Bacteriologic Analysis of Milk - CNM



Unit 15: Bacteriologic Analysis of MilkReference: Morello, Mizer, Wilson and Granato. Microbiology in Patient Care, 6th edition, 1998. Chapters 8,9,10,16Modified by Patricia Wilber, Karen Bentz, Heather Fitzgerald, and Andrea Peterson, 2018Creative Commons Attribution-NonCommercial 4.0 International License.Introduction:Milk as secreted by the lactating glands of healthy mammals is normally sterile. From that point on, however, it is subject 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 have no pathogenic importance. Milk handlers and their equipment may also introduce these and other microorganisms that are equally harmless, except that they can spoil the milk. Milk is an excellent growth medium for pathogenic bacteria 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. Human diseases that may become milk-borne via infected milk handlers include streptococcal infections, shigellosis, and salmonellosis. 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 the 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 lab you will culture milk to quantitatively assess bacterial load in the milk. You get to bring in your own milk (or another product suggested by your instructor) to test!! Your instructor will help organize who is bringing what. DAY 1: InoculationYou will work in groups of 3-4 for this exercise.Materials:1 – 5 mL sample (provided by student(s))1 tube of milk sample as a back-up provided by lab4 sterile water blanks (9 mL water per tube)5 sterile 1 mL or 5 mL pipettesPipette pumpMedia (for groups of 3-4 students)4 Sterile nutrient agar tubes (9 mL per tube) held at 45oC until use (so they are liquid).4 Sterile petri dishesProcedure:Choose a sample of milk. Make serial dilutions as follows (and shown):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.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. 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. 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.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)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.Using the same pipette, repeat step 3 for each diluted milk sample, in descending order of dilution (1:1,000, 1:100, 1:10).Remove the melted agar tubes from the 45oC incubator.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. When each poured plate is completely solidified, invert it.Incubate all plates at 35oC for 24 to 48 hours.Use the Internet in class (if available) or at home to find the bacteriologic milk standards for the type of milk your group tested. Fill in the chart below in Day 2. If done at home, print the standards and bring to the next class.DAY 2: Results, Interpretation and Post Lab Name____________Count or estimate the number of colonies on each plate of your diluted milk samples. Record the values in Table 15.1The numbers might be ridiculously high. If so, estimate in the following way:Divide your plate in sections. This plate is divided into 16 sections.Count only the number of colonies in ONE of the sections. Multiply that number by the number of sections. If there were 300 colonies in one of the 16 sections, the total for this plate would be 300 x 16 = 4,800For each plate, calculate the number of organisms per milliliter of milk using the formula below.Then calculate the average.Table 15.1 The number of colonies/plate and calculations for the number of organisms/mL of milkPlate# of coloniesFormulaResult: # of organisms/mL milk1:10 plate:x1 (mL) x 10 =1:100 plate:x1 (mL) x 100=1:1,000x1 (mL) x 1,000=1:10,000x1 (mL) x 10,000=Average =From your own results and those of your neighbors, report final results for the samples tested.Sample SourceAverage Plate Count (organisms/mL of milk)Bacteriologic Standard for the milk type you tested(you have to look this up)If the results are either higher or lower than required bacteriologic standards for the milk type you tested, give one possible explanation for one specific deviation.Research one of the diseases mentioned in the introduction. Write a brief (1 paragraph) explanation of the disease.Explain why pasteurization of milk has reduced the incidence of this disease. ................
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