Microbiological Examination of Water (Drinking Water, Supply Water and Pond Water)

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In water there are vast number of microorganisms. It is important to perform microbiological examination of water before using it for various purposes.

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Most probable number or MPN is one of the methods used for estimation of number of viable microorganisms in a given water sample by replication of liquid broth growth done in ten fold dilutions. Mostly it is used in samples that contains various particulate material that can interfere with plat count enumeration methods.


Water from various sources is serially diluted and then inoculated in the lactose broth. If the water contains coliforms then they utilize lactose for the production of acid and gas. The acid production is identified by colour alteration of the medium and the gas is detected by gas bubbles in the durham tubes. The total count of coliform bacteria is estimated by counting the number of tubes giving positive reaction.

So, the basic objective of this experiment is to know how to perform microbiological examination of water from various sources like drinking water, supply water and pond water.


Medium:     Lactose broth or Mac Conkey Broth, EMB Agar plates

Glassware: Test tubes of various capacities (20 ml, 10 ml, 5 ml), Durham tube

Others:       Sterile pipettes


Microbiological examination of water is performed through three different tests

  1. Presumptive test (Screening test for determining the presence of coliform organisms)
  2. Confirmatory test
  3. Completed test

Presumptive test

Prepare medium either Mac conkey broth or lactose broth in a single and double strength concentration.1 For polluted water put the double strength medium in 10 tubes and single strength medium in 5 tubes and then add durham tubes in inverted position to each of these tubes.

For unpolluted or treated water transfer double strength medium in 5 tubes and 50 ml of single strength medium in 1 bottle and to each bottle add a durham tube in inverted position.

Make it sure that the inner vial is filled with liquid and do not have any air bubbles. Sterilize every bottle. For water that is not treated at first take 5 tubes containing double strength medium and 10 tubes of single strength medium for each of the water sample that is to be tested.

Now take a sterile pipette and add 10 ml of water to 5 tubes that contains 10 ml double strength medium. Then add 1 ml of water to tubes that contains 10ml double strength medium and 0.1 ml of water to other 5 tubes that contains 10 ml double strength medium.

Then keep the tubes in incubator at 370C for about 24 hours. If there is no positive reaction in any tube then again keep it in incubator for another 48 hours.2 Now compare the number of tubes that gives a positive reaction to a common standard chart and then record the total number of bacteria present.

Confirmed test

Other microorganisms in addition to coliform bacteria also produce acid as well as gas from lactose fermentation. Therefore, to confirm the presence of coliform bacteria, a confirmatory test is done. The tubes that gives positive reaction in presumptive test add one loopful of medium to 3ml of lactose broth, to an agar slant and 3ml tryptone water.

Then keep the tube containing the lactose broth at 37oC and watch out for gas formation after 24 hours.3 If there is no gas production then again incubate it for about 48 hours for checking for gas production. Incubate the agar slants at 37oC for 24 hours and gram stain the organisms taken from slant to examine it microscopically.

If there is formation of gas in the lactose broth and gram staining shows gram negative bacteria and non-spore forming bacilli, then it confirms the presence of coliform bacteria. If there is no gas formation and gram staining doesn’t show gram negative and non-spore forming bacteria then it is a negative test.

Completed test

Some of the positive test of confirmatory test may be also wrong, therefore it is better to do completed test. For this you have to streak the inoculum in an EMB or Endo agar plate. Then incubate this plate at 37oC for about 24 hours. Appearance of growth confirms the presence of coliform bacteria.4

Microbiological examination of water


Performing three tests will confirm and give information about the quality of water being tested.


  1. Fenwick A. Waterborne Diseases—Could they be Consigned to History? Science. 2006; 313:1077–1081.
  2. George I, Crop P, Servais P. Use of β-D-Galactosidase and β-D-Glucuronidase Activities for Quantitative Detection of Total and Faecal Coliforms in Wastewater. Can. J. Microbiol. 2001; 47:670–675.
  3. Grabow WOK. Waterborne Diseases: Update on Water Quality Assessment and Control. Water SA. 1996; 22:193–202.
  4. Seas C, Alarcon M, Aragon JC, Beneit S, Quiñonez M, Guerra H, Gotuzzo E. Surveillance of Bacterial Pathogens Associated with Acute Diarrhea in Lima, Peru. Int. J. Infect. Dis. 2000; 4:96–99.

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