MEMBRANE ENDO AGAR LES

Code: MM0551

A membrane filtration medium requiring Basic Fuchsin for enumeration of coliform organisms in water, using a 2-stage enrichment technique.

Basic Fuchsin to be added at 1.2 gm/litre.

BASIC FUCHSIN

Code: BR0050
For each litre of medium use 12 ml of a 10% w/v solution of this dye dissolved in 50:50 ethanol:distilled water.

Directions
Suspend 45 grams in 1 litre of distilled water. Add 12ml of a 10% w/v solution of Basic Fuchsin. Heat gently with frequent agitation until the medium boils. DO NOT AUTOCLAVE. Cool to 45°C and dispense into 50-60 mm dishes in 4 ml volumes. For larger dishes use sufficient medium to give an equivalent depth (approx. 1.5 mm).
Plates should be protected from light and may be stored for up to two weeks in the refrigerator.

WARNING
Basic Fuchsin is a potential carcinogen and care must be taken to avoid inhalation of the powdered dye and contamination of the skin.

Description
M-Endo Agar LES is prepared according to the Lawrence Experimental Station formulation of McCarthy, Delaney and Grasso¹ and used for the enumeration of coliform organisms in water².
The value of the membrane filter technique for the enumeration of coliform organisms in water lies in its greater reliability and precision when compared with the MPN multiple tube test³.
McCarthy, Delaney and Grasso1 have recommended a two-stage process of enrichment to provide a non-toxic environment for maximal resuscitation of the coliforms.
Calabrese and Bissonnette⁴found that supplementation of M-Endo medium with catalase and sodium pyruvate resulted in improved recovery of coliform bacteria from chlorinated sewage effluent.
Experiments carried out by Noble⁵ indicated that sodium sulphite and Basic Fuchsin can be extremely detrimental to the stressed coliforms, reducing the total count.
Enrichment for a period of 2 hours ± 0.5 hours in single strength Lauryl Tryptose Broth CM0451 will give adequate resuscitation to the stressed coliform organisms and provide the best assessment of the quality of the drinking water.
Enrichment is usually not necessary for the examination of non-potable waters and sewage effluents.
Selection of the sample volume is governed by the expected bacterial density. An ideal quantity will result in growth of more than 50 coliform colonies and less than 200 colonies of all types.
All organisms which produce a colony with a golden-green metallic sheen within 24 hours incubation are considered members of the coliform group. The sheen may cover the entire colony or be restricted to the central area or the periphery. The recommended depth of M-Endo Agar LES in plates restricts the colony size and hence facilitates carrying out the colony count.

Technique
The water sample is filtered through a sterile membrane filter⁶.
For the first stage of enrichment, place a sterile incubating pad in the upper half of a sterile petri dish and pipette onto this 2 ml of Lauryl Tryptose Broth CM0451. Aseptically place the filter membrane on to the incubating pad and incubate, without inverting the dish, for 1-1.5 hours at 35°C in an atmosphere having 100% humidity. Place petri dishes of M-Endo Agar LES in the incubator for the entire period so that they will be at the correct temperature when required for the second stage of enrichment. The first stage enrichment culture is removed from the incubator and the filter membrane is stripped aseptically from the incubating pad and transferred to the surface of the petri dish of M-Endo Agar LES. It is important that complete contact is made between the membrane and the agar surface. The plate is inverted and incubated for 22-24 hours at 35°C.
Alternatively, the membrane filter incubating pad can be placed inside the lid of the petri dish of M-Endo Agar LES and 2 ml of Lauryl Tryptose Broth CM0451 pipetted onto the pad. The filter membrane is placed face upwards on the pad and incubated for 1-1.5 hours at 35°C.
To carry out the second stage of enrichment, the first-stage enrichment is removed from the incubator and the filter membrane is stripped from the pad and placed face upwards on the surface of the M-Endo Agar LES medium. The incubating pad is left in the lid and the plates are incubated in the inverted position for 24 hours at 35°C.
If preferred the second stage only may be used. The prepared membrane filter is placed directly on the agar surface and incubated as described.
All the organisms which produce a colony with a golden-green metallic sheen within 24 hours incubation may be considered as presumptive coliforms.

Calculation of Coliform Density
Report the coliform density in terms of total coliform/100 ml. Compute the count using those membrane filters with 20-80 coliform colonies and not more than 200 of all types per membrane.

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 in the dark and at 2-8°C.

Precautions
Use care when handling basic fuchsin to avoid inhaling the powder and staining the skin.

References
1. McCarthy J.A., Delaney J.E., Grasso R.J. (1961) `Measuring Coliforms in Water’, Water and Sewage Works, 108. 238-243.
2. American Public Health Association (1980) Standard Methods for the Examination of Water and Wastewater. 15th Edn, APHA Inc, Washington DC.
3. McCarthy J.A., Thomas H.A.J., Delaney J.E. (1958) `Evaluation of the Reliability of Coliform Density Tests’. AJPH, 48. 16-28.
4. Calabrese J.P. and Bissonnette G.M. (1990) Appl. Env. Microbiol. 56. 3558-3564.
5. Noble R.E. (1960) `Reliability of MPN Indexes for Coliform organisms’. JAWWA, 52. 803.
6. 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.