Microbiology of milk

A very concise description of Milkborne diseases and bacteriological analysis of milk for UG Medical, Nursing and Para Medical students.

1. Microbiology of Milk Dr. Suprakash Das M.D (Microbiology)

2. Types of Bacteria in Milk Acid forming bacteria- S. lactis , E. faecalis , Lactobacilli spp . These ferment lactose in the milk –lactic acid- smooth gelatenous curd. Alkali forming bacteria- Alcaligenes spp , ASBs, Achromobacter spp. -> render milk alkaline. Gas forming bacteria- Coliforms , Cl. p erfringens , Cl. b utyricum .-> Acid & Gas is formed-> Smooth gelateous card with gas. Proteolytic bacteria- ASBs such as B. subtilis , B. cereus, P. vulgaris , Staphylococci spp , Micrococci spp. Inert bacteria- Achromobacter spp and Most human pathogenic bacteria in milk. Human breast milk- S. epidermidis , S. mitis , S. aureus .

3. Milkborne diseases by Infected Cow Organism Disease Bacteria- Diseases in Human M. bovis Tuberculosis B. anthracis Anthrax B. abortus Undulent fever Streptococcus spp. Sore throat/ Scarlet fever S. aureus Diarrhrea / Vomiting Salmonella spp. Enteric fever/ Food poisoning Coxiella burnetii Q- Fever Viruses Cow pox Lesion on skin on milker Foot mouth disease virus Fever and vesicles

4. Milkborne diseases- External source Pathogenic Bacteria Human Disease Shigella spp. Dysentrry V. cholerae Cholera C. diphtheriae Diphtheria S. pyogens Scarlet fever Diarrheagenic E. coli Diarrhea Salmonella spp Enteric fever S. aureus Food poisoning Pathogenic viruses Polioviruses Paralytic poliomyelitis Hep . A viruses Hepatitis

5. Bacteriological Examination of Milk Viable count (Standard plate count)-  Introduction The standard plate count (SPC) is suitable for estimating bacterial populations in most types of dairy products, and it is a reference method specified in the Grade A Pasteurized Milk Ordinance to be used to examine raw and pasteurized milk. This procedure is also recommended for application in detecting sources of contamination by testing line-samples taken at successive stages in the processing . Principle The test employs  a serial  dilution technique for easy quantification of the micro-organisms. The appropriate dilutions of the milk sample are mixed with a sterile nutrient medium that can support the growth of the micro-organisms, when incubated at a suitable temperature. Each bacterial colony that develops on the plate is presumed to have grown from one bacterium or clump of bacteria in the inoculums. The total number of colonies counted on the plates multiplied by the dilution factor to represent the number of viable micro-organisms present in the sample tested.

6. Bacteriological Examination of Milk Test for Coliform Bacilli ( Coliform counts)-   Coliforms , that are often used to monitor the quality of milk, are not a single species of microorganism. These are a group of Gram negative rod-shaped bacteria that have similar biochemical characteristics being able to ferment lactose with the production of acid and gas within 48 hr at 35C and grow with or without oxygen. These are usually present in small number in raw milk.  Coliforms  count is simple and easy to conduct; hence, it can be used as a hygienic indicator to reflect the general microbiological quality in routine test. As   coliforms  can be easily killed by heat, these bacteria can also be used as an indicator of heat treatment failure as well as post heat treatment contamination. Absence of  coliforms  in 1:100 dilutions in raw milk and in 1:10 dilution of pasteurized milk is accepted as a satisfactory quality.   The presence of  E. coli  - contamination from excreta has occurred.

7. Bacteriological Examination of Milk Presumptive test Commonly used for the detection of  coliforms  in milk and helps in evaluation of its hygienic quality. When a sample of milk is inoculated into  MacConkeys  broth or Bile salt lactose peptone broth, and incubated at 37 C , the production of acid and gas in Durhams tube within 24-48 h. is regarded as presumptive evidence of  coliforms . The test is called as presumptive  coliform  test because, in addition to the presence of fecal  coliforms , the fecal streptococci and  Clostridium  perfringens  are likely to exist in the gut of warm-blooded animals, and their presence in milk will also indicate the source of fecal contamination. Hence , the test used to find out all the microorganisms of fecal origin is called presumptive test .

