Enumeration of Total Aerobic Microorganisms in Foods by ......35 C) was compared with the AOAC Official Method 966.23 (AOAC 35 C) for enumeration of to-tal aerobic microorganisms in

FOOD BIOLOGICAL CONTAMINANTS

Enumeration of Total Aerobic Microorganisms in Foods bySimPlate® Total Plate Count–Color Indicator Methods andConventional Culture Methods: Collaborative StudyPHILIP T. FELDSINE, STEPHANIE C. LEUNG, ANDREW H. LIENAU, and LINDA A. MUI

BioControl Systems Inc., 12822 SE 32nd St, Bellevue, WA 98005DAVID E. TOWNSEND

IDEXX Laboratories Inc., One IDEXX Dr, Westbrook, ME 04092

Collaborators: V. Arling; L. August; D. Barham; M. Bohnert; A. Boville; S. Brookman; C. Chavey; S. Clements; R. Davis;S. Devane; S. Dissel; S. Gartside; D. Hagel; C. Hernandez; S. Hopkins; M. Howell; F. Humbert; J. Hunsucker; J. Jackson;S. Koch; C. Kuber; J. Lamb; L. Lewis; B. Lightfoot; W. Lin; S. Musch; K. Nieves; M. Poumeyrol; S. Qvist; J. Rice;D. Solis; J. Terry; P. in’t Veld; R. Voermans; D. Warburton; J. Welch

The relative efficacy of the SimPlate® Total PlateCount–Color Indicator (TPC–CI) method (SimPlate35�C) was compared with the AOAC OfficialMethod 966.23 (AOAC 35�C) for enumeration of to-tal aerobic microorganisms in foods. The SimPlateTPC–CI method, incubated at 30�C (SimPlate 30�C),was also compared with the International Organi-zation for Standardization (ISO) 4833 method(ISO 30�C). Six food types were analyzed: groundblack pepper, flour, nut meats, frozen hamburgerpatties, frozen fruits, and fresh vegetables. Allfoods tested were naturally contaminated. Nine-teen laboratories throughout North America andEurope participated in the study. Three methodcomparisons were conducted. In general, therewas <0.3 mean log count difference in recoveryamong the SimPlate methods and their corre-sponding reference methods. Mean log counts be-tween the 2 reference methods were also very sim-ilar. Repeatability (sr) and reproducibility (sR)standard deviations were similar among the 3method comparisons. The SimPlate method (35°C)and the AOAC method were comparable for enu-merating total aerobic microorganisms in foods.Similarly, the SimPlate method (30�C) was compa-rable to the ISO method when samples were pre-pared and incubated according to the ISO method.

Current methods for enumeration of total aerobic micro-organisms include the AOAC culture method (1) andthe International Organization for Standardization

(ISO) method (2). Both methods use pour plates that are incu-bated for 48 h (AOAC) to 72 h (ISO) before obtaining results.The SimPlate� Total Plate Count–Color Indicator (TPC–CI)uses binary detection technology (BDT) to enumerate totalaerobic microorganisms in foods after only 24–28 h of incuba-tion, thereby offering a significant timesaving advantage overthe current culture methods. The SimPlate device also elimi-nates uncertainty in enumeration of plate counts caused byovercrowding, spreading colony types, and food particulateinterference on conventional pour plate methods. In theSimPlate multiple test format, prepared food test portions areplaced onto the center of a SimPlate device and TPC–CI liquidmedium is added. For the single test format, a premixed testportion/medium homogenate is dispensed into the test device.The test portion/medium homogenate is distributed into afixed number of individual incubating wells: 84 for normalcounting range SimPlate and 198 for the high counting range.

With SimPlate, foodborne microorganisms are suspendedin a nutritionally defined growth medium. Discrete aliquotsare separately compartmentalized and isolated from eachother in the incubating wells where biochemical activities ofviable microorganisms are monitored in a liquid environment.Detection by this biochemical process requires fewer microor-ganisms to produce a detectable signal in a SimPlate well thanthe number required to form a clearly visible colony in an agarplate. Enumeration is measured by a simple binary reaction;each well is either positive or negative. Any color change fromthe original background color in each well or in the sponge isinterpreted as a positive reaction. Aerobic microorganisms areenumerated by counting the numbers of wells in each platethat exhibit a color change (positive wells) after incubation.The final count per plate is derived from Table 1, which isbased upon the principle of the Poisson distribution. The large

FELDSINE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 2, 2003 257

Submitted for publication November 2002.The recommendation was approved by the Methods Committee on

Microbiology and Extraneous Materials as First Action. See “OfficialMethods Program Actions,” (2003) Inside Laboratory Management,January/February issue.

Corresponding author’s e-mail: [email protected].

number of wells allows accurate measure of the true microbialpopulation in a test sample.

A recent methods comparison study compared theSimPlate TPC–CI method and the AOAC method for enumer-ation of total aerobic microorganisms in 20 different foods(unpublished). Overall, the SimPlate method produced equiv-alent or statistically greater mean log counts for all food typesanalyzed, except for one lot of fresh fruit and one lot of freshjuice. The results indicated that SimPlate TPC–CI is equiva-lent to the pour plate reference method. A separate interna-tional multilaboratory collaborative study compared the rela-

tive effectiveness of the SimPlate TPC–CI method with that ofthe AOAC pour plate method for recovery of total aerobic mi-croorganisms. The SimPlate method and the ISO pour platemethod were also evaluated in this same study.

Collaborative Study

Study Design

Six food types representative of a wide range of food cate-gories were evaluated: ground black pepper, frozen fruits,flour, nut meats, frozen hamburger patties, and fresh vegeta-bles. Two test portions from 3 different lots were analyzed foreach food type (total of 6 test portions per food type). Allfoods tested were naturally contaminated. Collaborators weresent 2 sets of randomized test portions; one set was used foranalysis by the SimPlate method incubated at 35�C(SimPlate 35) and the AOAC pour plate method incubated at35�C (AOAC 35). The other set was used for analysis by theSimPlate method incubated at 30�C (SimPlate 30) and the ISOpour plate method incubated at 30�C (ISO 30). The black pep-per, flour, and nut meats were stored at room temperature untilthe day of analysis. Fresh vegetables were kept refrigerateduntil analysis. Frozen fruit and frozen hamburger patty testportions were kept frozen (–20�C) and tested unthawed.

For one set of test portions, collaborators were instructed toprepare the initial suspension and further decimal dilutions asrecommended by AOAC Official Method 966.23 (1). The ap-propriate dilutions were analyzed by the AOAC pour platemethod (AOAC 966.23) and the SimPlate method, both incu-bated at 35�C. For the second set of test portions, collaboratorsprepared the initial suspension and further decimal dilutionsaccording to ISO 6887 (3). The appropriate dilutions were an-alyzed according to ISO 4833 (2) and the SimPlate method,both incubated at 30�C. Results obtained were submitted onsummary forms with the appropriate raw data. A minimum of8 laboratories submitted valid data for each food type.

Test Portion Preparation

The naturally contaminated foods were purchased at the re-tail level in Seattle, WA. The bulk foods were thoroughlymixed and then packaged into 50 g test portions for shipmentto collaborators.

Test Portion Distribution

Each collaborator received 2 sets of 6 test portions for eachweek the study was conducted. Test products were distributedby overnight delivery from the United States to laboratoriesthroughout North America and Europe. Frozen foods wereshipped on dry ice; ground black pepper, flour, and nut meattest portions were shipped at room temperature. Fresh vegeta-bles were shipped on ice to North American collaborators only.

