitative detection of Aeromonas hydrophila. Appl. Environ.

Microbiol. 50(4):1027-1030.

Wang, W. L. L., N. W. Luechtefeld, L. B. Reller, and M. J. Blaser. 1980. Enriched Brucella

medium for storage and transport of cultures of Campylobacter fetus subsp. jejuni. J. Clin.Microbiol. 12:479-480.

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products may be available for laboratory use.

_________________________________________________________________________________

United States

Department of

Agriculture

Food Safety

and Inspection

Service

Office of

Public Health

Science



Athens, GA 30605

_______________________________________________________________________________________________


Notice of Change

Chapter new, revised, or archived: MLG Appendix 2.03

Title: Most Probable Number Procedure and Tables



The MPN procedure used by the FSIS Field Service Laboratories to enumerate

Listeria monocytogenes, Salmonella spp. and E. coli O157:H7 product sample isolates

was added.

A transcription error in Table 4 was corrected.



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MLG Appendix 2.03 Page 1 of 8




APP 2 Procedure

APP 2.1 Introduction

APP 2.1.1 For the convenience of analysts using the procedures in this Guidebook, tables of

Most Probable Numbers (MPN) are provided in this appendix.

APP 2.1.2 MPN is a procedure to estimate the population density of viable microorganisms in a

test sample. It is based upon the application of the theory of probability to the

numbers of observed positive growth responses to a standard dilution series of

sample inoculum placed into a set number of culture media tubes. Positive growth

response after incubation may be indicated by such observations as gas production infermentation tubes or visible turbidity in broth tubes, depending upon the type of

media employed. The sample should be diluted in such a manner that higher dilutions of the sample will result in fewer positive culture tubes in the series. The

number of sample dilutions to be prepared is generally based on the expected

population contained within the sample. If particularly high microbial populationsare expected, the sample must be diluted to a range where the MPN can be obtained.

Most reliable results occur when all tubes at the lower dilution are positive and all

tubes at the higher dilution are negative. Generally tenfold serial dilutions are used in either a 3, 5 or 10 tube MPN series. When a higher number of tubes are

inoculated in the series, the confidence limits of the MPN are narrowed. For

particularly high microbial populations, the values obtained by MPN are generallynot considered to be as precise as population numbers derived from direct platingmethods; however, it should be emphasized that MPN values are only estimates

while plate counts are direct counts of living organisms expressed in cfu/ml. MPN

values are, however, particularly useful when low concentrations of organisms(<100/g) are encountered in such materials as milk, food, water and soil where

particulate matter of the matrix may interfere with obtaining accurate colony counts.

APP 2.1.3 In application of probability theory to the determination of MPN values, it should be

kept in mind that the following assumptions are generally considered to be accepted:(a) the organisms are randomly and evenly distributed throughout the sample, (b) the

organisms exist as single entities, not as chains, pairs or clusters and they do notrepel one another, (c) the proper growth medium, temperature and incubationconditions have been selected to allow even a single viable cell in an inoculum to

produce detectable growth and (d) the population does not contain viable, sub-

lethally injured organisms that are incapable of growth in the culture medium used.

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APP 2.1.4 The following 4 tables present MPN values and corresponding 95% confidencelimits for a 3 tube test series using 4 different, commonly used sets of inoculum

quantities that should be particularly useful relative to performing the

microbiological analyses previously described in various chapters of this Guidebook.These MPN tables may be considered to be abbreviated since not all possible

combinations of positive and negative tubes within a series are presented. Those

combinations that occur often enough to have statistical significance are included,while those that are improbable have been omitted. If laboratory analyses produce

combinations that are not included in the tables, then one should repeat the test on

another portion of the original sample (assuming the microbiological integrity of the

sample has not been compromised) as a possible performance error or contamination

is indicated. If this is not possible and an MPN is imperative, then more completetables should be consulted from other reference sources or the MPN can be

calculated by equation (other reference sources) on the basis of the observed results.

APP 2.1.5 On occasions when more than three dilutions of a sample are used in a decimal

series of dilutions of a 3-tube MPN determination; the following guidelines should be followed. Results from only three consecutive dilutions are used to determine

the MPN. If one or more dilutions have all tubes positive, select the highest

dilution (smallest sample quantity) with positive results in all tubes and the nexttwo higher dilutions, as shown in examples a and b below. When none of the

dilutions yield all tubes positive, select the three lowest dilutions for which the

middle dilution contains the positive result, as shown in example c below. If a positive result occurs in a higher unselected dilution, add the number of positivetubes in this dilution to the results of the highest dilution of the three selected, as

shown in example d below. When all dilutions tested yield all tubes positive,

select the three highest dilutions (example e below). For additional information onMPN estimations, consult the APHA’s Compendium of Methods for the

Microbiological Examination of Foods (3rd

edition, 1992, chapter 6).

