Module 4. Microbiological methods of milk testing

Lesson 19

COLIFORM COUNTS IN MILK

19.1 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 35°C 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.

19.2 Enumeration of Coliforms and E. coli

The coliforms is 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.

19.2.1 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

19.2.2 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 Durham’s 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.                           .
19.2.3 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 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.

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.

19.2.4 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.

19.2.5 Test for fecal coliforms (Eijkman’s 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 44.5 °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.

19.2.6 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.

Inoculate 0.1, 1.0 and 10ml of water samples respectively into three sets of five MacConkeys broth tubes (single strength for 0.1 and 1.0 ml and double strength for 10 ml samples) each. Incubate at 37°C for 24 – 48 h. Note down the number of positive tubes showing acid and gas production in each set and calculate MPN of coliforms in the standard MacConkeys MPN table and interpret the results.

19.2.7 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.

19.2.7.1 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 group to indole. Trytophan is an essential amino acid that can undergo oxidation by some bacteria that possess trytpphanase 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.

19.2.7.2 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.

19.2.7.3 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

19.2.7.4 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.

Table 19.1 Differential characteristics of Escherichia coli and Enterococcus aerogenes

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