Monitoring of bacteria with culture-independent techniques In this study… i) Bacterial abundance:...
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![Page 1: Monitoring of bacteria with culture-independent techniques In this study… i) Bacterial abundance: total direct counting (TDC) following fluorescent staining.](https://reader035.fdocuments.in/reader035/viewer/2022062314/56649e885503460f94b8bebc/html5/thumbnails/1.jpg)
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
![Page 2: Monitoring of bacteria with culture-independent techniques In this study… i) Bacterial abundance: total direct counting (TDC) following fluorescent staining.](https://reader035.fdocuments.in/reader035/viewer/2022062314/56649e885503460f94b8bebc/html5/thumbnails/2.jpg)
Direct counting of bacterial cells
・ epifluorescence microscopy (EFM)
Staining(3 min)
Filtration
Observation
Sample: Neyagawa RiverStaining: DAPI (DNA-binding dye)
![Page 3: Monitoring of bacteria with culture-independent techniques In this study… i) Bacterial abundance: total direct counting (TDC) following fluorescent staining.](https://reader035.fdocuments.in/reader035/viewer/2022062314/56649e885503460f94b8bebc/html5/thumbnails/3.jpg)
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.
![Page 4: Monitoring of bacteria with culture-independent techniques In this study… i) Bacterial abundance: total direct counting (TDC) following fluorescent staining.](https://reader035.fdocuments.in/reader035/viewer/2022062314/56649e885503460f94b8bebc/html5/thumbnails/4.jpg)
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
![Page 5: Monitoring of bacteria with culture-independent techniques In this study… i) Bacterial abundance: total direct counting (TDC) following fluorescent staining.](https://reader035.fdocuments.in/reader035/viewer/2022062314/56649e885503460f94b8bebc/html5/thumbnails/5.jpg)
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
![Page 6: Monitoring of bacteria with culture-independent techniques In this study… i) Bacterial abundance: total direct counting (TDC) following fluorescent staining.](https://reader035.fdocuments.in/reader035/viewer/2022062314/56649e885503460f94b8bebc/html5/thumbnails/6.jpg)
We can visualize bacterial cells based on their characters
“Bio-imaging”
![Page 7: Monitoring of bacteria with culture-independent techniques In this study… i) Bacterial abundance: total direct counting (TDC) following fluorescent staining.](https://reader035.fdocuments.in/reader035/viewer/2022062314/56649e885503460f94b8bebc/html5/thumbnails/7.jpg)
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
![Page 8: Monitoring of bacteria with culture-independent techniques In this study… i) Bacterial abundance: total direct counting (TDC) following fluorescent staining.](https://reader035.fdocuments.in/reader035/viewer/2022062314/56649e885503460f94b8bebc/html5/thumbnails/8.jpg)
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
![Page 9: Monitoring of bacteria with culture-independent techniques In this study… i) Bacterial abundance: total direct counting (TDC) following fluorescent staining.](https://reader035.fdocuments.in/reader035/viewer/2022062314/56649e885503460f94b8bebc/html5/thumbnails/9.jpg)
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