APP 2.2 SAMPLE PREPARATION FOR MOST PROBABLE NUMBER (MPN)

A series of 3 sequential MPN tubes may be inoculated directly from the 1:10

sample-culture medium homogenate if a low level of organisms is expected.Prepare the sample in the initial culture medium as directed for the analysis. Set up3 empty tubes and 6 tubes that contain 9 ml of the appropriate culture medium.

Add 10 mL of the homogenate to each of 3 empty tubes, 1 mL of the homogenate

to each of 3 tubes of culture medium, and 0.1 mL to each of the last 3 tubes of

culture medium. This series of tubes represents 1.0, 0.1 and 0.01 g of sample. Theremainder of the sample homogenate should also be cultured.

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Alternatively, prepare serial 10-fold dilutions of the sample in the appropriateculture medium and follow the outline below. This is the preferred method if it is

anticipated that the level of organisms may exceed 10 growth units per gram.

Volume of Original

Sample

Source of

Inoculum

Quantity of

Inoculum

Quantity of

CultureMedium

Number of

Tubes

10 mL Sample 10.0 mL 90 mL 3

1.0 mL Sample 1.0 mL 9.0 mL 3

0.1 mL Dilution Tube 10-1 1.0 mL 9.0 mL 3

0.01 mL Dilution Tube 10-2 1.0 mL 9.0 mL 3

0.001 mL Dilution Tube 10-3 1.0 mL 9.0 mL 30.0001 mL Dilution Tube 10-4 1.0 mL 9.0 mL 3

To increase sensitivity in samples expected to have a low level of the organism of

interest, the FSIS laboratories perform the following procedure to prepare most

product samples for pathogen MPN analysis.

Start with a 65 ± 2 g test portion (representative of the entire sample) taken from

the sample reserve. Add 585 ml enrichment broth and stomach for 2 minutes.

Set up a 3-tube 5-dilution MPN (i.e., 15 individual dilutions for each sample)representing 10, 1, 0.1, 0.01, and 0.001 g of sample. Three containers will hold 100 ml homogenate each to represent the 10 g sample dilution. Three containers

will hold 10 ml homogenate each to represent the 1 g sample dilution. Three

containers will hold 1 ml homogenate plus 9 ml enrichment broth each to

represent the 0.1 g sample dilution. Three tubes will contain 0.1 ml homogenate plus 9.9 ml enrichment broth each to represent the 0.01 g sample dilution; and add

0.1 homogenate to 9.9 ml enrichment, vortex, and add 1.0 ml from that dilution to

each of three tubes containing 9.0 ml enrichment for the 0.001 g sample dilution.

Analyze each dilution (and the remainder of the sample homogenate) per

appropriate MLG chapter. MPN dilutions and the remainder of the samplehomogenate that screen positive will be confirmed.

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APP 2.3 Completion of the MPN

After all MPN dilutions have been completed, refer to the MLG for each pathogen being analyzed to determine incubation times and temperatures and procedures

for completion of the analyses.

APP 2.4 Example calculations

Sample quantities (g or ml)1

Example 10 1 0.1 0.01 0.001

Reported

positive

values

MPN

estimate/

g or mla

b

c

d e

3/32

3/3

0/3

3/33/3

3/3

3/3

0/3

3/3

3/3

2/3

3/3

1/3

2/3

3/3

0/3

2/3

0/3

1/3

3/3

0/3

0/3

0/3

1/3

3/3

3-2-0

3-2-0

0-1-0

3-2-23-3-3

9.3

93.

0.31

21.

>1100

1The analyst should make sure that ALL sample dilution factors (including

the preparation of any sample homogenate) are correctly applied in

calculating the actual sample quantities subjected to MPN analysis.

b2Numerator No. positive tubes

___________ = ____________________

Denominator No. tubes inoculated

APP 2.5 Reference(s):

Blodgett, R. FDA Bacteriological Analytical Manual Online, Appendix 2, Most

Probable Number Determination from Serial Dilutions, 2006.

Swanson, K.M.J, R. L. Petran and J. H. Hanlin. Chapter 6, Culture Methods for

Enumeration of Microorganisms in “Compendium of Methods for theMicrobiological Examination of Foods” Fourth Edition, 2001. Edited by F. P.

Downes and K. Ito. Published by American Public Health Association.

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MLG Appendix 2.03 MPN Table 1. Page 5 of 8


Table 1. MPN Index and 95% Confidence Limits for Various

Combinations of Positive Tubes in a 3 Tube Dilution Series Using

Inoculum Quantities of 10, 1 and 0.1 g (ml).

