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Part VIII Public Healthand EnvironmentalMicrobiology

y now, it should be clear that public health is concerned withcommunicable disease. Its history grew out of the IndustrialRevolution of the mid-1800s when large populations of people

migrated to large cities (as is the case in many developing countries today).These migrations brought uncontrolled urban growth and indescribablygrim conditions. Garbage and dead animals littered the streets; humanfeces and sewage stagnated in open sewers; rivers were used for washing,drinking, and excreting; and filth was rampant.

However, sanitary reformers spoke up for effective public health mea-sures. Working with bacteriologists, they pressed the government for bet-ter public health measures and the development of better methods forsewage treatment, water purification, and food preservation. In Part V of thelab manual, exercises examined the physical and chemical agents, andantibiotic treatments, that have been developed over the last century tolimit the spread of infectious and communicable disease.

Long before the present era, people had used salt as a food preservationagent. The effectiveness of salt and the antimicrobial effects of other foods canbe clearly demonstrated (Exercise 31). Food preservation also advanced by theestablishment of standard plate counts to monitor contamination in foodsand the addition of preservatives to other foods to prevent microbial growth(Exercise 31). However, not all microorganisms are bad. For example, specificbacteria have been used to ferment cabbage to sauerkraut and the yeastshave been used for millennia in the fermentation of beer (Exercise 31).

Microorganisms also are critical to the production of milk and otherdairy products. Standard plate counts, coliform plate counts, and proce-dures such as the methylene blue reduction test are used to monitor thenatural microorganisms in milk and any unnatural ones that should con-taminate it (Exercise 32). Other microbes also are involved in the produc-tion of an assortment of foods, including cheeses and yogurt.

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The purity of water also is reflected in public health measures. Coliformbacteria can contaminate water and make it unfit for human consumption.A series of water quality tests can be performed to detect coliform bacteriaand to estimate their numbers if present (Exercise 33). Aquatic environ-ments are also home to many nonpathogenic microorganisms and thesehave recently become of great interest to microbiologists since these organ-isms usually exist as a biofilm (Exercise 33).

Plate counts demonstrate that the soil is almost bursting with bacteria(Exercise 34). Some play a dominant role in numerous biogeochemicalcycles and forge links between what is useless and what is useful to otherliving organisms. For example, Rhizobium and other soil bacteria areessential for the conversion of useless nitrogen gas into ammonia andnitrates, useful products for plants and other organisms (Exercise 34).Other soil bacteria and fungi recycle carbon, while several genera of Strep-tomyces are involved in antibiotic production (Exercise 34). Importantly,the populations of bacteria in the environment can fluctuate over time as theenvironment changes. This can be elegantly demonstrated by constructingand observing a Winogradsky column (Exercise 34).

earning Objectives

When you have completed the exercises in Part VIII, you should be capable of:• Assessing the role of salt and other preservatives in food preservation. • Carrying out a standard plate count and coliform plate count of food and dairy

products. • Analyzing the importance of the fermentation process to food and beverage

products. • Employing the methylene blue reduction test to determine the bacterial content

of a milk sample.• Demonstrating the role of microorganisms in such food processes as cheese and

yogurt production.• Explaining the natural progression of bacterial populations under specific envi-

ronmental conditions.• Completing the series of tests (presumptive, confirmed, and completed) to

detect coliform bacteria and to estimate their numbers.• Carrying out a membrane filtration procedure and using selective and differential

media to determine coliform numbers in a water sample.• Constructing an apparatus to obtain and study a biofilm.• Isolating and performing an examination of Rhizobium from legume roots.• Employing the diagnostic test to detect bacterial ammonification.• Isolating Streptomyces from soil and assessing its capability to produce antibiotics.• Carrying out a plate count of soil bacteria.• Constructing and analyzing the changing patterns of microbial succession in a

Winogradsky column.• Designing an exercise to assess the ability of soil bacteria to recycle carbon-

containing materials.

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Microbiology of Foods

ost foods provide an excellent growth medium for micro-organisms. The supply of organic matter is plentiful, the watercontent is usually sufficient, and the pH is generally neutral or

only slightly acidic. The result is food spoilage, which leads to an economicloss to the manufacturer and a waste of money to the consumer, as well asa threat to health. In this exercise, tests will be conducted to determinehow spoilage may be prevented with salt and garlic and how micro-organisms may be detected in spoiled foods.

