Bacteriological Analysis of Milk

Milk is normally sterile as secreted by the lactating glands of healthy animals. From that point on, however, it is subjected to contamination from two major sources:

1. the normal flora of the mammary ducts, and
2. flora of the external environment, including the hands of milkers, milking machinery, utensils, and the animal’s coat (human skin in the case of the nursing mother).

The bacterial genera most frequently found in mammary ducts are Streptococcus, Lactobacillus, and Micrococcus. Species of these are most frequently found in milk and have no pathogenic importance. Milk handlers and their equipment may also introduce these and other microorganisms that are equally harmless, except that their activities in milk may spoil its qualities.

Milk is an excellent medium for pathogenic bacteria also and may be a reservoir of infectious disease. Milkborne infections may originate with diseased animals, or with infected human handlers who contaminate milk directly or indirectly. Important animal diseases transmitted to human beings through milk are tuberculosis, brucellosis, listeriosis, and yersiniosis. Streptococcal infections of animals and Q fever are also transmissible through milk.

Human diseases that may become milkborne via infected milk handlers include streptococcal infections, shigellosis, and salmonellosis. (These diseases, as well as staphylococcal food poisoning, can also be transmitted through other foods handled by infected people.)

Pasteurization is a means of processing raw milk before it is distributed to assure that it is relatively free of bacteria and safe for human consumption. It is a heat process gentle enough to preserve the physical and nutrient properties of milk, but sufficient to destroy pathogenic microorganisms (with the possible exception of hepatitis A virus). The two methods most commonly used for pasteurization of milk are

1. heating at 62.9°C (145°F) for 30 minutes, or
2. heating to 71.6°C (161°F) for a minimum of 15 seconds.

Bacteriologic standards for milk include:

1. total colony counts,
2. coliform tests,
3. cultures for pathogens, and
4. testing for the heat-sensitive enzyme phosphatase, normally present in raw milk (this enzyme is destroyed by adequate pasteurization and should not be detectable in properly processed milk).

In the laboratory session, you will learn how a total colony count of milk is determined.

Purpose	To illustrate a method for quantitative culture of milk
Materials	Tubes of pasteurized milk diluted 1:10 Tubes of raw milk diluted 1:10 Sterile water blanks (9 ml water per tube) Sterile tubed agar (9 ml per tube) Sterile 5.0-ml pipettes Pipette bulb or other aspiration device Sterile petri dishes Thermometer

Procedures

1. Review Experiment 15.2 for serial dilution technique.
2. Set up a boiling water bath. Place four tubes of nutrient agar in it.
3. You will be assigned a sample of either pasteurized or raw milk diluted 1:10, from which you will make further serial dilutions as follows:
1. Using a sterile 5-ml pipette, transfer 1 ml of the 1:10 milk sample into a water blank (9 ml water). Label the tube “1:100” and discard the pipette.
2. Use a second sterile pipette to transfer 1 ml of the 1:100 milk dilution to another water blank. Label the new dilution “1:1,000” and discard the pipette.
3. With a third pipette, transfer 1 ml of the 1:1,000 dilution to a water blank, label it “1:10,000,” and discard the pipette.
4. Take four sterile petri dishes. Mark the bottom of each, respectively, 1:10, 1:100, 1:1,000, 1:10,000.
4. With a sterile 5-ml pipette, measure 1 ml of the highest milk dilution (1:10,000) and deliver it into the bottom of the petri dish so marked.
5. Using the same pipette, repeat step 6 for each diluted milk sample, in descending order of dilution (1:1,000, 1:100, 1:10).
6. If the tubes of agar are melted, remove them from the water bath and place them in a beaker of lukewarm water (about 45°C). Using a thermometer in this cooling water bath, and testing both water and tubes with your hands, make certain the agar has cooled to 45°C.
7. Pour each tube of cooled agar into one of the petri dishes containing a milk dilution. Cover the plate and rotate it gently to assure distribution of the milk in the melted agar.
8. When each poured plate is completely solidified, invert it.
9. Incubate all plates at 35°C for 24 to 48 hours.

Results

1. Count the number of colonies on each plate of your diluted milk sample. For each plate, calculate the number of organisms per milliliter of milk. Average the four figures and report a final plate count. If the number of colonies on a plate is too numerous to count (more than 300 per plate), select for counting only those plates that have between 30 and 300 colonies on them. Use these plate counts to perform your calculation.
1. 1:10 plate: # colonies × 1 (ml) × 10 = _____ organisms/ml
2. 1:100 plate: # colonies × 1 (ml) × 100 = _____ organisms/ml
3. 1:1,000 plate: # colonies × 1 (ml) × 1,000 = _____ organisms/ml
4. 1:10,000 plate: # colonies × 1 (ml) × 10,000 = _____ organisms/ml

Final plate count =
# organisms/ml, 1:10 plate _____ +
# organisms/ml, 1:100 plate _____ +
# organisms/ml, 1:1,000 plate _____ +
# organisms/ml, 1:10,000 plate _____ +

Total ____________ ÷ 4 =
average plate count ____________ organisms/ml


2. From your own results and those of your neighbors, report final results for tested milk samples.
   Pasteurized milk, total plate count ______ organisms/ml
   Raw milk, total plate count ______ organisms/ml


3. State your interpretation of these results in terms of required bacteriologic standards for grade A milk.