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MEMBRANE LAURYL SULPHATE BROTH

Code: MM0615

A replacement medium for Membrane Enriched Teepol Broth for the enumeration of coliform organisms and Escherichia coli in water.

Directions
Dissolve 76.2 grams in 1 litre of distilled water. Distribute into final containers, e.g. 100 ml screw cap bottles. Sterilise by steaming for 30 minutes on three consecutive days or by autoclaving at 121°C for 15 minutes.

Description
In formulating Membrane Enriched Teepol Broth, Burman¹ substituted Teepol in place of bile salts in the membrane filtration test medium used to detect coliform organisms in water. The use of Teepol in place of bile salts had been previously recommended by Jameson and Emberley² and its value was confirmed by other workers (Jebb³ and Windle- Taylor^4,5). It is essential to use one standard grade of Teepol and Teepol 610 (BDH Ltd.) has been recommended.
Burman⁶ showed that resuscitation media are not required with Membrane Enriched Teepol Broth if a preliminary incubation is carried out at a lower temperature. Thus non-chlorinated organisms benefit from 4 hours incubation at 30°C, but chlorinated organisms require 6 hours incubation at 25°C.
Membrane Lauryl Sulphate Broth is similar to Membrane Enriched Teepol Broth except that the selective agent Teepol 610 has been replaced by sodium lauryl sulphate.
In 1976 the production of Teepol 610 ceased and studies were carried out to identify a suitable alternative selective agent that could be incorporated into the basal medium. As a result of this work it was recommended⁷ that Teepol 610 should be replaced by sodium lauryl sulphate (BDH No.44244) at a concentration of 0.1% w/v.
Membrane Lauryl Sulphate Broth CM0615 has been recommended^7,8 as a standard medium for the enumeration of coliform organisms and Escherichia coli from water and sewage by the membrane filtration technique.
The Medium and method are fully described in The Bacteriological Examination of Water Supplies Report 71⁹.

Technique
The coliform and Escherichia coli count are made on separate volumes of water. The volumes should be chosen so that the number of colonies to be counted on the membrane lies between 10 and 100. If the water is suspected to contain less than 100 coliform organisms per 100 ml, then a 100 ml sample should be filtered.
The water samples are filtered through a sterile membrane filter (Report 71⁹) and the membrane filter is placed face upwards on an absorbent pad previously saturated with Membrane Lauryl Sulphate Broth. The pad and membrane filter should be incubated in a vapour-tight container to prevent evaporation.
Membranes to be incubated at 44°C should be placed in watertight heavy containers and immersed in a closely controlled water-bath.
Burman recommends the following incubation periods and temperatures:

Unchlorinated waters

If rapid results are required, the membrane may be examined after a total incubation time of 12 hours. If no colonies are present, a nil count can be assumed. If small colonies of indeterminate colour are present, then the membranes must be returned to the incubator for the full period.

Presumptive coliform organisms
After incubation, yellow colonies from membranes incubated at 35°C should be sub-cultured to Lactose Peptone Water to confirm that they will produce gas at 35°C after 43 hours incubation.

Presumptive Escherichia Coli
Yellow colonies from membranes in 44°C should be sub-cultured to Lauryl Tryptose Mannitol Broth CM0831, incubated at 44°C to confirm gas production and indole production at this temperature after 24 hours incubation.

Storage conditions and Shelf life
Store the dehydrated medium below 25°C and use before the expiry date on the label.
Store the prepared medium at 2-8°C.

Quality Control
Positive control:
Escherichia coli ATCC® 25922

Negative control:
Bacillus subtilis ATCC® 6633

Precautions
Avoid overheating.

References
1. Burman N. P. (1967a) Proc. Soc. Wat. Treat Exam. 16. 40.
2. Jameson J. E. and Emberley N. W. (1956) J. Gen. Microbiol. 15. 198-204.
3. Jebb W. H. H. (1959) J. Hyg. Camb. 57. 184-192.
4. Windle Taylor E. (1959--60) `Glutamic acid media’ 39th Ann. Rep. Dir. Water Exam. Met. Water Board, London, pp. 27-30.
5. Windle Taylor E. (1961--62) `Glutamic acid medium’ 40th Ann. Rep. Dir. Water Exam. Met. Water Board, London, pp. 18-22.
6. Burman N. P. (1967b) `Rec. Adv. in Bacteriological Examination of Water. Progress in Microbiological Techniques’ edited by C. H. Collins, London, Butterworth, p. 185.
7. Joint Committee of PHLS and The Standing Committee of Analysts (1980) J. Hyg. Camb. 85. 181.
8. Stanfied G. and Irving T. E. (1981) Water Research 15. 469-474.
9. Departments of the Environment, Health & Social Security and PHLS (1982) The Bacteriological Examination of Drinking Water Supplies. Report on Public Health and Medical Subjects No.71, HMSO, London.