Test Portion Analysis

The AOAC and ISO culture methods use different diluentsfor initial suspension and decimal dilutions of test portions.This required collaborators to set up separate paired samples

258 FELDSINE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 86, NO. 2, 2003

Table 1. SimPlate conversion table

Wells = populationa

1 = 2 31 = 76 61 = 216

2 = 4 32 = 80 62 = 224

3 = 6 33 = 84 63 = 232

4 = 8 34 = 86 64 = 240

5 = 10 35 = 90 65 = 248

6 = 12 36 = 94 66 = 256

7 = 14 37 = 96 67 = 266

8 = 16 38 = 100 68 = 276

9 = 18 39 = 104 69 = 288

10 = 22 40 = 108 70 = 298

11 = 24 41 = 112 71 = 312

12 = 26 42 = 116 72 = 324

13 = 28 43 = 120 73 = 338

14 = 30 44 = 124 74 = 354

15 = 32 45 = 128 75 = 372

16 = 36 46 = 132 76 = 392

17 = 38 47 = 136 77 = 414

18 = 40 48 = 142 78 = 440

19 = 42 49 = 146 79 = 470

20 = 46 50 = 150 80 = 508

21 = 48 51 = 156 81 = 556

22 = 50 52 = 160 82 = 624

23 = 54 53 = 166 83 = 738

24 = 56 54 = 172 84 = >738

25 = 58 55 = 178 If there are no positivewells, and the sponge

is positive,population is 1

26 = 62 56 = 184

27 = 64 57 = 190

28 = 68 58 = 196 If there are no positivewells, and the sponge

is negative,population is <1

29 = 70 59 = 202

30 = 74 60 = 208

a The population reflects the number of microorganisms per plate.To determine the number of microorganisms per g (mL) foodproduct, refer to I(c), Reading and Interpretation of Results.

with one test portion prepared with the AOAC diluent and theother test portion prepared with the ISO diluent.

Designated food test portions were prepared as specifiedby AOAC Method 966.23. For all food types, except nutmeats, 50 g test portions were added to 450 mL Butterfield’sphosphate buffered diluent (BPBD) water and homogenizedby blending for 2 min. For nut meats, 50 g test portions wereadded to 50 mL BPBD and vigorously shaken to produce the100 dilution. After homogenization, test portions were seriallydiluted into BPBD. The number of dilutions necessary to per-form the test depended on the food type being analyzed. Sub-sequent 10-fold dilutions were prepared by adding 10 mL ofthe previous dilution to 90 mL sterile diluent and shaken25 times in a 30 cm arc. BioControl provided the suggested di-lution series for each food type the week before analysis wasinitiated. From each appropriate dilution, 1 mL aliquot wastransferred to a sterile Petri dish in duplicate. A 12–15 mLamount of Standard Methods Agar (SMA) tempered to42–45�C was added and swirled to evenly distribute thedilution and agar. Plate counts of each dilution level were per-formed in duplicate. All plates were incubated at 35�C for48 � 2 h. All plates were counted after the required incubation.Data were recorded onto appropriate worksheets. For theAOAC method, plates containing 30–300 colonies werecounted.

Designated food test portions were also prepared as speci-fied by ISO method 6887. For all food types, 50 g test portionswere added to 450 mL peptone salt solution and homogenizedby macerating for 1 min. After homogenization, test portionswere serially diluted into peptone salt solution. The number ofdilutions necessary to perform the test depended on the foodtype being analyzed. Subsequent 10-fold dilutions were pre-pared by adding 10 mL of the previous dilution to 90 mL ster-ile diluent and mixed well. BioControl provided the suggesteddilution series for each food type the week before analysis wasinitiated. From each appropriate dilution, 1 mL aliquot wastransferred to a sterile Petri dish in duplicate. A 15 mL amountof SMA tempered to 44.5–45.5�C was added and swirled toevenly distribute the diluted food test portion and agar. Platecounts of each dilution level were performed in duplicate. Allplates were incubated at 30 � 1�C for 72 � 3 h. All plates werecounted after the required incubation. Data were recordedonto appropriate worksheets.

Diluted test portions prepared for the AOAC and ISO ref-erence methods were also used for SimPlate analyses. One setof SimPlates was set up using the dilutions from the AOACmethod. A separate set of SimPlates was set up using the dilu-tions from the ISO method. A 1 mL amount of the diluted testportion was transferred to the center of the SimPlate device.The SimPlate was overlayed with 9 mL rehydrated TPC–CImedium, for a final volume of 10 ± 0.2 mL. After the plate wascovered with the lid, the food/medium homogenate was mixedand swirled into the wells. Excess liquid was decanted into theintegrated collection sponge. The SimPlate devices from theAOAC test portions were incubated upright at 35 � 1�C for24–28 h. The SimPlate devices from the ISO test portionswere incubated upright at 30 � 1�C for 24–28 h. After incuba-

tion, the number of wells with a color change from the originalbackground color was counted. Table 1 was used to correlatethe number of positive wells to the total aerobic microorgan-isms per SimPlate device by matching the number of positivewells counted to the corresponding microbial populationgiven in Table 1. If a dilution was made to the original foodtest portion before inoculation of the SimPlate device, then thetotal aerobic microorganisms per gram of food was calculatedby multiplying the population from Table 1 by the dilutionfactor.

Statistical Analysis

For each lot of food, duplicate plate counts were averagedand reported as aerobic plate count per gram (APC/g) for theAOAC or ISO reference methods, and the SimPlate methods.The base 10 logarithms of SimPlate counts and APC/g foreach of the reference culture methods were used for statisticalanalysis. Repeatability (sr) and reproducibility (sR) standarddeviations, relative standard deviations of repeatability(RSDr) and reproducibility (RSDR), and repeatability (r) andreproducibility (R) values were calculated according to themethods of Youden and Steiner (4) after outliers were elimi-nated. The Cochran test was used to remove laboratoriesshowing significantly greater variability among replicateanalyses than other laboratories for a particular set of test por-tions. An F statistic that computes the ratio of the 2 varianceswas used to compare repeatability and reproducibility vari-ances. Mean responses between the 2 methods were comparedby using a 2-sample (paired) t-test.

AOAC Official Method 2002.07Detection and Quantification

of Total Aerobic MicroorganismsSimPlate Total Plate Count–Color Indicator (TPC–CI) Method

First Action 2002

(Applicable to detection and quantification of total aerobicmicroorganism populations in milk chocolate, cake mix,ground pepper, nut meats, dairy foods, red meats, poultrymeats, seafoods, lunch meat, frozen pot pies, cereals, pasta,egg products, flour, hash brown potatoes, vegetables, fruits,and fruit juice.)

Caution: Test portion dilutions and incubated SimPlate de-vices from food products could contain patho-genic bacteria if the particular test portion wascontaminated. Standard aseptic microbiologicallaboratory techniques, including decontaminationof any spills with disinfectant, are recommended.

See Tables 2002.07A–C for the results of theinterlaboratory study supporting acceptance of the method.

A. Principle

SimPlate Total Plate Count–Color Indicator (TPC–CI)method is used for detection and quantification of total aerobicpopulations. The TPC–CI medium and food mixture is dis-pensed into a SimPlate device and incubated for 24–28 h. The



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medium changes color in the presence of aerobic microorgan-isms. The total aerobic microorganisms count is determinedby counting the wells with changed color and correlating thenumber of positive wells with the number of total aerobic mi-croorganisms found in Table 1.

B. Media and Reagents

(a) Dehydrated Total Plate Count–Color Indicator(TPC–CI) medium.—In individually packaged single or mul-tiple test format.

(b) Supplement A (optional).—Add 1.0 mL sterile Sup-plement A solution per 100 mL sterile deionized H2O. Alter-natively, add 1.0 mL Supplement A to 100 mL deionized H2Oand autoclave for 15 min at 121�C.

(c) Butterfield’s phosphate buffered diluent(BPBD).—See 966.23(m).

(d) Peptone salt solution.—Dissolve 1.0 g enzymatic di-gest of casein and 8.5 g NaCl in 1 L deionized H2O. Autoclavefor 15 min at 121�C. Final pH, 7.0 � 0.2 at 25�C.

(e) SimPlate devices.—Twenty devices per package.Items (a), (b), and (e) are available from BioControl Sys-

tems, Inc. (12822 SE 32nd St, Bellevue, WA 98005).