Certain microorganisms may be beneficial to the food industry becausethey bring about fermentation in the food and lead to consumable products.This aspect also will be demonstrated by using organisms to produce sauer-kraut and fermented beverages.

Food Preservation with Salt and Garlic

High-salt environments exert an inhibitory effect on bacterial growth bystimulating the flow of water out of the organisms by the process of osmo-sis. This causes the microorganisms to shrink and disintegrate. Foods suchas salted beef and cod, bacon, and ham are preserved in this way. In this sec-tion, a piece of food will be suspended in broths containing various con-centrations of salt, and the growth of bacteria will be determined. Inaddition, the antimicrobial effect of garlic will be tested.

pecial Materials

• Raw hamburger• Tubes of normal nutrient broth• Tubes of nutrient broth containing 1%, 5%, and 10% salt• Raw garlic and garlic press

rocedure

I. Effectiveness of Salt1. Select one tube of normal nutrient broth and tubes of nutrient broth con-

taining 1%, 5%, and 10% salt. Label each of the tubes with your name, thedate, and the salt concentration of the broth.

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31

M

PURPOSE: to examine theeffects of salt and garlic aspreservatives.

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2. Aseptically inoculate each tube with approximately 1 gram of raw ham-burger meat, being careful to minimize airborne contamination. Themeat need not be weighed, but the sample should be about the size of apea. A clean and lightly flamed spatula or knife should be used. Incubatethe four tubes at 37º C for 24 to 48 hours.

3. Observe the tubes for the presence of bacteria, and note your results inTable 31.1 of the Results section, using (���) for heavy growth, (��) formoderate growth, (�) for trace of growth, and (�) for absence of growth.Be careful to distinguish bacterial growth from meat particles. Notewhether the amount of growth decreases as the salt concentrationincreases, and write your observations on the effect of salt as a foodpreservative. Prepare Gram stains (Exercise 6) from loopfuls of the var-ious broths, and observe the types of bacteria present in the hamburgermeat. Note whether the type of bacteria changes as the salt concentrationincreases. Representations may be placed in the appropriate spaces.

II. Effectiveness of Garlic1. Much has been written in recent years about the inhibitory effects of

garlic and how garlic can be used therapeutically to kill bacteria. Totest this principle, you will need two tubes of nutrient broth, a sample ofhamburger meat, and a sample of freshly-squeezed garlic.

2. As in steps 1 and 2 above, inoculate two tubes of nutrient broth with1-gram samples of raw hamburger meat. One tube is the control andwill receive no further treatment. The second tube, the experimental,will receive approximately 0.25 gram of fresh garlic, including the juiceand pulp. The tubes should then be incubated at 37ºC for approximately48 hours.

3. Examine the tubes for the presence or absence of bacterial growth andrecord your observations in the Results section. The absence of bacteriain the experimental tube provides evidence for the inhibitory effect of thegarlic. The presence of equal amounts of growth in the control and exper-imental tubes indicates noninhibition. The reduction of growth in theexperimental tube indicates some inhibition. Note that the experimentalconditions may be varied to provide different results than you obtained.You should suggest variations in the procedure, and indicate how they willprovide further information on garlic’s effects on bacteria.

Standard Plate Count of Food Products

The extent of bacterial contamination in foods may be determined by thestandard plate count procedure. In this process, food is ground withfluid in a blender to suspend the microorganisms. Samples of the fluid arethen diluted and placed in Petri dishes with growth medium. After incu-bation, the number of colonies is counted and multiplied by the dilution fac-tor to yield the total number of bacteria per gram of food sample. Theunderlying principle is that each bacterium will form a visible colony. The

B.

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!Treat incubated tubescarefully because bacte-rial pathogens may have increased in numbers todangerous levels.

PURPOSE: to determine the bacteria present in rawhamburger and other foods.

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test is similar to that performed with milk samples in Exercise 34A. In thisexercise, samples of foods including hamburger meat and mushroomswill be tested for their bacterial content.

pecial Materials

• Raw hamburger • Other food samples, such as potato salad, fresh vegetables, cold cuts, egg salad,

rice pudding, or salad fixings• Sterile Petri dishes and blenders• Sterile 1.1-ml pipettes and mechanical pipetters• Sterile 180-ml water samples• Sterile 99-ml water dilution blanks• Weighing apparatus• Melted nutrient agar• 10 fresh mushrooms and a bottle of salad dressing

rocedure

I. The Bacterial Flora of Hamburger Meat

1. To ensure reliable results, aseptic procedures should be followed through-out this procedure. Sterile instruments should be used whenever possible,and precautions should be taken to limit airborne contamination. Theprocedure may be performed in pairs due to the volume of materials nec-essary. The instructor will assign the type of food to be tested, and may givespecial directions on the use of the 1.1-ml pipettes that are commonly usedfor this type of test.

