Most probable number (MPN) method for counting coliform

• MPN method utilizes 3 set method or 4 set method for counting coliform in water. However three set method is commonly used.
• MPN method for counting coliform in water is completed in three steps:

step I: Presumptive test:

• At first three set of 5 test tubes are taken.
• 10 ml of double strengthen liquid media (MacConkey broth) is placed in each test tubes of 1^st set
• Similarly, 10 ml of single strengthen liquid media is placed in each test tubes of 2^nd set and 3^rd
• ** double strengthen broth refers to broth made up using twice the normal amount of broth powder.
• Lactose broth or lauryl sulphate broth or tryptose lauryl broth is used as liquid media for the test.
• Then, Durham’s tube is inserted in inverted position in each test tubes of all sets.
• All the test tubes are then cotton plugged and sterilized using autoclave for 15 minutes at 15 lb/inc pressure at 121 °
• After cooling water sample is added in each test tubes as follows;
  • Add 10 ml water sample in each test tubes of 1^st set
  • Add 1 ml water sample in each test tubes of 2^nd set
  • Add 0.1ml water sample in each test tubes of 3^rd
• Then incubate all test tubes at 35.5 °C for 24 hours. After incubation gas production in Durham’s tubes is observed.
• Tubes in which gas production is 10% or more is recorded as positive tube and tubes in which gas production is less than 10% is recorded as doubtful.
• Doubtful test tubes are further incubated for 24 hours and again gas production is noted. If gas production is still less than 10%, then tube is recorded as negative and are discarded
• ** doubtful result is given by other gas producing lactose fermenting bacteria other than coliforms such as Lactobacillus, Streptococci, Bacillus, Clostridium Clostridium produces more than 10% gas but only after 48 hours of incubation.
• All the positive test tubes are taken for confirmatory test.

step II: Confirmatory test for MPN method:

• Positive tubes obtained from presumptive test are now confirmed for coliform.
• For confirmation of coliform, brilliant green lactose bile (BGLB) broth is used as culture media, because BGLB broth inhibits growth of gram positive bacteria such as lactobacillus, Streptococci, Bacillus and Clostridium
• Coliforms can grow in this BGLB medium
• For confirmation, one loopful of sample from each positive tubes obtained from presumptive test is inoculated in respective tubes containing Brilliant green lactose bile broth and incubated for 24 hours at 35.5 °
• Gas production 10% or more are recorded as positive while less than 10% is recorded as doubtful. Doubtful tubes are again incubated and the result is recorded.
• All the positive test tubes are now confirmed for presence of coliforms.
• Finally the number of bacteria present in water sample is determined from previous MPN chart. Alternatively number of coliforms can also be calculated by the formula;
  • Coliforms/100ml = numbers of positive tubes
• _/ volume of samples in negative tubes * volume of samples in whole experiments

step III: Completed test for MPN method:

• It is a final test in which a loopful of sample from positive confirmatory tubes is streaked on Eosin methylene blue agar or M-endo agar and incubated for 24 hours.
• Three types of colonies are obtained in culture media;
  • Typical colony: they are pink colored with greenish metallic appearance or nucleated colony. Coliforms gives typical colony
  • Atypical colony: they are pink and non-nucleated colony. Coliforms as well as other lactose fermenting organisms gives atypical colony
  • Non-typical colony: they are non-pink colony given by non-coliforms.

MPN method for faecal coliforms; Eijkman test:

• Eijkman test or confirmed E.coli test or differential coliform test is MPN test used to detect faecal coliforms in water,
• This test is similar to MPN test for total coliforms but incubation temperature is slightly higher.
• Faecal coliforms are thermotolerant and can grow at temperature of 44.5° Therefore if incubation temperature is maintained at this point, only faecal coliforms can grow, but not other coliforms.

step I: Presumptive test:

• Media: lactose broth or Macconkey broth
• Incubation temperature: 35.5°C

step II: Confirmatory test:

• Media used: E. coli media (EC media)
• Incubation temperature: 44.5 °C

step III: Completed test:

• Media used: M-endo agar or Eosin methylene blue agar
• Incubation temperature: 44.5 °C