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Find Coliform Bacteria By Multiple Tube Fermentation Technique



Many bacteria are found in water. most of them are totally harmless (non pathogenic) and few are harmful (pathogenic), which causes diseases e.g. typhoid, fever, parathyphoid, dysentery, and cholera etc. The ground water at great depths is free from these bacteria. The sanitary engineer is not concerning all of them. The Coliform group is one of the most important types and includes aero genes, Acrobatic Cloace, eschroica coli. Therefore Coliform may be define in part  as including all of the aerobic and facultative green non-spore bacilli, which formate lagtode with gas formation within 48 hours at 3.5 C. Coliform themselves are harmless bacteria. But they are not indication of bacteria  pollution of water , but also because their absence or presence and their number  can be determine by routine laboratory test.

The number of Coliform May be found by following test:

  • Pour plate total amount method
  • Membrane filter method
  • Multiple tube fermentation method

The last method based on the Coliform ferment lactose with gas formation. Appropriate quantity of water to be tested is placed in sterile tube containing lactose. The Tubes are incubated for 24 hours and then examined in the presence or absence of gas is noted and recorded. If no gas is formed within 24 hours then wait for 48 hours. If the gas is formed then Coliform is confirmed. To find the number of Coliform from this method the result from various size of portion if the sample are noted the most probable number (MPN) of the Coliform in the water is obtained by applying the laws of the statics to the result of the test. For this purpose the most provable number charts are available.

WHO Guideline Value for Bacteria Coliform

According to WHO the water is divided into the following classes depending upon the amount of Coliform bacteria present in it.

Class Status Coliform per 100ml
01 Excellent 0
02 Satisfactory 1-3
03 Suspicious 4-10


Fermentation tube, Durham,s tube, Cotton, Beakers, autoclave (steam sterilizer) and pippete filter.


Water samples, lactose, and bullion solution.


This test is carried in three stages: We will confine our selves to the first stage (Presumptive test) which is performed in the following steps.

  1. Take 15 test tubes and make 3 sorts of them each having 5 test tubes

  2. Fill each of them with 10ml of lactose broth solution

  3. Insert Durham,s tubes upside down in all test tubes and they are gently shaken to remove air.

  4. Clog all the tes tubes with cotton

  5. Sterelize all the test tubes at 121C"in autoclave for minute.

  6. Take out the tube after sterilization and the tube is cooled down

  7. 1ml and 0.1 ml of sample is added respectively to 2nd and 3rd set of tubes.

  8. Incubate all these test tubes at 350" for 24 hours in an incubator.

  9. After 24 hours each test tube it is said to be positive presumptive test other wise negative.


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