2. Obtain four sterile Petri dishes and label them on the bottom side withyour name, the date, and the designations 1:10, 1:100, 1:1000, and1:10,000. Select one sterile 180-ml water sample, one sterile 99-ml waterdilution blank, one sterilized blender, and a 1.1-ml pipette and mechan-ical pipetter.

3. Aseptically weigh 20 grams of the food sample to be tested. Combinethe food with 180 ml of sterile water in a sterile blender as shown in Fig-ure 31.1A. Blend for 3 minutes or as directed by the instructor. The result-ing fluid represents a 1:10 dilution of the food sample. Allow the largeparticles of food to settle before proceeding. If a blender is not available, vig-orous shaking will provide adequate suspension of the organisms.

4. Using a mechanical pipetter, pipette 0.1 ml of the blended material to the1:100 plate and 1.0 ml to the 1:10 plate, as shown in Figure 31.1B. Be care-ful to avoid airborne contamination of the plate by lifting the lid only highenough to permit entry of the pipette.

5. Pipette 1.0 ml of the blended material to the 99-ml water dilution blank (Fig-ure 31.1C), and draw up and release some material several times to washout the pipette. Shake the bottle for 2 minutes to effect even distribution.Aseptically pipette 0.1 ml of this material to the 1:10,000 plate, and 1.0 mlto the 1:1000 plate (Figure 31.1D).

6. Aseptically pour into each plate enough melted nutrient agar to cover thebottom of the plate. Rotate the plates ten times in a wide arc on the labo-ratory desk to ensure even mixture of the sample and nutrient agar. Allow

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Quick ProcedureStandard PlateCount (Food)

1. Blend food sample withsterile water.

2. Pipette 1.0 and 0.1 mlsamples to Petri dishes.

3. Pipette 1.0 to a 99-mlwater blank and shake.

4. Pipette 1.0 and 0.1 mlsample of diluted foodto Petri dishes.

5. Add liquid nutrientagar to all Petri dishesand mix.

6. Incubate.

7. Perform colony countsand locate valid count.

8. Multiply valid count bydilution factor.

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the agar to harden, and incubate the plates in the inverted position at 37º Cfor 24 to 48 hours.

7. The instructor will demonstrate the use of the Quebec colony counter forperforming plate counts. Place each plate on the colony counter, deter-mine the number of colonies per plate, and enter your results in thechart. Be sure to count surface as well as subsurface colonies. Be carefulto avoid counting food particles, which generally appear as irregularspots on the plates. If a plate contains significantly more than 300 colonies,it is unnecessary to obtain an exact count. In such a case the acronymTNTC (too numerous to count) may be entered in Table 31.2 in theResults section.

8. From your results, select the colony count that falls between 30 and 300.This is the “valid” count. Counts over 300 are considered invalid becauseovercrowding may cause two or more bacteria to form a single colony;counts under 30 are invalid because the chance of sampling error is sig-nificant. Multiply the valid count by the dilution factor of the plate (i.e., 10,100, 1000, or 10,000). This is the total plate count per gram of food sample.Consult with the instructor if more than one colony count falls between 30and 300, if all colony counts are below 30, or if all colony counts are over300. Enter your observations on the bacterial contamination of the food inTable 31.2. Obtain plate counts for other foods from fellow students, andenter these results in Table 31.3. At the direction of the instructor, Gramstains may be prepared from the colonies on the plates to examine thebacterial content of the food. Examination results can be recorded in theResults section.

II. The Bacterial Content of Marinated Mushrooms1. Mushrooms are a valuable food sample for bacterial testing because they

are grown in rich organic soil, where the bacterial content is nor-mally high. A plate count, as described above, is a useful way of deter-mining the number of bacteria per gram of mushrooms. In addition, the

282 31 M I C R O B I O L O G Y O F F O O D SA

B

C

D

Combine 20 g foodand 180 ml water in a sterile blender.

Pipette

Pipette 1.0 ml

Pipette

1:10,000 dilution

1:1000 dilution

0.1 ml 0.1 ml

1:100 dilution

1.0 ml 1.0 ml1:10 dilution

99 mlwater

Dilutionblank

F I G U R E 3 1 . 1Standard plate countprocedure using a food sample.