C. Apparatus

(a) Incubator.—Maintaining 35–37�C.(b) Micropipetor.—Accurately dispensing 0.1 and

1.0 mL.(c) Pipets.—Accurately dispensing 1.0 and 10 mL.(d) Blender/stomacher.—Waring, or equivalent, for

blending test portions; IUL Instruments (Cincinnati, OH)masticator, or equivalent, for macerating test portions. Note:A blender is used if testing in accordance with the AOACmethod; a stomacher is used if testing in accordance with theISO method.

D. General Instructions

Do not use expired media. Store reconstituted medium be-tween 15 and 25�C in the dark and use within 12 h. Dispose ofmedium in a decontamination container, and sterilize beforediscarding.

E. Test Portions Preparation

(a) Weigh 50 g test portion into 450 mL sterile diluent, e.g.,BPBD (AOAC method) or peptone salt solution (ISO method).This is a 1:10 dilution. Macerate or blend to homogenize.

(b) If alternative test portion size is specified in testingprocedure, prepare 10% (w/v) suspension.

(c) If necessary, prepare 10-fold serial dilutions appropri-ate for the anticipated population of the test portion.

F. TPC–CI Test Procedure, Single Test Medium

(a) For 1.0 mL test portion size.—Resuspend powderedmedium with 9.0 mL sterile deionized water containing 1 mLSupplement A per 100 mL water. Add 1.0 mL prepared test por-tion and mix well. Do not count this reconstitution as a dilution.

(b) For 0.1 mL test portion size.—Resuspend powdered me-dium with 9.9 mL sterile deionized water containing 1 mL Sup-

plement A per 100 mL water. Add 0.1 mL prepared test por-tion and mix well. This is an additional 1:10 dilution from E.

Note: The final volume of test portion/medium mixture inthe container should be 10 � 0.2 mL.

(c) Remove lid from SimPlate device and transfer test por-tion/medium mixture onto center of plate. Immediately re-place lid. Continue with H(a).

G. TPC–CI Test Procedure, Multiple Test Medium

(a) Empty contents of one container into 100 mL steriledeionized water containing 1 mL Supplement A per 100 mLwater. Shake to completely dissolve.

(b) Remove lid from SimPlate device. Pipet prepared testportion onto center of plate. If prepared test portion size is1.0 mL, overlay test portion with 9.0 mL medium. Do notcount this media addition as a dilution.

(c) For 0.1 mL prepared test portion, overlay with 9.9 mLmedium; this is an additional 1:10 dilution of test portion from E.

Note: The final volume of test portion/medium mixture onthe plate should be 10 � 0.2 mL.

(d) Immediately replace the lid. Continue with H(a).

H. Test Procedure for Single and Multiple Tests

(a) Gently swirl to distribute test portion/medium mixtureinto all wells. Hold plate with both hands, tilted slightly tohelp distribute medium into wells.

(b) Pour off excess medium by holding lid against plate oneither side of sponge cavity. Tip plate toward you to allow liq-uid to drain into sponge. Observe background color of wells.Background is defined as color of the test portion/mediummixture inside the wells before incubation.

(c) If testing in accordance with AOAC/BAM/USDAmethods, incubate SimPlate devices in an upright position inthe dark for 24–28 h at 35 ±1�C (32 ± 1�C for dairy products).If testing in accordance with ISO procedures, incubate devicesin an upright position in the dark for 24–28 h at 30 ± 1�C.

I. Reading and Interpretation of Results

(a) After incubation, observe color change of liquid inwells. Disregard particulate matter if present. Count numberof wells showing a color change from the background color.The most common color change produced by microorganismsis pink, but orange, peach, red, brown, and white may also beobserved.

(b) To determine the population, perform the followingcalculations: (1) Count the number of positive wells on theplate; (2) use Table 1 to determine total number of microor-ganisms per plate.

(c) To calculate number of microorganisms per gram (mL),multiply the count in I(b)(2) by the appropriate dilution factor(see E and F for Single test, or E and G for Multiple test).

Ref.: J. AOAC Int. 86, 259–263(2003)

Results

Four methods for enumerating total aerobic microorgan-isms in foods were compared in this collaborative study:


AOAC pour plate method incubated at 35�C (AOAC 35), ISOpour plate incubated at 30�C (ISO 30), SimPlate method incu-bated at 30�C (SimPlate 30), and SimPlate method incubatedat 35�C (SimPlate 35). Nineteen laboratories throughoutNorth America and Europe participated in the study (Table 2).Eight laboratories analyzed all 6 food types, 6 laboratories an-alyzed 5 food types, one laboratory analyzed 4 food types, and4 laboratories analyzed one food type (Table 2). The platecounts for individual test portions are presented in Tables 3–8.Repeatability and reproducibility analyses are presented inTables 2002.07A–C.

Ground Black Pepper

Three lots of ground black pepper (Lots A–C) were ana-lyzed (Table 3). Throughout the analysis of ground black pep-per, some laboratories reported aerobic plate counts for certaintest portions that were not in the suitable counting range forAOAC or ISO methods (Table 3). Data from these paired testportions were not used for statistical analysis.

Data generated by the AOAC 35 and the SimPlate 35methods were compared statistically. Laboratory 7 was deter-mined to be an outlier for Lot A by the Cochran test; therefore,the paired data were excluded from statistical analysis. Over-

all, there were 12, 13, and 14 laboratories that submitted validdata for Lots A–C, respectively. Mean log counts recoveredfrom Lot A were similar for the 2 methods. Lots B and C con-tained higher mean log counts as measured by the SimPlatemethod (p < 0.01). The repeatability (sr) and reproducibility(sR) standard deviations (SDs) of the 2 methods were statisti-cally analyzed for the 3 lots (Table 2002.07A). For Lots A andC, the AOAC method reported better SDr values. However,the SimPlate method showed better SDR values for Lots A–C.

Data generated by the ISO 30 and SimPlate 30 methodswere compared statistically. Overall, 14 laboratories submit-ted valid data for the 3 lots of pepper analyzed. The mean logcounts for the ISO 30 and the SimPlate 30 methods were notsignificantly different for the 3 lots analyzed. The ISO methodreported better SDr values for Lots B and C test portions. TheISO method also reported better SDR values for Lot B(p < 0.01). The SDR values were not significantly different forLots A and C between the 2 methods (Table 2002.07B).

Data generated by the ISO 30 and the AOAC 35 methodswere compared statistically. Overall, 14 laboratories submit-ted valid data for Lots A and C, and 13 laboratories submittedvalid data for Lot B. The mean log counts from the AOAC 35and the ISO 30 methods were not significantly different for


Table 2. Collaborator participation for SimPlate TPC–CI interlaboratory study by food typea

Lab Ground black pepper Flour Nut meats Frozen hamburger patties Frozen fruits Fresh vegetables

1 Y Y Y Y Y Y

2 Y Yb Y Y Y Y

3 Y Y Y Y Y Y

4 Y Y Y Y Y Y

5 Y Y Y Y Y Y

6 N Y Y Y Y Y

7 Y Y Y Y Y Y

8 Y Y Y Y Yb Y

9 Y Y Y Y Y N

10 Y Y Y Y Y N

11 Y Y Y Y Y N

12 Y Y N Y Y N

13 Y Y Yb Y Y N

14 Y Y Y Y Y N

15 Y Y Y Y Y Y

16 N N N N N Y

17 N N N N N Y

18 N N N N N Y

19 N N N N N Y

Totalc 14 15 14 15 15 13

a Y = Collaborator analyzed this food type; N = collaborator did not analyze this food type.b Laboratory did not follow study instructions. Results were not included in the statistical analysis for the designated food types.c Total number of laboratories participating in analysis of this food type.


Tab

le3.

