Variation in colony counts of total viable anaerobic rumen bacteria as influenced by media and cultural methods

Abstract

Volume and type of medium, carbohydrate concentration, carbohydrate ratios, and inoculum level were investigated as possible factors influencing total colony counts of anaerobic rumen bacteria obtained in roll tubes (18 by 150 mm). Colony counts were lower when the rumen fluid was clarified by centrifugation before inclusion in the medium; however, decreasing the volume of 40% rumen fluid glucose cellobiose starch agar medium (RGCSA) from 9 to 4 ml per tube did not affect total counts. With 4 ml of RGCSA per roll tube, total carbohydrate concentrations of 0, 0.1, and 0.3% affected total colony counts as follows: 0.1%>0.3%>0%. Also, if the ratio of equal parts glucose plus cellobiose:starch was varied from 1:1 to 9:1, at a total carbohydrate concentration of 0.1%, higher counts were obtained at the 1:1 ratio. When colony counts exceeded a range of 80 to 100 colonies per 4 ml roll tube, in many instances the relationship between inoculum concentration and total number of viable colonies was no longer linear. The present method (RGCSA medium with 0.025% each of glucose and cellobiose and 0.05% starch, 4 ml per tube) was compared to the clarified rumen fluid medium and non rumen fluid medium (medium 10) of Caldwell and Bryant, 9 ml of each per tube. Total counts of rumen contents from sheep consuming four different types of rations were higher with the 4 ml of RGCSA medium than with the other two media. Dilution of the basal medium as a result of inoculum volume, as much as 1.5 ml per 4 ml of medium, did not appear to affect total counts. Colony counts and the simplicity of medium preparation and inoculation would favor the present method for routine use in estimating numbers of total viable anaerobic rumen bacteria, especially when large numbers of samples are involved.