Differential carbohydrate media and anaerobic replica plating techniques in delineating carbohydrate-utilizing subgroups in rumen bacterial populations

Abstract

A basal (BC) medium devoid of added carbohydrates, a complete (CC) medium containing nine carbohydrates, and a variety of differential (DC) media containing one or two carbohydrates were developed for enumerating rumen bacteria. The colony counts on the BC medium were 85 to 100% of those obtained on the CC medium. These colonies were pinpoint size (≤1 mm in diameter) but increased in size (2 to 5 mm in diameter) when carbohydrates were subsequently added. With the CC medium or other media tested, the colony counts were 20 to 50% higher on plates than on roll tubes and were about 35% of the direct cell counts. The lower colony counts on roll tubes were shown to result primarily from the loss of viability due to heat stress. The DC media were found by plating techniques to be suitable for differentiating mixed rumen bacterial populations into subgroups based upon carbohydrate utilization as shown by differences in subgroup profiles found within solid and liquid fractions of rumen contents, within rumen contents from animals fed high-forage and high-grain diets, and by correct colony formations by pure cultures of rumen bacteria on appropriate DC media. With simple modifications and use of an anaerobic glove box, replica plating methods and the CC and DC media were found to be a suitable means of rapidly determining the range of utilizable carbohydrate energy sources of rumen bacteria.