Inte

rlab

ora

tory

resu

ltsfo

rae

rob

icp

late

cou

nts

for

gro

un

db

lack

pep

per

sam

ple

s(lo

g10

CF

U/g

)by

Sim

Pla

te35

,Sim

Pla

te30

,AO

AC

35,a

nd

ISO

30m

eth

od

s

Lab

LotA

LotB

LotC

Tes

tpor

tion

1T

estp

ortio

n2

Tes

tpor

tion

3T

estp

ortio

n4

Tes

tpor

tion

5T

estp

ortio

n6

SIM

35�C

AO

AC

35�C

SIM

30�C

ISO

30�C

SIM

35�C

AO

AC

35�C

SIM

30�C

ISO

30�C

SIM

35�C

AO

AC

35�C

SIM

30�C

ISO

30�C

SIM

35�C

AO

AC

35�C

SIM

30�C

ISO

30�C

SIM

35�C

AO

AC

35�C

SIM

30�C

ISO

30�C

SIM

35�C

AO

AC

35�C

SIM

30�C

ISO

30�C

16.

206.

245.

916.

036.

066.

145.

955.

997.

657.

627.

367.

367.

767.

778.

697.

427.

036.

896.

806.

367.

016.

776.

686.

39

26.

045.

906.

455.

905.

875.

926.

745.

897.

65—

a7.

377.

217.

76—

a7.

387.

196.

446.

546.

496.

366.

896.

576.

526.

30

36.

026.

175.

886.

036.

066.

065.

795.

937.

417.

596.

966.

947.

547.

546.

966.

976.

566.

686.

155.

946.

776.

526.

185.

95

46.

256.

015.

495.

805.

996.

035.

585.

867.

447.

537.

127.

147.

567.

657.

277.

266.

616.

656.

346.

416.

616.

636.

116.

38

56.

165.

835.

585.

325.

995.

735.

775.

257.

546.

957.

187.

007.

727.

117.

207.

156.

686.

136.

316.

126.

486.

136.

316.

15

76.

11b

4.67

b5.

464.

645.

88b

4.65

b5.

464.

747.

596.

757.

136.

727.

536.

707.

056.

706.

585.

816.

375.

756.

735.

716.

115.

75

86.

045.

915.

955.

916.

005.

995.

785.

527.

497.

307.

627.

297.

557.

397.

537.

376.

676.

356.

476.

346.

536.

376.

456.

69

96.

176.

186.

006.

176.

006.

085.

876.

097.

227.

117.

497.

567.

617.

558.

397.

606.

706.

696.

716.

797.

006.

626.

576.

72

106.

025.

765.

455.

816.

226.

015.

545.

717.

537.

277.

887.

927.

497.

267.

297.

136.

546.

616.

025.

996.

596.

546.

075.

91

116.

176.

125.

695.

996.

236.

125.

726.

047.

587.

667.

317.

537.

587.

657.

207.

386.

736.

757.

327.

356.

766.

606.

366.

85

126.

315.

835.

675.

826.

205.

625.

665.

617.

226.

727.

387.

377.

526.

717.

327.

266.

926.

086.

536.

106.

616.

546.

606.

43

135.

43b

5.17

b5.

485.

474.

57—

a4.

945.

037.

766.

926.

517.

007.

417.

177.

166.

716.

185.

806.

016.

006.

455.

946.

316.

20

146.

666.

614.

855.

376.

096.

425.

005.

317.

267.

186.

236.

517.

167.

256.

326.

696.

266.

335.

645.

796.

446.

405.

635.

91

156.

236.

206.

116.

036.

206.

185.

975.

967.

797.

727.

437.

607.

697.

727.

407.

566.

736.

786.

506.

676.

996.

906.

616.

67

aC

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Tab

le4.

Inte

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ora

tory

resu

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rob

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late

cou

nts

for

flou

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10C

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/g)b

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met

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tpor

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5T

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n6

SIM

35�C

AO

AC

35�C

SIM

30�C

ISO

30�C

SIM

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AO

AC

35�C

SIM

30�C

ISO

30�C

SIM

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AO

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35�C

SIM

30�C

ISO

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SIM

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AO

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35�C

SIM

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ISO

30�C

SIM

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AO

AC

35�C

SIM

30�C

ISO

30�C

SIM

35�C

AO

AC

35�C

SIM

30�C

ISO

30�C

15.

985.

815.

615.

545.

645.

675.

625.

543.

54—

a3.

583.

733.

433.

493.

593.

413.

813.

804.

094.

153.

523.

544.

204.

32

2—

b—

b—

b—

b—

b—

b—

b—

b—

b—

b—

b—

b—

b—

b—

b—

b—

b—

b—

b—

b—

b—

b—

b—

b

35.

665.

635.

975.

995.

385.

406.

005.

973.

20—

a4.

324.

153.

52—

a4.

284.

204.

594.

264.

694.

684.

204.

044.

724.

75

45.

575.

665.

565.

525.

525.

635.

405.

523.

743.

573.

403.

613.

903.

683.

083.

514.

044.

154.

364.

284.

264.

084.

004.

15

56.

045.

235.

985.

265.

985.

205.

825.

284.

434.

083.

893.

474.

293.

863.

763.

304.

804.

004.

513.

924.

864.

634.

483.

93

65.

885.

79c

5.88

5.87

c6.

005.

65c

6.84

6.86

c4.

263.

833.

963.

884.

263.

813.

283.

534.

434.

204.

544.

404.

464.

364.

584.

34

75.

675.

935.

725.

765.

695.

825.

986.

003.

203.

604.

113.

993.

623.

933.

893.

903.

914.

00c

4.52

4.84

c3.

664.

32c

4.34

4.34

c

85.

795.

865.

815.

865.

945.

886.

265.

833.

233.

643.

583.

853.

083.

573.

853.

714.

364.

234.

434.

263.

233.

114.

264.

26

95.

866.

086.

085.

946.

086.

086.

116.

084.

084.

114.

004.

004.

084.

083.

974.

044.

234.

204.

574.

384.

324.

234.

634.

56

105.

735.

585.

474.

695.

994.

955.

745.

203.

71—

a3.

464.

564.

09—

a4.

114.

794.

034.

564.

534.

113.

523.

534.

234.

08

115.

895.

866.

006.

045.

865.

866.

006.

044.

454.

484.

234.

114.

564.

614.

324.

204.

814.

534.

864.

804.

824.

684.

974.

78

126.

015.

905.

325.

185.

815.

575.

605.

363.

814.

063.

18—

a4.

043.

763.

453.

414.

67—

a4.

344.

094.

34—

a4.

024.

06

136.

235.

895.

835.

936.

616.

045.

765.

864.

43—

a3.

563.

724.

453.

613.

613.

764.

623.

834.

304.

044.

584.

114.

404.

08

145.

155.

825.

835.

965.

725.

775.

836.

042.

90—

a3.

903.

973.

00—

a3.

813.

934.

114.

044.

654.

484.

084.

084.

744.

64

155.

785.

825.

595.

795.

915.

986.

005.

884.

103.

813.

493.

633.

523.

813.

943.

914.

614.

454.

514.

404.

414.

414.

454.

26

aC

ount

sw

ere

noti

nra

nge.

The

paire

dte

stpo

rtio

nw

asex

clud

edfr

omst

atis

tical

anal

ysis

.b

Did

notf

ollo

wst

udy

inst

ruct

ions

.c

Out

lier;

data

notu

sed

inan

alys

isfo

rm

etho

dco

mpa

rison

ofA

OA

C(3

5�C

)an

dIS

O(3

0�C

)m

etho

ds.


Tab

le5.

Inte

rlab

ora

tory

resu

ltsfo

rae

rob

icp

late

cou

nts

for

nu

tmea

tssa

mp

les

(log

10C

FU

/g)b

yS

imP

late

35,S

imP

late

30,A

OA

C35

,an

dIS

O30

met

ho

ds

Lab

Pea

nuts

Alm

onds

Haz

elnu

ts

Tes

tpor

tion

1T

estp

ortio

n2

Tes

tpor

tion

3T

estp

ortio

n4

Tes

tpor

tion

5T

estp

ortio

n6

SIM

35�C

AO

AC

35�C

SIM

30�C

ISO

30�C

SIM

35�C

AO

AC

35�C

SIM

30�C

ISO

30�C

SIM

35�C

AO

AC

35�C

SIM

30�C

ISO

30�C

SIM

35�C

AO

AC

35�C

SIM

30�C

ISO

30�C

SIM

35�C

AO

AC

35�C

SIM

30�C

ISO

30�C

SIM

35�C

AO

AC

35�C

SIM

30�C

ISO

30�C

14.

