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Gamma-proteobacteria in seawater. Sidhartha Goyal Hyunsoo Na. Contents. Introduction on (CARD-)FISH Procedures Results Discussion. Fluorescent in-situ hybridization. Detection and localization - U nculturable bacteria - Specific mRNAs in tissue - PowerPoint PPT Presentation
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Gamma-proteobacteria in sea-water
Sidhartha GoyalHyunsoo Na
Contents• Introduction on (CARD-)FISH• Procedures• Results• Discussion
Fluorescent in-situ hybridization
• Detection and localization- Unculturable bacteria- Specific mRNAs in tissue
• Capture live bacteria by targeting rRNA
CARD-FISH
Cy3 DAPI
Procedures1. Sample preparation
- Fixation and immobilization2. Permeabilization & inactivation of endogenous perox-
idases3. Hybridization4. CARD5. Microscopy
Results – relative abun-dance
• Requires training!• Can
Gamma/DAPI
EUB/DAPI NON/DAPI0
102030405060708090
Relative abundance of bacteria of interests
Row1Row2Row3Row4(%
)
Beach sample
Gamma/DAPI
EUB/DAPI NON/DAPI0
20
40
60
80
100
120
C1-150m (row1)C1-150m (row2)M1-150m (row3)M1-150m (row4)
(%)
Seawater taken from 200 m depth
Results – cells/ ml at C1/M1 - 200m
• Requires training!• Can
Results – cells/ml at Hopkins beach
• Requires training!• Can
Distribution of cells on the filter
Cy3 DAPI
Discussion
`
• Training - Distinguish between cells and particles
• Probe design may be challenging• Consortium analysis– Physical association of cells
• Community composition…?• Cheap and quick!
Thank you