COLIFORM COUNTS IN MILK

Introduction
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.
Coliforms have been used as an indicator for pathogens, but these are no longer
recommended for this purpose due to the fact that coliforms are present as normal
inhabitants of soil and water, not just specifically from feces. Hence, there is a co-
relationship between, the presence of pathogens from fecal source,
especially Salmonella and Shigella species, and the presence of coliforms.
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 is a proof that
contamination from excreta has occurred.

Enumeration of Coliforms and E. coli

The coliforms are not a valid taxonomic distinction, but is defined functionally i.e., by
the fermentation of lactose. Coliforms are Gram-negative, oxidase-negative, aerobic or
facultative anaerobic non-spore-forming rods, able to grow in the presence of bile salts
that ferment lactose to produce acid and gas within 48 h at 37 C. Different genera
of coliforms are Citrobacter, Enterobacter, Escherichia and Klebsiella.

Principle
The test is mainly based on the principle that the members of this group are capable of
producing acid and gas from lactose in the presence of bile salts and basic dyes.
Presence of typical coliforms colonies in Petri plates is indicator
of coliforms contamination.

Following protocol is used for determining the presence of coliforms:

 Presumptive test
 Confirmatory test
 Completed test
 Test for fecal coliforms
 Most probable number (MPN) for enumeration of low counts
 Differentiation of Escherichia coli and Enterobacter aerogenes

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 37C, 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. .

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. The plates are incubated
at 37C 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.
 b. 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.

Completed test
Broth tubes showing gas production in confirmatory test or typical or atypical colonies
from the Eosin Methylene Blue or Endo agar plates should be subjected to completed
test. A loop full from positive Brilliant Green Lactose Bile broth tubes should be
streaked on Eosin Methylene Blue or Endo agar plates and incubated at 37 C for 24 h.
The plates should be once again looked for typical or atypical coliform colonies. The
typical colonies should be once again transferred to MacConkeys broth tubes as well as
on nutrient agar slants. Acid and gas production in MacConkeys broth after 24 - 48 h.
at 37 C indicates the termination of completed test. Similarly, Gram’s stain preparation
from the nutrient agar slants showing Gram negative, non-spore forming cocco-
bacillary rods, demonstrates the definite presence of coliforms.

Test for fecal coliforms

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 44.5 C 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.

Differentiation of Escherichia coli and Enterococcus aerogenes (IMViC tests)

Escherichia coli, Enterococcus aerogenes, the two types of coliforms in milk can be
differentiated on the basis of biochemical tests, collectively known as IMViC test.

Indole test
It is based on the ability of microbes to degrade the amino acid tryptophan into indole.
Test is used to determine the tryptophan oxidizing ability of number of coliform groups
to indole. Trytophan is an essential amino acid that can undergo oxidation by some
bacteria that possess tryptophanase to indole. The presence of indole is detected
by Kovac’s reagent composed of p-dimethyl amino-benzaldehyde, butanol and
hydrochloric acid. Indole is extracted from the medium into the reagent layer by the
acidified butanol and forms a complex with p-dimethyl amino benzaldehyde to give
cherry red colour. Therefore, positive test shows development of cherry red colour
using Kovac's reagent.

Methyl red test

It determines the ability of microbes to ferment glucose with the production and
stabilization of acid end products. Glucose is oxidized by all intestinal organisms for
energy production. The end products vary depending on the specific enzymatic
pathways present in the bacteria. Escherichia coli and Enterobacter aerogenes both
produce organic acids during early incubation period. The low acid pH (4.0) is
stabilized and maintained by Escherichia coli during later incubation period
but Enterococcus aerogenes coverts acids to non-acid end products such as ethanol
and acetoin resulting in elevated pH (6.0). The pH indicator, methyl red detects the
acids and presents by the formation of red colour. In this test methyl red is used as pH
indicator to detect a large concentration of acid as end product. This indicator turns to
red in the pH range of 4.0 and yellow in the pH range of 6.0.

Voges-Proskaur test
The test is based on the ability of microorganism to produce acetyl
methyl carbinol from glucose on fermentation, acetylmethylcarbinol, the neutral or
non-acidic end product from glucose metabolism, releases a compound known
as diacetyl and this is detected in the presence of Barritt's reagent producing a deep rose
colour.
 Few microorganisms are capable of producing more acidic or neutral end products such
as acetyl methyl carbinol or 2-3 butylene glycol from the organic acids which results
from glucose metabolism. These compounds are detected by Barritt’s reagent which
consists of α-napthol and 40 per cent KOH. Detection of AMC requires that this end
product be oxidized to a diacetyl compound which occurs in the presence of α-
napthol and a guanidine group of peptone present in the medium. As a result pink colour
complex is formed

Citrate test
The test determines the ability of microorganism to use citrate as a carbon source.
Citrate inside the bacterial cell undergoes enzymatic degradation and finally
produces pyruvic acid and carbon dioxide. This carbon dioxide combines with sodium
to form sodium carbonate and converting medium of reaction into
alkaline. Bromothymol blue indicator previously incorporated into the medium turns
the medium from green into deep Prussian blue.
Some microorganisms are capable of utilizing citrate as a carbon source in the absence
of glucose or lactose. This depends on the presence of citrate permease that facilitates
its transport into the cell. Citrate is the first major intermediate in the Kreb’s cycle and
is produced by the condensation of active acetyl Co A with oxaloacetic acid. During its
conversion to pyruvic acid, the medium becomes alkaline and CO2 generated combines
with sodium and water to form Na2 CO3 whose presence is detected
by bromothymol blue indicator which changes green color into deep blue.

Differential characteristics of Escherichia coli and Enterococcus aerogenes

Test Escherichia coli Enterococcus aerogenes
Mac Conkeys broth Acid and gas production Acid and gas production
2% BGLB broth Gas production Gas production
EMB agar Dark colonies with green Pink colonies without any
metallic sheen metallic sheen
Indole production + -
Methyl red test + -
Voges- - +
Proskauer test
Citrate utilization - +
test