OPTIMIZATION OF MEDIA FOR ENUMERATION AND ISOLATION OF AEROBIC HETEROTROPHIC BACTERIA FROM THE DEEP TERRESTRIAL SUBSURFACE

The objective of this study was to develop an agar plate-count medium to maximize the number of colony-forming units (CFU) recovered from terrestrial subsurface sediment samples. A Plackett-Burman experimental design was used to test the effects of 13 possible medium variables, including various ingredients and culture conditions. These experiments used sediment samples from different surface and subsurface areas to test the resulting formulations. Microorganisms in different sediments did not respond uniformly to any variable. Variables that always had a positive effect, when significant, included addition of vitamins, addition of activated charcoal (added as a scavenger of toxic oxygen forms), use of pour plates rather than spread plates. and elevated, rather than in situ, incubation temperature. Lowered water potential always led to a negative response, where significant. Varying concentrations of most common medium components, including dilution of carbon sources, had few significant effects over the ranges tested. A medium formulation for enhanced recovery of aerobic heterotrophic bacteria from subsurface sediments was developed, based on these results.