Session Title: Novel Technologies and Methods: Functional Community Analysis
Presentation Date: Thursday, August 21, 2008
Poster Board Number: 0262
HIGH TEMPERATURE-DIRECT VIABLE COUNT METHOD FOR ENUMERATION OF METABOLICALLY ACTIVE THERMOPHILIC BACTERIA IN COMPOST
K. Shiotsuka, K. Sakai
Kyushu University, Department of Plant Pesources, Graduate School of Bioresource and Bioenvironmental Sciences, Fukuoka, Japan
Aims: We aimed to enumerate metabolically active (extremely) thermophilic bacteria in composts by improving the direct viable count (DVC) method reported by Kogure et al. (Can. J. Microbiol. 25, 415, 1979), establishing high temperature-DVC (HT-DVC) method.
Methods: Shell compost was used as a sample for developing the DVC method. The sample was pretreated at 60 °C for 24 h and diluted 100-fold. With yeast extract (0.02 %) and thermostable DNA replication inhibitors (pipemidic acid and norfloxacin, each 50 µg ml-1), the diluted sample was further incubated at 60 °C for 24 h. Bacterial cells in the mixture were stained with ethidium bromide and observed using an epifluorescent microscope. Sewage sludge compost was used for investigating application of HT-DVC method to detect metabolically active thermophiles growing at 80 °C.
Results: In the shell compost, elongated cells over 4 µm length of 6.1 × 109cells g-1 were detected at 60 °C. The number was over 5,000 times higher than that estimated by plate count method, indicating that nonculturable thermophilic bacteria would be detected by the HT-DVC method. In the sewage sludge compost, elongated cells of 2.6 × 108 cells g-1 were detected maximally at 80 °C. This indicated that HT-DVC method could be applied to enumerate thermophiles growing at 80 °C which is too high to apply the quantitative culture-dependent method.
Conclusion: HT-DVC method would be useful for counting metabolically active thermophilic bacteria in composts.