The document discusses various types of bacteria found in milk, milkborne diseases caused by bacteria in infected cows or external sources, and several bacteriological examination methods for milk including standard plate count, coliform count tests, alkaline phosphatase testing to check for adequate pasteurization, and reduction tests to classify milk quality.

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. perfringens, Cl.
butyricum.-> 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.

8. 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.

9. 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 370C, 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.

11. Bacteriological Examination of Milk
Confirmatory test
 The positive presumptive coliform tubes showing acid and gas
production is selected for to the confirmatory test.
a. A loop full of inoculums from the positive presumptive tubes is
streaked on the surface of Eosin Methylene Blue or Endo agar
plates.
b. 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.
c. 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.
d. The formation of gas in the tubes within the incubation period is
considered as confirmatory test for coliforms.
•

12. 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.

13. 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.

15. 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

17. 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.

18. 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.