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effectiveness of the acid in a marinade can be demonstrated to be a methodfor lowering the bacterial count.

2. Select ten fresh unprocessed mushrooms and divide them into two equalgroups. Slice the first group of five and place them in a clean plastic bag.Then cover the mushrooms in the bag with any commercial dressing to beused as a marinade. The dressing should contain vinegar (Italian dressingis a good choice). The mushrooms should be left undisturbed for one hour.During that time interval, the other five mushrooms should be sliced andset aside.

3. At the end of the hour, weigh out one gram of the marinated mushroomsand add it to a sterile 99-ml water dilution blank. Perform a plate count asdescribed in steps 1 to 8 in the previous procedure. Then weigh out onegram of the unmarinated mushrooms and add it to another sterile 99-mlwater dilution blank. Perform a plate count on this sample as explained inPart B. Set all the plates aside to incubate in the inverted position for 24 to48 hours.

4. Perform calculations as cited previously to complete the plate counts.Then compare the results and draw your conclusions on the effect of themarinade on the bacterial count. Your conclusions may be entered inTable 31.4 and Table 31.5 of the Results section. Consider other preserv-atives that can be used to reduce bacterial counts and suggest variationsof this procedure.

Fermentation of Sauerkraut

The word sauerkraut is derived from German roots meaning “sour-cabbage.” This food product is formed by species of Lactobacillus and Leu-conostoc that normally occur in cabbage and ferment it. The acid they pro-duce inhibits the growth of other organisms and provides a naturalpreservative in the product. The high salt content enhances preservation.This section will demonstrate the process for sauerkraut production.

pecial Materials

• Heads of cabbage• Shredder and waxed paper• Sterile 1000-ml beakers, foil-covered• Noniodized salt and Petri dish lids• Weighing apparatus• Large stones or bricks covered with foil• Gram stain reagents

rocedure

1. Remove the core and outer leaves from a quarter of a head of cabbage andweigh it on a scale. Calculate 3% of this figure, and weigh out that amountof salt. Noniodized salt is recommended to avoid iodine, which mayinterfere with bacterial growth.

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PURPOSE: to use sauerkrautproduction as an example ofmicrobial fermentation ofcommon foods.

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2. Shred the cabbage on waxed paper and mix it thoroughly with the salt.Tightly pack the cabbage into a sterile 1000-ml beaker. Cover the cabbagewith a clean Petri dish lid or a sterile watch glass, and add a foil-wrappedstone or brick to weigh down the cover. The tight packing will encouragethe anaerobic conditions necessary for fermentation of the cabbage.Replace the foil over the beaker, and set the cabbage aside at approxi-mately 30° C for 1 to 2 weeks.

3. Observe the cabbage at regular intervals, and note the accumulation offluid as water flows out of the plant cells by osmosis caused by the salt.Gram stains of fluid samples may be prepared to observe the changingflora. Gram-positive rods of the genus Lactobacillus and Gram-positivecocci belonging to the genus Leuconostoc should be evident. At the con-clusion of the fermentation period and at the suggestion of the instructor,taste the sauerkraut to determine the success of the fermentation. Enter yourobservations in the Results section.

Fermentation of Wine and Beer

The production processes for wine and beer are highly specialized andsophisticated. However, the fundamental process may be observed in thelaboratory by fermenting grape juice and malt extract broth with yeasts, asshown in this section.

pecial Materials

• Cotton-plugged tubes of grape juice supplemented with 5% sugar• Tubes of malt extract broth with cotton plugs• Cultures of Saccharomyces cerevisiae or commercial yeast

rocedure

I. Basic Fermentation1. Select tubes of sterile grape juice and malt extract broth containing cotton

plugs. The cotton plug will permit the carbon dioxide that is produced toescape. Inoculate each tube with a heavy loopful of Saccharomyces cere-visiae or commercial yeast, and incubate the tubes at approximately 30° Cfor several days to a week as directed by the instructor. Uninoculated con-trol tubes should be included.

2. Shake the experimental tubes lightly and note the foaming at the surface.The foaming indicates that carbon dioxide has been produced and is nowescaping from the liquid. Note the winelike aroma in the grape juice and thebeerlike odor of the malt extract broth. At the instructor’s direction, taste theliquid to determine its quality, and prepare stains of the sediment at the bot-tom of the tubes to observe the yeast cells. Enter your observations in theResults section.

