Bacteriologic Analysis Of Milk

Unit 15

Unit 15. Bacteriologic Analysis of Milk Reference: Morello, Mizer, Wilson and Granato. Microbiology in Patient Care, 6th edition, 1998. Chapters 8,9,10,16 Modified by Patricia G. Wilber, Karen Bentz and Heather Fitzgerald, 2015 Introduction: Milk is normally sterile as secreted by the lactating glands of healthy mammals. 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. These species are 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. Milk-borne 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, and yersiniosis. Streptococcal infections of animals and Q fever are also transmissible through milk. Human diseases that may become milk-borne 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 microorganism (with the possible exception of hepatitis A virus). The two methods commonly used for pasteurization of milk are (1) heating at 62.9oC (145oF) for 30 minutes, or (2) heating to 71.6oC (161oF) 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 this laboratory session, you will learn how a total colony count of milk is determined. You get to bring in your own milk to test!! Your instructor will help organize who is bringing what.

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DAY 1: Inoculation. In this lab you will culture milk to quantitatively assess bacterial load in the milk. You will work in groups of 3-4 for this exercise. Materials:  1 – 5 mL milk sample (provided by student(s))  1 tube of milk sample as a back-up provided by lab  4 Sterile water blanks (9mL water per tube)  5 Sterile 1 mL or 5 mL pipettes  Pipette pump  Media (for groups of 3-4 students) o 4 Sterile nutrient agar tubes (9 mL per tube) held at 45oC until use (so they are liquid). o 4 Sterile petri dishes

Procedure 1. Choose a sample of milk, from which you will make serial dilutions as follows: a. Using a sterile pipette, transfer 1 mL of the milk sample into a water blank (9 mL of water). Label the tube “1:10” and discard the pipette. Mix the diluted sample before proceeding to the next step. b. Use a second sterile pipette to transfer 1 mL of the 1:10 milk dilution to another water blank. Label the new dilution “1:100” and discard the pipette. Mix the diluted sample before proceeding to the next step. c. Use a third 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. Mix the diluted sample before proceeding to the next step. d. With a fourth pipette, transfer 1 mL of the 1:1,000 milk dilution to another water blank. Label the new dilution “1:10,000” and discard the pipette. Mix the diluted sample. 2. Take four sterile petri dishes. Mark the bottom of each, respectively, 1:10, 1:100, 1:1,000, 1:10,000. (also put the date, your names, milk) 3. With a fifth sterile pipette, measure 1 mL of the highest milk dilution (1:10,000) and transfer it into the bottom of the petri dish so marked. 4. Using the same pipette, repeat step 3 for each diluted milk sample, in descending order of dilution (1:1,000, 1:100, 1:10). 5. Remove the melted agar tubes from the 45oC incubator. 6. Pour each tube of 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. 7. When each poured plate is completely solidified, invert it. 8. Incubate all plates at 35oC for 24 to 48 hours. 9. Use the Internet in class (if available) or at home to find the bacteriologic milk standards for Grade A milk. If available in class, fill in the chart below in Day 2. If done at home, print the standards and bring to the next class.

DAY 2: Results and Interpretation 1. Count the number of colonies on each plate of your diluted milk samples. For each plate, calculate the number of organisms per milliliter of milk. Average the four figures and report a final plate count.

a. 1:10 plate: #colonies x 1(mL) x 10 = ______organisms/mL b. 1:100 plate: # colonies x 1(mL) x 100 = ______organisms/mL c. 1:1,000 plate: # colonies x 1 (mL) x 1,000 = ______organisms/mL d. 1:10,000 plate: #colonies x 1 (mL) x 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. Milk Source Average Plate Count Bacteriologic Standard (organisms/mL) for Grade A milk (you have to look this up)

3. If your results are either higher or lower than required bacteriologic standards for Grade A milk give one possible explanation for each deviation.

Insert Photos of Results Here:

The authors of this lab unit would like to thank Andrea Peterson and Deyanna Decatur for testing new media and organisms, our associate dean Linda Martin for many kinds of aid, Mike Jillson and Alex Silage for IT support, and our dean John Cornish.

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