Combination of MPN Index 95% Confidence Limits

Positives per g (ml) Lower Upper

0-0-0 <0.03 --- 0.095

0-0-1 0.030 0.0015 0.0960-1-0 0.030 0.0015 0.11

0-1-1 0.061 0.012 0.18

0-2-0 0.062 0.012 0.18

0-3-0 0.094 0.036 0.38

1-0-0 0.036 0.0017 0.18

1-0-1 0.072 0.013 0.18

1-0-2 0.11 0.036 0.38

1-1-0 0.074 0.013 0.20

1-1-1 0.11 0.036 0.38

1-2-0 0.11 0.036 0.421-2-1 0.15 0.045 0.42

1-3-0 0.16 0.045 0.42

2-0-0 0.092 0.014 0.38

2-0-1 0.14 0.036 0.42

2-0-2 0.02 0.045 0.42

2-1-0 0.15 0.037 0.42

2-1-1 0.20 0.045 0.42

2-1-2 0.27 0.087 0.94

2-2-0 0.21 0.045 0.42

2-2-1 0.28 0.087 0.94

2-2-2 0.35 0.087 0.94

2-3-0 0.29 0.087 0.94

2-3-1 0.36 0.087 0.94

3-0-0 0.23 0.046 0.94

3-0-1 0.38 0.087 1.1

3-0-2 0.64 0.17 1.8

3-1-0 0.43 0.09 1.8

3-1-1 0.75 0.17 2.0

3-1-2 1.2 0.37 4.2

3-1-3 1.6 0.40 4.2

3-2-0 0.93 0.18 4.23-2-1 1.5 0.37 4.2

3-2-2 2.1 0.40 4.3

3-2-3 2.9 0.90 10.

3-3-0 2.4 0.42 10.

3-3-1 4.6 0.90 20.

3-3-2 11. 1.8 41.

3-3-3 >11. 4.2 ---

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Inoculum Quantities of 1, 0.1 and 0.01 g (ml).



0-0-0 <0.3 ----- 0.95

0-0-1 0.30 0.015 0.960-1-0 0.30 0.015 1.1

0-1-1 0.61 0.12 1.8

0-2-0 0.62 0.12 1.8

0-3-0 0.94 0.36 3.8

1-0-0 0.36 0.017 1.8

1-0-1 0.72 0.13 1.8

1-0-2 1.1 0.36 3.8

1-1-0 0.74 0.13 2.0

1-1-1 1.1 0.36 3.8

1-2-0 1.1 0.36 4.21-2-1 1.5 0.45 4.2

1-3-0 1.6 0.45 4.2

2-0-0 0.92 0.14 3.8

2-0-1 1.4 0.36 4.2

2-0-2 2.0 0.45 4.2

2-1-0 1.5 0.37 4.2

2-1-1 2.0 0.45 4.2

2-1-2 2.7 0.87 9.4

2-2-0 2.1 0.45 4.2

2-2-1 2.8 0.87 9.4

2-2-2 3.5 0.87 9.4

2-3-0 2.9 0.87 9.4

2-3-1 3.6 0.87 9.4

3-0-0 2.3 0.46 9.4

3-0-1 3.8 0.87 11.

3-0-2 6.4 1.7 18.

3-1-0 4.3 0.90 18.

3-1-1 7.5 1.7 20.

3-1-2 12. 3.7 42.

3-1-3 16. 4.0 42.

3-2-0 9.3 1.8 42.3-2-1 15. 3.7 42.

3-2-2 21. 4.0 43.

3-2-3 29. 9.0 100.

3-3-0 24. 4.2 100.

3-3-1 46. 9.0 200.

3-3-2 110. 18. 410.

3-3-3 >110. 42. ---

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Inoculum Quantities of 0.1, 0.01 and 0.001 g (ml).



0-0-0 <3.0 --- 9.5

0-0-1 3.0 0.15 9.6

0-1-0 3.0 0.15 11.0-1-1 6.1 1.2 18.

0-2-0 6.2 1.2 18.

0-3-0 9.4 3.6 38.

1-0-0 3.6 0.17 18.

1-0-1 7.2 1.3 18.

1-0-2 11. 3.6 38.

1-1-0 7.4 1.3 20.

1-1-1 11. 3.6 38.

1-2-0 11. 3.6 42.

1-2-1 15. 4.5 42.

1-3-0 16. 4.5 42.

2-0-0 9.2 1.4 38.

2-0-1 14. 3.6 42.

2 0 2 20 4 5 42

Documents

Binder2[1]