864.

114.

754.

514.

574.

324.

264.

232.

622.

512.

722.

652.

892.

872.

462.

343.

123.

114.

714.

833.

523.

362.

932.

88

23.

383.

544.

734.

644.

384.

514.

494.

262.

402.

152.

452.

402.

182.

182.

622.

324.

264.

184.

854.

644.

113.

733.

944.

00

34.

363.

893.

513.

344.

113.

923.

943.

782.

412.

541.

851.

302.

412.

761.

781.

702.

983.

184.

304.

083.

003.

002.

452.

28

44.

003.

573.

083.

933.

953.

823.

343.

483.

202.

382.

602.

402.

412.

412.

742.

265.

365.

231.

902.

813.

543.

432.

722.

20

54.

203.

773.

08—

a4.

544.

043.

30—

a2.

412.

082.

411.

302.

542.

112.

541.

004.

153.

303.

653.

484.

714.

631.

60—

a

63.

873.

694.

434.

434.

434.

264.

284.

432.

342.

36—

a—

a2.

662.

54—

a—

a2.

71—

a—

a—

a2.

40—

a—

a—

a

74.

324.

303.

203.

083.

854.

153.

833.

542.

261.

701.

301.

462.

461.

952.

08—

a3.

183.

083.

413.

544.

674.

433.

343.

18

84.

494.

433.

724.

844.

344.

044.

234.

042.

562.

632.

852.

202.

852.

632.

792.

153.

363.

202.

322.

152.

693.

762.

382.

38

94.

414.

344.

824.

594.

233.

854.

704.

402.

462.

492.

812.

882.

672.

493.

002.

652.

36—

a3.

263.

453.

082.

915.

154.

83

104.

324.

204.

404.

524.

484.

004.

884.

582.

151.

932.

152.

641.

782.

112.

722.

714.

003.

784.

264.

083.

483.

522.

762.

76

113.

723.

544.

924.

184.

653.

405.

345.

202.

652.

582.

812.

402.

282.

562.

152.

513.

403.

112.

932.

923.

764.

572.

892.

66

13—

b—

b—

b—

b—

b—

b—

b—

b—

b—

b—

b—

b—

b—

b—

b—

b—

b—

b—

b—

b—

b—

b—

b—

b

143.

404.

154.

945.

184.

684.

745.

086.

042.

762.

802.

812.

832.

842.

842.

852.

863.

203.

204.

264.

343.

183.

263.

263.

30

154.

324.

204.

664.

544.

324.

233.

113.

672.

632.

602.

542.

642.

862.

723.

022.

994.

113.

795.

46—

a4.

594.

683.

112.

87

aC

ount

sw

ere

noti

nra

nge.

The

paire

dte

stpo

rtio

nw

asex

clud

edfr

omst

atis

tical

anal

ysis

.b

Did

notf

ollo

wst

udy

inst

ruct

ions

.


Tab

le6.

Inte

rlab

ora

tory

resu

ltsfo

rae

rob

icp

late

cou

nts

for

fro

zen

ham

bu

rger

pat

ties

sam

ple

s(lo

g10

CF

U/g

)by

Sim

Pla

te35

,Sim

Pla

te30

,AO

AC

35,a

nd

ISO

30m

eth

od

s

Lab

LotA

LotB

LotC

Tes

tpor

tion

1T

estp

ortio

n2

Tes

tpor

tion

3T

estp

ortio

n4

Tes

tpor

tion

5T

estp

ortio

n6

SIM

35�C

AO

AC

35�C

SIM

30�C

ISO

30�C

SIM

35�C

AO

AC

35�C

SIM

30�C

ISO

30�C

SIM

35�C

AO

AC

35�C

SIM

30�C

ISO

30�C

SIM

35�C

AO

AC

35�C

SIM

30�C

ISO

30�C

SIM

35�C

AO

AC

35�C

SIM

30�C

ISO

30�C

SIM

35�C

AO

AC

35�C

SIM

30�C

ISO

30�C

12.

792.

742.

913.

122.

792.

762.

903.

103.

943.

813.

93a

3.95

a3.

813.

593.

88a

4.90

a2.

412.

642.

683.

152.

542.

622.

723.

08

22.

512.

582.

832.

632.

46—

b2.

922.

833.

703.

115.

074.

723.

433.

274.

994.

852.

36—

b2.

992.

942.

382.

542.

902.

95

33.

482.

953.

283.

363.

112.

733.

143.

354.

244.

115.

255.

014.

233.

754.

865.

032.

852.

692.

943.

183.

132.

702.

543.

09

43.

473.

053.

263.

103.

163.

113.

323.

424.

004.

025.

375.

303.

863.

815.

355.

393.

393.

253.

203.

133.

893.

453.

323.

15

52.

612.

532.

963.

043.

392.

663.

092.

914.

584.

055.

374.

954.

002.

915.

304.

962.

542.

57—

b—

b2.

682.

593.

183.

00

63.

722.

923.

493.

323.

163.

013.

263.

134.

834.

63—

b—

b4.

814.

42—

b—

b3.

012.

89—

b—

b2.

752.

624.

875.

86

72.

812.

773.

022.

812.

32—

b2.

902.

902.

963.

044.

994.

882.

992.

794.

954.

912.

412.

542.

842.

862.

592.

582.

492.

78

82.

762.

853.

483.

283.

182.

822.

943.

134.

254.

423.

865.

144.

314.

443.

945.

252.

382.

813.

083.

162.

712.

903.

043.

05

93.

393.

413.

193.

403.

483.

463.

403.

814.

314.

165.

375.

564.

123.

915.

505.

543.

083.

063.

343.

632.

362.

573.

303.

54

103.

482.

69c

3.22

a3.

90ac

3.61

2.60

c3.

52a

2.77

ac4.

373.

195.

545.

104.

324.

035.

485.

262.

952.

993.

012.

792.

953.

093.

212.

96

113.

033.

233.

193.

233.

123.

263.

063.

134.

213.

995.

445.

204.

153.

945.

305.

163.

023.

383.

322.

952.

993.

112.

943.

08

123.

443.

013.

063.

063.

19—

b3.

143.

124.

114.

185.

015.

094.

664.

865.

195.

212.

802.

722.

743.

112.

973.

042.

703.

02

133.

432.

873.

463.

243.

462.

733.

383.

164.

694.

335.

255.

434.

534.

125.

255.

383.

162.

822.

983.

133.

182.

912.

933.

13

142.

432.

692.

633.

142.

452.

632.

813.

263.

223.

294.

615.

403.

333.

304.

665.

442.

732.

883.

053.

132.

712.

853.

043.

18

152.

892.

923.

183.

223.

193.

443.

343.

394.

013.

725.

455.

373.

893.

895.

445.

443.

032.

923.

173.

113.

243.

033.

213.

18

aO

utlie

r;da

tano

tuse

din

anal

ysis

for

met

hod

com

paris

onof

ISO

(30�

C)

and

SIM

(30�

C).

bC

ount

sw

ere

noti

nra

nge.

The

paire

dte

stpo

rtio

nw

asex

clud

edfr

omst

atis

tical

anal

ysis

.c

Out

lier;

data

notu

sed

inan

alys

isfo

rm

etho

dco

mpa

rison

ofIS

O(3

0�C

)an

dA

OA

C(3

5�C

).


Tab

le7.

