Improved methods for determining the most probable number ofBacterium coliand ofStreptococcus faecalis

Abstract

Three methods have been investigated for determining Most Probable Numbers, using lactose-bile salts broth forBacterium coliand glucose-yeast extract-azide broth forStreptococcus faecalis. In the ‘direct’ method inoculated tubes of medium are incubated directly in a water-bath, at 44° C. forBact. colior at 45° C. forStr. faecalis. In the ‘subculture’ method primary incubation at 37° C. is followed by subculture of positive tubes to tubes of fresh media which are incubated at the higher temperature. In the ‘resuscitation’ method each medium is divided into 2 parts. The inoculum is mixed, in McCartney bottles or tubes, with the first part of the medium, containing the sugar and the peptone, and the mixture is held at 37° C. for 1–2 hr. to resuscitate organisms weakened by age or by long immersion in water. The second part of the medium, containing the bile salts or the azide, is then added and the bottles or tubes are incubated at 44 or 45° C. With samples of polluted waters the ‘subculture’ method gives significantly higher counts than the ‘direct’ method forStr. faecalisbut not forBact. coli.The ‘resuscitation’ method gives significantly higher results than the ‘subculture’ method for both organisms, it requires less time for completion, and is specific for the organism concerned.These results were obtained with media containing 0.003% brom-cresol purple as indicator; neutral red was found to be inhibitory.This paper is published by permission of the Department of Scientific and Industrial Research.