Burman (1) substituted bile salts with teepol in Membrane Enriched Teepol Broth, the membrane filtration test medium used to detect coliform organisms in water. Membrane or M-Lauryl Sulphate Broth recommended (2, 3) is similar to M-Enriched Teepol Broth except teepol has been replaced by sodium lauryl sulphate. Sodium lauryl sulphate was found to be an adequate reproducible substitute. Recently ISO Committee (4) has recommended a slight modification (M1023I) of M-Lauryl Sulphate Broth for enumeration of coliforms in water. E. coli and coliform count should be made on separate samples of water. The volumes should be chosen so as the number of colonies on the membrane lies between 10 and 100. The water samples are filtered through sterile membrane filter and then placed face upward on an absorbent pad saturated with M-Lauryl Sulphate Broth. The membranes should be incubated in a container which does not allow evaporation to occur. Water-tight metal containers placed in an accurate water bath are required for incubation of membranes at 44°C. Burman (5) recommended the following incubation temperatures and durations.
Unchlorinated waters:
Coliform organisms: 4 hours at 30°C followed by 14 hours at 35°C
Escherichia coli: 4 hours at 30°C followed by 14 hours at 44°C
Non-chlorinated organisms benefit from 4 hours incubation at 30°C but chlorinated organisms require 6 hours incubation at 25°C. After incubation, yellow colonies are formed which should be confirmed further. No colonies should be assumed on appearance of as nil count. On appearance of small colonies of an intermediate colour, the membrane should be returned for incubation for full period. E. coli forms yellow coloured colonies on membranes when incubated at 44°C. Subculture to Lactose Broth (M1003) and Tryptone water (M463I) to confirm gas and indole production respectively after 24 hours incubation at 44°C. Coliform organisms which give yellow colonies on membranes incubated at 35-37°C should be subcultured in Lactose Broth (M1003). This can be incubated at 37°C for 48 hours to check gas production.
Storage and Shelf-life:
Store below 30°C in tightly closed container and the prepared medium at 2 - 8°C. Use before expiry date on the label.
References:
1. Burman N. P., 1967, Proc. Soc. Wat. Treat. Exam., 16:40.
2. Joint Committee of PHLS and the Standing Committee of Analysis, 1980, J. Hyg. Camb., 85:181.
3. Stanfied G. and Irving T. E., 1981, Water Research, 15:469.
4. International Organization for Standardization, 1990, Draft ISO/DIS: 9308-1.
5. Burman N. P., 1967, Rec. Adv. in Bacteriological Examination of waters; C.H. Collins (Ed.), Butterworth, London.