Inte

rlab

ora

tory

resu

ltsfo

rae

rob

icp

late

cou

nts

for

fro

zen

fru

itssa

mp

les

(log

10C

FU

/g)b

yS

imP

late

35,S

imP

late

30,A

OA

C35

,an

dIS

O30

met

ho

ds

Lab

Pea

ches

Str

awbe

rrie

sB

lueb

errie

s

Tes

tpor

tion

1T

estp

ortio

n2

Tes

tpor

tion

3T

estp

ortio

n4

Tes

tpor

tion

5T

estp

ortio

n6

SIM

35�C

AO

AC

35�C

SIM

30�C

ISO

30�C

SIM

35�C

AO

AC

35�C

SIM

30�C

ISO

30�C

SIM

35�C

AO

AC

35�C

SIM

30�C

ISO

30�C

SIM

35�C

AO

AC

35�C

SIM

30�C

ISO

30�C

SIM

35�C

AO

AC

35�C

SIM

30�C

ISO

30�C

SIM

35�C

AO

AC

35�C

SIM

30�C

ISO

30�C

12.

833.

103.

45—

a2.

993.

043.

293.

043.

15—

a3.

26—

a3.

28—

a3.

41—

a2.

802.

922.

812.

793.

042.

912.

672.

63

22.

982.

973.

743.

742.

993.

073.

503.

684.

263.

724.

28—

b4.

403.

974.

18—

b2.

26—

a2.

61—

a2.

58—

a2.

54—

a

33.

532.

874.

253.

673.

473.

003.

933.

974.

403.

944.

224.

004.

464.

134.

093.

993.

513.

112.

492.

723.

182.

872.

362.

53

44.

013.

023.

603.

193.

35—

a4.

063.

414.

003.

503.

894.

843.

863.

443.

904.

813.

052.

662.

742.

482.

942.

812.

972.

94

52.

86—

a3.

673.

232.

92—

a3.

633.

284.

263.

753.

973.

794.

193.

444.

193.

872.

61—

a2.

612.

242.

612.

542.

952.

63

63.

233.

204.

134.

112.

933.

064.

304.

134.

864.

164.

053.

784.

654.

204.

133.

793.

202.

663.

062.

643.

352.

932.

732.

62

72.

18c

2.99

c3.

503.

393.

61c

3.44

c3.

543.

584.

13—

a4.

31—

a4.

06—

a3.

88—

a2.

68—

a2.

283.

172.

48—

a2.

70—

a

8—

d—

d—

d—

d—

d—

d—

d—

d—

d—

d—

d—

d—

d—

d—

d—

d—

d—

d—

d—

d—

d—

d—

d—

d

93.

353.

504.

034.

043.

543.

584.

164.

054.

183.

904.

153.

964.

194.

114.

273.

833.

222.

692.

412.

673.

393.

153.

032.

95

104.

39—

a4.

112.

623.

56—

a4.

632.

794.

203.

994.

163.

934.

484.

154.

173.

922.

83—

a2.

852.

282.

15—

a2.

41—

a

113.

303.

063.

763.

983.

473.

434.

203.

954.

094.

024.

223.

914.

264.

074.

494.

233.

243.

033.

142.

803.

233.

053.

412.

85

123.

603.

624.

064.

243.

653.

764.

134.

114.

464.

013.

823.

444.

183.

604.

183.

462.

53—

a2.

232.

392.

652.

512.

402.

17

133.

873.

363.

654.

053.

463.

293.

914.

024.

363.

874.

183.

634.

483.

964.

023.

602.

63—

a2.

882.

532.

83—

a2.

692.

42

142.

36—

a3.

613.

632.

32—

a3.

983.

844.

534.

424.

364.

174.

364.

234.

023.

982.

762.

752.

672.

732.

772.

682.

812.

81

153.

363.

184.

264.

143.

513.

544.

064.

084.

374.

224.

153.

944.

404.

184.

294.

063.

463.

163.

152.

943.

102.

883.

262.

90

aC

ount

sw

ere

noti

nra

nge.

The

paire

dte

stpo

rtio

nw

asex

clud

edfr

omst

atis

tical

anal

ysis

.b

Spr

eadi

ngm

icro

orga

nism

s;un

able

toen

umer

ate

plat

es.

cO

utlie

r;da

tano

tuse

din

anal

ysis

for

met

hod

com

paris

onof

SIM

(35�

C)

and

AO

AC

(35�

C).

dD

idno

tfol

low

stud

yin

stru

ctio

ns.


Tab

le8.

Inte

rlab

ora

tory

resu

ltsfo

rae

rob

icp

late

cou

nts

for

fres

hve

get

able

ssa

mp

les

(log

10C

FU

/g)b

yS

imP

late

35,S

imP

late

30,A

OA

C35

,an

dIS

O30

met

ho

ds

Lab

Car

rot

Bro

ccol

iC

eler

y

Tes

tpor

tion

1T

estp

ortio

n2

Tes

tpor

tion

3T

estp

ortio

n4

Tes

tpor

tion

5T

estp

ortio

n6

SIM

35�C

AO

AC

35�C

SIM

30�C

ISO

30�C

SIM

35�C

AO

AC

35�C

SIM

30�C

ISO

30�C

SIM

35�C

AO

AC

35�C

SIM

30�C

ISO

30�C

SIM

35�C

AO

AC

35�C

SIM

30�C

ISO

30�C

SIM

35�C

AO

AC

35�C

SIM

30�C

ISO

30�C

SIM

35�C

AO

AC

35�C

SIM

30�C

ISO

30�C

16.

206.

187.

417.

647.

187.

197.

247.

646.

686.

626.

687.

186.

916.

847.

188.

007.

047.

066.

908.

477.

547.

337.

678.

66

27.

427.

797.

747.

627.

567.

747.

817.

627.

137.

357.

277.

267.

497.

647.

928.

18—

a—

a8.

678.

81—

a—

a8.

658.

81

37.

427.

457.

597.

597.

437.

537.

697.

597.

017.

017.

747.

697.

437.

417.

927.

737.

708.

388.

638.

728.

67—

a8.

668.

58

4—

a—

a—

a—

a—

a—

a8.

45—

a—

a—

a8.

808.

828.

178.

31—

a—

a—

a—

a—

a—

a—

a—

a—

a—

a

57.

086.

396.

786.

597.

226.

196.

946.

597.

035.

877.

907.

396.

965.

707.

817.

227.

346.

307.

707.

886.

976.

038.

368.

04

67.

507.

218.

107.

657.

707.

437.

887.

657.

447.

238.

278.

157.

387.

018.

047.

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the 3 lots analyzed. The AOAC method reported better SDr

values for Lot B test portions, but were not significantly dif-ferent for Lots A and C. The SDR values were not significantlydifferent for the 3 lots analyzed (Table 2002.07C).

Flour

Three types of flour (rye, wheat, and white) were analyzed(Table 4). Laboratory 2 did not follow study instructions. Thedata from this laboratory were excluded from statistical analy-sis. Throughout the analysis of flour, some laboratories re-ported aerobic plate counts for certain test portions that werenot in the suitable counting range for AOAC or ISO methods(Table 4). Data from these paired test portions were not usedfor statistical analysis.

Data generated by the AOAC 35 and the SimPlate 35methods were compared statistically. Overall, there were 11,13, and 14 laboratories that submitted valid data for wheat,white, and rye flour test portions, respectively. The mean logcounts from the AOAC 35 and the SimPlate 35 methods werenot significantly different for the 3 lots analyzed. The AOACmethod reported better SDr and SDR values for wheat test por-tions (p < 0.05). The SDr and SDR values for rye and white testportions were not significantly different between the 2 meth-ods (Table 2002.07A).

Data generated by the ISO 30 and SimPlate 30 methodswere compared statistically. Overall, 14 laboratories submit-ted valid data for all 3 types of flour for statistical analysis.The mean log counts from the ISO and the SimPlate methodswere not significantly different for the 3 lots analyzed. TheISO method reported better SDr values for the wheat test por-tions. However, the SDR was not significantly different be-tween the 2 methods for wheat test portions. The SDR valuesfor rye test portions were significantly better for the SimPlatemethod (p > 0.05). The white flour test portions reported simi-lar SDr and SDR values between the 2 methods (Ta-ble 2002.07B).

