Monitoring of bacteria with culture-independent techniques Importance of microbial monitoring ・Accurate estimation of bacterial abundance ・Community analysis In this study… i) Bacterial abundance: total direct counting (TDC) following fluorescent staining and quantitative real-time PCR (qPCR) ii) Bacterial community analysis: Denaturing gradient gel electrophoresis (DGGE) and phylogenetic analysis by cloning approach

Direct counting of bacterial cells ・epifluorescence microscopy (EFM) Staining (3 min) Filtration Sample: Neyagawa River Staining: DAPI (DNA-binding dye) Observation

Bacterial cells in natural mineral water UV excitation Microbes exist everywhere. Most of them are NOT harmful, while they sometimes cause opportunistic infections. Bacterial cells were stained with DAPI (1 µg/mL) filtration: 40 mL

Yamaguchi, et al. J. Antibact. Antifung. Agents，22: 65 (1994) Discrimination of active cells from inactive cells Cells in logarithmic phase Blue excitation CFDA, PI: 10 µg/mL (staining: 5 min) Sample: Bacillus megaterium double-stained with CFDA and PI Esterase active cells: green Inactive cells: red spore-forming bacteria Yamaguchi, et al. J. Antibact. Antifung. Agents，22: 65 (1994)

Detection of esterase active cells in natural river water Blue excitation UV excitation CFDA: 150 µg/mL DAPI: 1 µg/mL Sample: Kitahashi (Neyagawa River); staining: 5 min

We can visualize bacterial cells based on their characters “Bio-imaging”

Determination of abundant bacteria by PCR-DGGE Sample Bacterial cells collected by swabbing DNA extraction PCR amplification 16S rDNA Primers: EUB f933-GC-clamp, EUB r1387 DGGE ( Denaturing Gradient Gel Electrophoresis )

Primers for PCR-DGGE Bacterial 16S rDNA: 1,500 bp 500 bp EUBf933 conserved region (common to all bacteria) variable region (specific to bacterial species) EUBr1387 Different bacteria have different sequences in the PCR products

Comparison between PAGE and DGGE 1 2 3 4 1 2 3 4 40% Denaturant conc. Lane 1 : Al. calcoaceticus Lane 2 : E. coli Lane 3 : Ps. putida Lane 4 : DNA mixture 60% PCR products with same length and different sequences can be discriminated