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Fluorescent in-situ hybridization
• Detection and localization- Unculturable bacteria- Specific mRNAs in tissue
• Capture live bacteria by targeting rRNA
Procedures
1. Sample preparation - Fixation and immobilization
2. Permeabilization & inactivation of endogenous perox-idases
3. Hybridization4. CARD5. Microscopy
Results – relative abun-dance
• Requires training!• Can
Gamma/DAPI
EUB/DAPI NON/DAPI0
10
20
30
40
50
60
70
80
90
Relative abundance of bacteria of interests
Row1
Row2
Row3
Row4(%)
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
Discussion
`
• Training - Distinguish between cells and particles
• Probe design may be challenging• Consortium analysis– Physical association of cells
• Community composition…?• Cheap and quick!