Data generated by the ISO 30 and the AOAC 35 methodswere compared statistically. Laboratories 6 and 7 were deter-mined to be outliers by the Cochran test for rye flour and whiteflour test portions, respectively. These paired data were ex-cluded from statistical analysis. Overall, there were 10, 12,and 13 laboratories that submitted valid data for wheat, white,and rye flour test portions, respectively. Mean log counts be-tween the 2 methods were similar for rye and wheat flour testportions. The ISO method recovered higher mean log countsfor white flour test portions (p < 0.05). The SDr and SDR val-ues of the 2 methods were not significantly different for therye and wheat test portions analyzed. The ISO method re-ported better SDr values for white flour test portions. How-ever, the SDR values were not significantly different for whiteflour test portions (Table 2002.07C).

Nut Meats

Three types of nut meats (peanuts, almonds, and hazelnuts)were analyzed (Table 5). Laboratory 13 did not follow studyinstructions. The data from this laboratory were excludedfrom statistical analysis. Collaborators reported a higher inci-

dence of low aerobic plate counts that were not in the suitablecounting range for AOAC 35 or ISO 30 methods for almondtest portions. There may have been die off of microorganismsduring shipment before analysis. All data reported for almondtest portions were used for statistical analysis. For peanuts andhazelnuts, some laboratories reported aerobic plate counts forcertain test portions that were not in the suitable countingrange for AOAC or ISO methods (Table 5). Data from thesepaired test portions were not used for statistical analysis.

Data generated by the AOAC 35 and the SimPlate 35methods were compared statistically. Overall, 13 laboratoriessubmitted valid data for peanut and almond test portions, and12 laboratories submitted valid data for hazelnut test portions.The mean log counts for the AOAC and the SimPlate methodswere not significantly different for the almond and hazelnuttest portions analyzed. Higher mean log counts were recov-ered by the SimPlate method (p < 0.05) for peanut test por-tions. The peanut and almond test portions reported better SDr

values for the AOAC method, whereas the hazelnut test por-tions reported similar SDr values between the 2 methods. TheSDR values of the 2 methods were not significantly differentfor any of the 3 nut meats analyzed (Table 2002.07A).

Data generated by the ISO 30 and SimPlate 30 methodswere compared statistically. Overall, 12 laboratories submit-ted valid data for peanuts and almonds, and 11 laboratoriessubmitted valid data for hazelnuts for statistical analysis.Mean log counts between the 2 methods were equivalent forall 3 lots of nut meat test portions. The ISO method reportedbetter SDr values for almonds test portions (p < 0.05). The SDr

values were not significantly different between the 2 methodsfor peanut and hazelnut test portions. The SDR were not sig-nificantly different between the 2 methods for all 3 lots ana-lyzed (Table 2002.07B).

Data generated by the ISO 30 method and the AOAC 35method were compared statistically. Overall, 12 laboratoriessubmitted valid data for all 3 types of nut meats analyzed.Mean log counts between the 2 methods were equivalent forall 3 lots of nut meat test portions. The AOAC method re-ported better SDr values for almond test portions. The SDR

values were significantly better for the AOAC method forpeanut and almond test portions (p < 0.01). However, the SDr

and SDR values were not significantly different between the 2methods for the hazelnut test portions (Table 2002.07C).

Frozen Hamburger Patties

Three lots of frozen hamburger patties (Lots A–C) wereanalyzed (Table 6). Throughout the analysis of frozen ham-burger patties, some laboratories reported aerobic plate countsfor certain test portions that were not in the suitable countingrange for AOAC or ISO methods (Table 6). Data from thesepaired test portions were not used for statistical analysis.

Data generated by the AOAC 35 and the SimPlate 35methods were compared statistically. Overall, 15 laboratoriessubmitted valid data for all 3 lots of patties analyzed. Themean log counts between the AOAC and the SimPlate meth-ods were not significantly different for the 3 lots analyzed.The AOAC method reported better repeatability for Lots A


and C, whereas the SimPlate method reported better repeat-ability for Lot B. Lots A and C generated better SDR values forthe AOAC method, whereas for Lot B, the SDR was not signif-icantly different between the 2 methods (Table 2002.07A).

Data generated by the ISO 30 and SimPlate 30 methodswere compared statistically. Laboratory 10 was determined tobe an outlier for Lot A by the Cochran test; therefore, thepaired data were excluded from statistical analysis. Overall,there were 14, 13, and 15 laboratories that submitted validdata for Lots A–C test portions, respectively. The mean logcounts from the ISO and the SimPlate methods were not sig-nificantly different for the 3 lots analyzed. The ISO method re-ported better SDr for Lots B and C test portions. The SDr wasnot significantly different for Lot A between the 2 methods.The ISO method reported better SDR for Lot B test portions.The SDR was not significantly different for Lots A and C be-tween the 2 methods (Table 2002.07B).

Data generated by the ISO 30 and the AOAC 35 methodswere compared statistically. Laboratory 10 was determined tobe an outlier by the Cochran test for Lot A; therefore, thepaired data were excluded from statistical analysis. Overall,14 laboratories submitted valid data for Lots A and B test por-tions, and 15 laboratories submitted valid data for Lot C testportions. The ISO method recovered higher mean log countsfor all 3 lots of patties analyzed (p < 0.01). The ISO methodreported better SDr for Lots B and C test portions. The SDr

was not significantly different for Lot A between the 2 meth-ods. The ISO method reported better SDR for Lot B test por-tions. The methods were not significantly different for Lot A,and the AOAC method reported better SDR for Lot C (Ta-ble 2002.07C).

Frozen Fruits

Three types of frozen fruits (peaches, strawberries, andblueberries) were analyzed (Table 7). Laboratory 8 did notfollow study instructions. The data from this laboratory wereexcluded from statistical analysis. Throughout the analysis offrozen fruits, several laboratories reported aerobic platecounts for certain test portions that were not in the suitablecounting range for AOAC 35 or ISO 30 methods (Table 7).Data from these paired test portions were not used for statisti-cal analysis.

Data generated by the AOAC 35 and the SimPlate 35methods were compared statistically. Laboratory 7 was deter-mined to be an outlier by the Cochran test for peach test por-tions; therefore, the paired data were excluded from statisticalanalysis. Overall, 10 laboratories submitted valid data forpeaches and blueberries, and 12 laboratories submitted validdata for strawberries. The SimPlate method recovered highermean log counts for strawberries and blueberries (p < 0.01).The SDr and SDR values of the 2 methods were not signifi-cantly different for any of the 3 fruits analyzed (Ta-ble 2002.07A).

Data generated by the ISO 30 and SimPlate 30 methodswere compared statistically. Overall, 13 laboratories submit-ted valid data for peaches and blueberries, and 11 laboratoriessubmitted valid strawberry data for statistical analysis. Straw-

berries recovered higher mean log counts by the SimPlatemethod (p < 0.05). The ISO method reported better SDr val-ues for peach and strawberries test portions. However, theSimPlate method showed better SDR values for these 2 fruits.The SDr and SDR values for blueberries were not significantlydifferent between the 2 methods (Table 2002.07B).

Data generated by the ISO 30 and the AOAC 35 methodswere compared statistically. Laboratory 2 was unable to re-cord counts from the ISO 30 pour plates because of spreadingmicrobial growth. These paired test portions were excludedfrom statistical analysis. Overall, 11 laboratories submittedvalid data for peaches and strawberries, and 10 laboratoriessubmitted valid data for blueberries. Mean log counts from thepeach test portions were higher for the ISO method (p < 0.01),whereas the AOAC method recovered higher mean countsfrom the blueberry test portions (p < 0.05). The ISO methodreported better SDr values for peach and strawberry test por-tions. The SDR values were not significantly different for the 3lots of frozen fruits analyzed (Table 2002.07C).

