Combined Use of Trimethoprim with Nalidixic Acid for the Selective Isolation and Enumeration of Actinomycetes from Soil.

Abstract

The use of synthetic antibacterial compounds as selective supplements in agar media for the isolation and enumeration of soil actinomycetes is described. A preliminary survey revealed that the combined application of trimethoprim (TP) and nalidixic acid (NA) had no deleterious effect on most test actinomycetes (35 genera and 159 species), while the growth of most test non-filamentous soil bacteria (10 genera and 20 species) was inhibited or discouraged. A mixture of TP (20 mg/l) and NA (10 mg/l) was incorporated into humic acid-vitamin (HV) agar and four other isolation media. The efficiency of isolating and enumerating actinomycetes cultivated under these conditions was evaluated using 13 different natural soil samples. The TP-NA combination substantially reduced the occurrence of competing non-filamentous bacteria on the isolation plates without adversely affecting actinomycete growth, thus greatly facilitating the recognition, enumeration and pure isolation of actinomycete colonies. A detailed taxonomic examination showed that the antimicrobial combination allowed the recovery of varieties of actinomycete genera. At least 11 different genera were isolated from a garden soil sample on HV plates containing TP and NA. The most frequently isolated Streptomyces strains were further categorized into various species-groups. The results suggest that the TP-NA combination may prove to be a useful tool for autecological and enumeration studies of actinomycetes, and for the isolation of wide varieties of actinomycete strains.