Monitoring of bacteria with culture-independent techniques

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

Importance of microbial monitoring

・ Accurate estimation of bacterial abundance

・ Community analysis

Direct counting of bacterial cells

・ epifluorescence microscopy (EFM)

Staining(3 min)

Filtration

Observation

Sample: Neyagawa RiverStaining: DAPI (DNA-binding dye)

Bacterial cells in natural mineral water

Bacterial cells were stained with DAPI (1 µg/mL)filtration: 40 mL

UV excitation

Microbes exist everywhere.

Most of them are NOT harmful,

while they sometimes cause opportunistic infections.

Sample: Bacillus megaterium double-stained with CFDA and PI

Esterase active cells: greenInactive cells: red

CFDA, PI: 10 µg/mL (staining: 5 min)

Blue excitation

Cells in logarithmic phase

Discrimination of active cells from inactive cells

Yamaguchi, et al. J. Antibact. Antifung. Agents ， 22: 65 (1994)

spore-forming bacteria

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

DNA extraction

PCR amplification 16S rDNA

Primers: EUB f933-GC-clamp, EUB r1387

DGGE ( Denaturing Gradient Gel Electrophoresis )

Bacterial cells collected by swabbing

Primers for PCR-DGGE

Bacterial 16S rDNA: 1,500 bp

EUBf933

EUBr1387variable region(specific to bacterial species)

conserved region(common to all bacteria)

Different bacteria have different sequences in the PCR products

500 bp

Comparison between PAGE and DGGE

Lane 1 : Al. calcoaceticus Lane 2 : E. coli Lane 3 : Ps. putidaLane 4 : DNA mixture

40%

60%

Den

atu

ran

t co

nc.

1 2 3 4

DGGE

PCR products with same length and different sequences can be discriminated

1 2 3 4

PAGE