The Estimation of Numbers of Coli Bacteria in Foods by Means of A Deep Agar Tube Technique with A Modified Violet Red Bile Agar

Abstract

The author has investigated the possibilities of substituting the agar plate technique for a deep agar tube technique in estimating numbers of ooliform bacteria in foods. Especially when water bath incubation at 44°C is used for selective counting of E. coli I a tube technique would seem to offer considerable practical advantages over the ordinary Petri dish plate technique. The medium selected for the purpose is Violet Red Bile Agar. However, in order to be able to cultivate ooliform bacteria in VRB-deep agar tubes the formation of gas from fermentation of lactose must be suppressed. To achieve anaerogenic growth of ooliform bacteria the author has utilized the existing knowledge of the inhibitory effect of nitrate- and nitrite ions upon the formic acid hydrogenlyase enzyme which catalyses the decomposition by coliforms of formic acid into hydrogen and carbon dioxide. Nitrate was found preferrable to nitrite because of less inhibitory effect. The optimal concentration of nitrate in VRB Agar was determined to 0.5 % NaNO3. Flat Miller-Prickett-tubes with an inlaid white enamel glass plate were found to facilitate counting. To obtain well developed colonies with a typical zone of precipitation the agar contents of the medium was reduced to 0.9 per cent. Two series of comparative coli counts with the modified VRB-Nitrate agar in deep agar tubes against ordinary VRB-Agar in Petri dishes at 37° C and 44°C showed on the whole no statistically valid differences. The problem of small retarded colonies appearing in tube cultures and plate cultures as well, mostly when seeded with raw milk, was investigated. All of 108 such strains except two could be identified as members of the ooliform group. The selectivity of the medium was tested by plating a variety of non-ooliform organisms. Only a few strains of fecal streptococci, Proteus, Aloaligenes and Pseudomonas were capable of scanty growth, and in no instance the resulting colonies could be mistaken for colonies of ooliform bacteria.