The nature of milk which is secreted inside the secretory glands of the cow is ordinarily

sterile. Numerous types of microorganisms grow in raw as well as pasteurized milk. These
vigorously multiplying microorganisms retard the oxidation reduction potential of the milk
medium because of the exhaustion of oxygen by the microbial population. These
microorganisms, predominantly coliforms, thrive in this milk and spoil it to extent of change
in colour, odour, and taste of the milk. It becomes unconsumable by humans, and if
consumed, it can lead to serious consequences such as food poisoning. Usually, microbes like
E. coli, Streptococcus pyogenes, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus
spp., Paenibacillus spp., Enterobacter spp., and others, are the major cause of milk
contamination. The contaminated milk transfers diseases from bovines to humans. The main
reason for this contamination is the inadequate handling of milk. The good quality of milk
can be maintained initially by following good milk hygiene practices. Some of these practices
could include maintaining cleanliness and good health of cows, avoiding dust and dirt in the
milking area, making sure that the farmer milking the cow does not have any contagious
diseases like typhoid or diarrhoea, prevention of mixing colostrum with fore milk, keeping
hands clean with water and soap before milking, cleaning the udder of the cow with warm
water, and using clean containers and machines for milking (De Silva et al., 2016).

It has been seen that the microbiota of the raw milk change from significantly Gram positive
to Gram negative owing to the circumstances in which the milk is stored and transported in
refrigerated containers. The major organism responsible for spoiling the milk at a temperature
of 4oC is Pseudomonas because it has got a very short multiplication time. The other
organisms such as Enterobactericeae spoil the milk at 10oC. The milk that is commercially
available is routinely pasteurized so as to get rid of pathogenic bacteria which are resistant to
heat treatment. But still, some disease-causing bacteria are able to cope up with
pasteurization and survive. Hence, it becomes essential to study its bacteriological grade.
There are many factors that determine the expiry date of pasteurized milk. Some of them
include, quality and storage time of raw milk before processing, type of heat treatment
applied, number of thermophiles and hyperthermophiles, packaging technique used, storage
of milk after pasteurization and impact of light (Sarkar, 2015). Identification of initial
microbial load has a great significance, and various methods are applied at the levels of
laboratory and field.

The methylene blue reduction test, where methylene blue reductase is formed has become a
major mechanism for the determination of the bacteriological quality of the milk. The
fundamental of methylene blue reduction test is based on the fact that the colour taken up by
the milk on addition of a dye such as methylene blue will vanish either fast or slowly. This is
dependent on the quality of the milk sample to be studied. Methylene blue acts as a redox
indicator that loses its colour in an environment devoid of oxygen and is considered to be
reduced. The high rate of bacterial metabolism produces reducing substances that result in the
depletion of oxygen in the sample milk (Bongard et al., 1995; Merker et al., 1997). The dye
reduction time provides an evidence of microbial activity and hence the presence of
microbiota in the sample milk.
The standard plate count (SPC) is the method used for the estimation of microbiological
populations in milk samples (Barbano et al., 2006). The method is carried out through a serial
dilution technique for the straightforward quantification of the microbes. The exact dilutions
of the milk sample are prepared by mixing it with a sterile nutrient medium which can be
beneficial for the growth of the microorganisms, provided it is incubated at the desired
temperature. Every colony of bacteria that grows on the plate is thought to have multiplied
from one bacterium or clump of bacteria in the inoculating material. The total number of
colonies on the plates are counted and multiplied by the dilution factor. This number signifies
the number of potent micro-organisms present in the sample being tested. The formula used
to calculate SPC is:
N = C/[(l x n1) + (0.1 x n2)]d.

Where,
N = number of colonies per milliliter or gram of product,
C = sum of all colonies on all plates counted,
n1= number of plates in lower dilution counted,
n2 = number of plates in next higher dilution counted,
d = dilution from which the first counts were obtained.

Some aerobic bacteria that grow in ambient temperature and some facultative anaerobic
bacteria are able to proliferate easily on a non-selective microbiological medium. Standard
plate count and methylene blue reductase test are the reliable methods for measuring these
microbes. Positive standard plate count and methylene blue reductase test results depict
contamination of milk or failure in proper refrigeration due to high growth of
microorganisms. They produce enzymes such as lipase and protease as a result of microbial
metabolism that hydrolyse lipids and proteins. As a result, many by products are formed that
destroy flavour and odour of milk. Some of these enzymes and other products are heat
resistant, that means they can degrade the milk quality even after pasteurization. Nowadays,
people prefer consuming pasteurized and homogenized milk from supermarkets because it is
thought to be free from pathogenic bacteria.