Water and Milk Bacteriological Examination
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Direct answer
Water and milk bacteriological examination detects faecal contamination and hygiene failure by counting coliforms, with Escherichia coli as the key indicator of faecal pollution. Drinking water should show no coliforms in 100 millilitres, and tests use the most probable number method or membrane filtration. In milk, the methylene blue reduction test and phosphatase test assess bacterial load and pasteurisation.
What you must remember
- Indicator organism: Escherichia coli, because it is abundant in human and animal faeces and rarely multiplies in water, so its presence signals recent faecal contamination.
- Drinking water standard: the Bureau of Indian Standards and WHO require that E. coli and coliforms be absent from 100 millilitres of water.
- Most probable number: a statistical estimate from the pattern of positive tubes across serial dilutions, read from standard tables.
- Membrane filtration: water is filtered through a membrane, incubated on selective medium and counted directly as colonies.
- Presumptive test: acid and gas in lactose broth at 37 degrees Celsius within 48 hours suggests coliforms.
- Completed test: E. coli on eosin methylene blue agar gives a green metallic sheen, and its IMViC pattern is plus, plus, minus, minus.
- Methylene blue reduction test: the time taken for milk to decolourise reflects total bacterial load, so faster decolourisation means poorer quality.
- Phosphatase test: alkaline phosphatase is destroyed by adequate pasteurisation, so a positive test indicates under-pasteurisation or contamination after heating.
A staged sequence for the multiple tube coliform test
The most probable number test runs in stages, and each stage narrows the result. The steps below follow the usual sequence for drinking water.
- Prepare dilutions: make serial dilutions of the water sample and inoculate several tubes of lactose broth at each dilution.
- Incubate presumptively: incubate at 37 degrees Celsius for 24 to 48 hours, and record tubes showing acid and gas.
- Confirm coliforms: transfer positive tubes to brilliant green lactose bile broth at 37 degrees Celsius, where gas confirms coliforms.
- Read the MPN: convert the pattern of positive tubes at each dilution into a most probable number per 100 millilitres using standard tables.
- Complete the test: streak confirmed tubes onto eosin methylene blue agar, pick colonies with a green metallic sheen, and perform IMViC tests.
- Interpret against the standard: any E. coli or detectable coliform in 100 millilitres of drinking water makes it unsafe until it is treated.
Milk examination follows the same logic of screening first and confirming second, but it uses the methylene blue and phosphatase tests as its screening steps.
Where the exam tries to catch you
Water and milk questions often hinge on knowing which test measures what.
- Trap: calling water safe because the coliform count is low Correct: any E. coli in 100 millilitres signals faecal contamination, so the water is unsafe even at low counts.
- Trap: treating a slow methylene blue reduction time as proof that milk was pasteurised Correct: the test measures bacterial load, not heat treatment, and the phosphatase test is the check for adequate pasteurisation.
- Trap: reporting the most probable number as a direct colony count Correct: the MPN is a statistical estimate from the pattern of positive tubes, not a count of visible colonies.
Frequently asked questions
Why is Escherichia coli the indicator of faecal contamination?
Escherichia coli is used as the indicator because it lives in large numbers in the human and animal gut and is rarely found in water without faecal input. Its presence therefore signals recent faecal contamination and a possible risk from enteric pathogens such as Salmonella and Shigella.
What is the most probable number method?
The most probable number method estimates bacterial density from the pattern of positive tubes across serial dilutions, using statistical tables. It is used when colonies cannot be counted directly, as in the multiple tube coliform test for drinking water.
What does the methylene blue reduction test measure in milk?
The methylene blue reduction test measures how quickly bacteria in milk decolourise a blue dye, which reflects the total bacterial load. Faster decolourisation means more bacteria and poorer milk quality, so the test is a rapid hygiene screen rather than an organism test.
What does a positive phosphatase test indicate in milk?
A positive phosphatase test indicates that milk was inadequately pasteurised or contaminated after heating, because proper pasteurisation destroys alkaline phosphatase. A negative result is taken as evidence that the milk was adequately pasteurised.
What are the IMViC reactions of Escherichia coli?
Escherichia coli gives the IMViC pattern plus, plus, minus, minus, meaning positive indole, positive methyl red, negative Voges-Proskauer and negative citrate. Klebsiella and Enterobacter give minus, minus, plus, plus, which is how the pattern separates them from E. coli.