Fresh Vegetables

Three types of vegetables (carrots, broccoli, and celery)were analyzed (Table 8). Throughout the analysis of freshvegetables, some laboratories reported aerobic plate countsfor certain test portions that were not in the suitable countingrange for AOAC 35 or ISO 30 methods (Table 8). Data fromthese paired test portions were not used for statistical analysis.

Data generated by the AOAC 35 and the SimPlate 35methods were compared statistically. Overall, 12 laboratoriessubmitted valid data for carrot and broccoli test portions, and10 laboratories submitted valid data for celery test portions.The mean log counts between the AOAC 35 and the SimPlate35 methods were not significantly different for the 3 types ofvegetables analyzed. The SDr and SDR values for carrot testportions were not significantly different between the 2 meth-ods. The SimPlate method reported better SDr and SDR valuesfor broccoli test portions. The celery test portions reportedbetter SDR values for the SimPlate method (p < 0.01; Ta-ble 2002.07A).

Data generated by the ISO 30 and SimPlate 30 methodswere compared statistically. Overall, 11 laboratories submit-ted valid data for carrot and celery test portions, and 13 labora-tories submitted valid data for broccoli test portions. Themean log counts between the ISO 30 and the SimPlate 30methods were not significantly different for the 3 types of veg-etables analyzed. The SDr and SDR values were not signifi-cantly different between the 2 methods for carrot and broccolitest portions. The ISO method reported better SDr for the cel-ery test portions. The SDR, however, was not significantly dif-ferent between the 2 methods for the celery test portions (Ta-ble 2002.07B).

Data generated by the ISO 30 and the AOAC 35 methodswere compared statistically. Overall, 11 laboratories submit-ted valid data for carrot and broccoli test portions, and 9 labo-ratories submitted valid data for celery test portions. The ISOmethod recovered higher mean log counts for the broccoli andcelery test portions (p < 0.01). Mean log counts for carrot test


portions were not significantly different between the 2 meth-ods. The SDr and SDR values of the 2 methods were not signif-icantly different for the carrot test portions analyzed. Thebroccoli test portions reported similar SDr values between the2 methods. The AOAC method reported better SDR values forbroccoli and celery test portions. The ISO method showedbetter SDr for celery test portions (Table 2002.07C).

Discussion

In this international collaborative study, the SimPlatemethod was compared with the AOAC pour plate method(966.23) for enumeration of total aerobic microorganisms.The ISO method (ISO 4833) for enumerating total aerobic mi-croorganisms was also analyzed. The AOAC and ISO meth-ods use the same growth medium (Standard Methods Agar);however, they differ in the diluents used, the incubation tem-perature of the test, and the duration of incubation. The AOACmethod uses BPBD as the test diluent, whereas ISO usespeptone salt solution. The AOAC method incubates the Petriplates at 35�C for 48 h, whereas the ISO method incubates theplates at 30�C for 72 h. Because of these differences betweenthe 2 reference methods, an additional set of SimPlates was setup according to ISO requirements, for a total of 4 methods be-ing tested.

Six food types were evaluated by the 4 methods in thisstudy. Three lots of naturally contaminated test portions weretested for each food type, for a total of 18 food lots analyzed.Recovery (mean log counts) of aerobic microorganisms wasstatistically different for 5 of the 18 food lots analyzed by theSimPlate 35 and AOAC 35 methods. In actuality, the meanlog counts were similar for 4 of the 5 lots, with <0.30 mean logdifference in recovery. The SimPlate method recovered an av-erage mean log count 0.37 higher than the reference methodfor strawberries. A possible explanation for the higher recov-ery from the SimPlate method may be that incubation in a liq-uid medium is more suitable than an agar medium for thegrowth of microflora present in the strawberries.

The recovery of total aerobic microorganisms from theSimPlate devices incubated at 30�C correlated well with thatof the ISO method incubated at 30�C. Only one of the 18 foodlots analyzed (strawberries) had a statistically different recov-ery between the 2 methods (Table 2002.07B), with theSimPlate method recovering 0.18 mean log counts higher thanthe ISO method.

A comparison of the AOAC and ISO methods for recoveryof total aerobic microorganisms indicates a greater variabilityin results. Eight of the 18 lots analyzed had a statistically sig-nificant difference in recovery between the 2 methods, withthe ISO method recovering higher mean log counts for 7 ofthese lots. Two major differences between the 2 referencemethods are the incubation temperature and length of incuba-tion. Some microflora may favor the lower temperature usedby the ISO (30�C) method. Slow growing microflora wouldalso benefit from the extra day of incubation by the ISOmethod (72 h), contributing to the higher mean log counts.

Nonetheless, most food lots tested had similar mean logcounts between the 2 methods.

Conclusions

In general, there was <0.3 mean log count difference in re-covery between the AOAC 35 and the SimPlate 35 methods,the ISO 30 and the SimPlate 30 methods, and the AOAC 35 andthe ISO 30 methods. Higher mean log counts (>0.3 mean logcount difference) were recovered by the ISO 30 method for 2lots of frozen hamburger patties, peach test portions, broccolitest portions, and celery test portions compared with the AOAC35 method. The SDr and SDR values were similar between the 3method comparisons.

Recommendation

It is recommended that the SimPlate Total PlateCount–Color Indicator method be adopted First Action forenumeration of total aerobic microorganisms in foods incu-bated at 30 or 35�C.

Acknowledgments

The participation of the following collaborators is ac-knowledged with appreciation:

Mary Howell, Food Standards Agency, London, UKFlorence Humbert and Marylene Bohnert, Agence

Francaise de Securite Sanitaire des Aliments, Ploufragan,France

Martine Poumeyrol, Agence Francaise de SecuriteSanitaire des Aliments, Maisons-Alfort, France

Sandra Koch, Robert Voermans, Lynn Lewis, and JohnJackson, Analytical Laboratories, Inc., Boise, ID

Julia Terry and Stuart Hopkins, BioControl Systems, Inc.,Bellevue, WA

Victoria Arling and Bree-Ann Lightfoot, Canadian FoodInspection Agency, Calgary, Canada

Shawn Gartside and Sandra Clements, Central States Ana-lytical, Evansville, IN

Linda August, Samara Brookman, and Jessica Rice,Certispec Food Laboratory Inc., Quebec, Canada

Sven Qvist, Danish Veterinary and Food Administration,Soberg, Denmark

Robert Davis and Diane Barham, Davis Research, Avon,MS

Sharon Musch, Goldkist Laboratory, Boaz, ALCathy Chavey, Goldkist Laboratory, South Sumter, SCDon Warburton and Anne Boville, Health Canada, Ottawa,

CanadaPaul in’t Veld and Serge Dissel, Inspectorate for Health

Protection and Veterinary Public Health, Region South,Hertogenbosch, The Netherlands

Debra Hagel, Norpath Laboratories, Ltd., Seaham, CountyDurham, UK

Carol Kuber and Susan Devane, Universal Laboratories,Hampton, VA


Jeff Hunsucker, Kenneth Nieves, and Jacqueline Welch,U.S. Food and Drug Administration, Atlanta, GA

Wen Lin, Daniel Solis, Christopher Hernandez, andJennifer Lamb, U.S. Food and Drug Administration, Los An-geles, CA

References

(1) Official Methods of Analysis (2000) 17th Ed., AOAC IN-TERNATIONAL, Gaithersburg, MD, Method 966.23

(2) ISO 4833 (1991) International Organization for Standardiza-tion, Geneva, Switzerland

(3) ISO 6887-1 (1999) International Organization for Standard-ization, Geneva, Switzerland

(4) Youden, W.J., & Steiner, E.H. (1975) Statistical Manual ofthe AOAC, AOAC, Arlington, VA


Documents

Enumeration of Total Aerobic Microorganisms in Foods by ......35 C) was compared with the AOAC Official Method 966.23 (AOAC 35 C) for enumeration of to-tal aerobic microorganisms in