8. Bacteriological Examination of Milk Confirmatory test The positive presumptive  coliform  tubes showing acid and gas production is selected for to the confirmatory test. A loop full of inoculums from the positive presumptive tubes is streaked on the surface of Eosin  Methylene  Blue or Endo agar plates. The plates are incubated at 37 C   for 24-48 h. The typical colonies of  coliforms  developed on these selected media will appear pink with or without dark center and green metallic sheen on Eosin  Methylene  Blue and deep red on Endo agar. A loop full of inoculums from positive presumptive tubes should be transferred to Brilliant Green Lactose Bile broth tubes that are incubated at 37 C for 48 h. The formation of gas in the tubes within the incubation period is considered as confirmatory test for  coliforms .   

9. Bacteriological Examination of Milk Test for fecal  coliforms  ( Eijkmans test) Incubation at elevated temperature can lead to differentiation of fecal  coliforms  from non-fecal counter parts. In this test, inoculums from positive presumptive tubes are transferred to Brilliant Green Lactose Bile broth or  MacConkeys  broth tubes. The tubes should be incubated at 44.5C   for 24 h. Gas production in the inoculated tubes within 24 h. is considered a positive reaction indicating fecal origin of  coliforms .  A negative reaction suggests non-fecal origin of the  coliforms . Most probable number (MPN) The presumptive test also enables to obtain an idea of number of microorganisms present in milk by MPN. The tubes of lactose broth or  MacConkeys  broth inoculated with samples of milk are being tested and a count of the number of tubes, showing acid and gas production, are counted and that figure is compared with MPN table .

10. Bacteriological Examination of Milk Methylene Blue Reduction Test The methylene blue reduction test is based on the fact that the color imparted to milk by the addition of a dye such as methylene blue will disappear more or less quickly. The removal of the oxygen from milk and the formation of reducing substances during bacterial metabolism causes the color to disappear. The agencies responsible for the oxygen consumption are the bacteria. It is generally assumed that the greater the number of bacteria in milk, the quicker will the oxygen be consumed, and in turn the sooner will the color disappear. Classification .–The suggested classification is listed. Class 1 . Excellent, not decolorized in 8 hours. Class 2 . Good, decolorized in less than 8 hours but not less than 6 hours. Class 3 . Fair, decolorized in less than 6 hours but not less than 2 hours. Class 4 . Poor, decolorized in less than 2 hours.

11. Bacteriological Examination of Milk The Resazurin Test The resazurin test is conducted similar to the methylene blue reduction test with the judgement of quality based either on the color produced after a stated period of incubation or on the time required to reduce the dye to a given end-point. The two most commonly used TESTS are the "one-hour test" and the "triple-reading test" taken after one, two, and three hours of incubation. Tubes are examined and classified at the end of an hour in the "one-hour test" or at the end of three successive hourly intervals in the " triple reading test." The following relationships of color and quality are generally accepted: Color of Sample : Quality of Milk Blue (no color change): Excellent Blue to deep mauve: Good Deep mauve to deep pink: Fair Deep pink to whitish pink: Poor White: Bad

12. Bacteriological Examination of Milk Alkaline Phosphatase Test for Pasteurized Milk Alkaline Phosphatase is an enzyme which is naturally present in milk, but is destroyed at a temperature just near to the pasteurization temperature. Alkaline Phosphatase test is used to indicate whether milk has been adequately pasteurised or whether it has been contaminated with raw milk after pasteurisation . This test is based on the principle that the alkaline phosphatase enzyme in raw milk liberates phenol from a disodium para -nitro phenyl phosphate and forms a yellow coloured complex at alkaline pH . The intensity of yellow colour produced is proportional to the activity of the enzyme.

13. Bacteriological Examination of Milk The colour intensity is measured by direct comparison with standard colour discs in a Lovibond comparator. The test is not applicable to sour milk and milk preserved with chemical preservatives. Results of this procedure are expressed as micrograms of phenol per mL of milk. A value of greater than 1 microgram is indicative of improper pasteurization.

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