The impact of sample processing and media chemistry on the culturable diversity of bacteria isolated from a cave

Although molecular approaches can identify members of microbial communities in the environment, genomic information does not necessarily correlate with environmental phenotype. Understanding functional roles can be done by cultivating representative species, yet the culturablility of bacteria from caves remains low, at 0.02%, limiting our understanding of microbial community interactions and processes. We have investigated several factors influencing culturability of bacteria from a single sample location in Maxwelton Sink Cave, WV, USA. Extended incubation of inoculated plates showed a significant increase in colony counts from two to four weeks, indicating that extended incubations increase culturability. There were no significant differences in plate counts or diversity measures when the sample was suspended in different buffers prior to cultivation, while samples plated immediately after collection demonstrated higher culturability. Although supplementing the media with antibiotics reduced colony counts and cultured diversity, these plates did appear to contain a higher proportion of slow-growing oligotrophs. Finally, among a selection of culture media used, pyruvate agar showed the highest culturability and bacterial diversity, which may be a result of the oxygen radical scavenging effects of pyruvate. By identifying methods that improve culturable diversity, we hope to further understand the roles played by bacteria in cave communities, and test hypotheses that are best assessed using culture-based methods, such as screening for bioactive compounds or confirming in situ metabolic strategies.