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Quick ProcedureSauerkrautFermentation

1. Shred cabbage and mixwith 3% salt.

2. Pack into containerand place weight ontop.

3. Incubate for 1 to2 weeks.

PURPOSE: to examine thefundamental process of wineand beer fermentation.

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II. Quantitative Estimation of Fermentation1. The extent of fermentation over a period of time can be studied by mea-

suring the amount of gas produced in the fermentation flask. For this exer-cise you will need a balloon and a flask of grape juice inoculated withyeast. The balloon should be stretched out to loosen it. Graph paper also willbe needed for the calculations.

2. Place the balloon over the mouth of the flask and secure it tightly with awire, rubber band, or other device. Set the flask aside to allow the juice toferment and observe the balloon at regular time periods over a number ofhours. The balloon will open and expand with gas. The circumference ofthe balloon should be measured and noted in Table 31.6 of the Resultssection. The expansion will be rapid at first as the oxygen is used up andcarbon dioxide is quickly produced. It will eventually slow as thechangeover from respiration to fermentation occurs. (Less carbon dioxideis produced during fermentation than during respiration.)

3. A graph should be prepared comparing the circumference of the balloon asit relates to time. This graph will help quantitate the progress of the fer-mentation.

4. The above exercise can be varied in numerous ways to study variousaspects of fermentation. For example, different carbohydrate sources canbe used (grape juice, apple juice, orange juice) to see whether there is a dif-ference in the rate of fermentation and whether the yeast shows a prefer-ence. The fermentation can be conducted at different temperatures(refrigerator, room, 37° C, 55° C) to determine the optimum temperature forthe fermentation. The concentration of the grape juice can be varied tostudy the effect of substrate concentration on fermentation activity.Inhibitors such as metals or drugs can be incorporated into the grape juiceto study their effect on the fermentation. The instructor may suggest oth-er variables to be tested.

uestions

1. Why must a plate be considered invalid if it contains less than 30 ormore than 300 colonies?

2. Does a high standard plate count in food indicate that the food should notbe eaten?

3. Explain some common errors that may cast doubt on the accuracy of aplate count.

4. What precautions must be taken to ensure a successful sauerkraut fer-mentation?

5. Suppose a balloon were tied over the mouth of a tube of grape juiceinoculated with yeast. What would take place? Why?

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Table 31.1. Presence of Growth in Nutrient Broth Tubes

Salt Concentration No Added Salt 1% Salt 5% Salt 10% Salt

Growth

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Name

Date Section

Exercise Results

Microbiology of Foods

A. Food Preservation with Salt and Garlic

I. Effectiveness of Salt

31

Observations and Conclusions:

Salt conc.:

Magnif.:

Stained Smears from Nutrient Broth Tubes

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Growth of Bacteria in Nutrient Broth

Food Alone Food plus Garlic

II. Effectiveness of Garlic

Observations and Conclusions:

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M I C R O B I O L O G Y O F F O O D S 31 289

� � bacteria/gram of hamburger(valid count) (dilution factor)

B. Standard Plate Count of Food Products

I. The Bacterial Flora of Hamburger Meat

Table 31.2. Plate Counts at Various Dilutions

Dilution 1:10 1:100 1:1000 1:10,000

Plate Count

Magnif.:

Stained Smears of Flora from Hamburger Meat

Table 31.3. Summary of Standard Plate Counts

Student Standard Plate Count Sample Tested

1.

2.

3.

4.

5.

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Table 31.5. Plate Counts at Various Dilutions

Dilution 1:10 1:100 1:1000 1:10,000

Plate count

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Table 31.4. Plate Counts at Various Dilutions

Dilution 1:10 1:100 1:1000 1:10,000

Plate count

� � bacteria/gram of food(valid count) (dilution factor)

� � bacteria/gram of food(valid count) (dilution factor)

II. The Bacterial Content of Mushrooms

Unmarinated Mushrooms

Marinated Mushrooms

Observations and Conclusions:

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M I C R O B I O L O G Y O F F O O D S 31 291

C. Fermentation of Sauerkraut

Observations and Conclusions:

Observations and Conclusions:

I. Basic Fermentation

D. Fermentation of Wine and Beer

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292 31 M I C R O B I O L O G Y O F F O O D S

Magnif.:

MicrobialFlora in Sauerkraut

MicrobialFlora in Wine/Beer

Table 31.6. Balloon Size at Various Time Periods

Experimental Conditions Time Circumference

(describe)

II. Quantitative Estimation of Fermentation

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