1. 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

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

3. 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

4. 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)

5. 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

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

7. 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 )

8. 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

9. Comparison between PAGE and DGGE1 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