M-Endo HiVeg™ Agar LES

Principle and Interpretation

M-Endo HiVeg Agar LES is prepared by completely replacing animal based peptones with vegetable peptones which make the medium free of BSE/TSE risks. M-Endo HiVeg Agar LES is the modification M-Endo Agar LES (Lawrence, Experimental Station) which is formulated according to the formulation of McCarthy, Delaney and Grasso (1) and is used for the enumeration of coliforms in water (2). Membrane filter technique for coliform enumeration is more reliable and precise than MPN multiple tube test. A two-stage process has been suggested for enrichment to get a non-toxic environment for maximum revival of the coliforms. This medium, like the conventional medium is based on the medium described by Endo for the differentiation of lactose fermenters from non-fermenters (3).

HiVeg hydrolysate, HiVeg hydrolysate No.1, HiVeg peptone and yeast extract provide essential nutrients especially nitrogenous for the coliforms. Lactose is the fermentable carbohydrate. Sodium sulphite, Synthetic detergent No. III and basic fuchsin inhibit the growth of gram-positive organisms. Phosphates buffer the medium. Coliforms ferment the lactose and form red colonies and similar colouration of the medium. Lactose non-fermenters form colourless colonies.

In the first step of enrichment, the pad is impregnated with Lauryl Tryptose HiVeg Broth (MV080). Membrane filter through which water sample is passed is aseptically placed on it and incubated without inverting for 2 hours at 35°C in a humid atmosphere. After incubation, the membrane filter is aseptically transferred to the M-Endo HiVeg Agar LES plate and incubated at 35°C for 24 hours. Alternatively membrane filter pad can be placed inside the lid of petri plate of M-Endo Agar LES and then impregnated with 2 ml Lauryl Tryptose HiVeg Broth (MV080) and incubated for 1 - 1½ hour at 35°C. In the second step, the prepared membrane filter is kept directly on the agar surface and incubated as described above. Presumptive coliforms produce golden green colonies with metallic sheen within 24 hours of incubation. If the inoculum is too heavy, the sheen will be suppressed. Sometimes non-coliform organisms may produce typical colonies with sheen, coliforms may also occasionally produce atypical colonies (dark red without sheen).

Coliform density calculation: Note the coliform density in terms of total coliforms/100 ml. Extrapolate the count using membrane filters with 20-80 coliform colonies but not more than 200 of all types per membrane.

The formula for calculating the count is as follows:

Total coliform = coliform colonies x 100

colonies/100 ml ml of sample filtered