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

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

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

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• Training - Distinguish between cells and particles

• Probe design may be challenging• Consortium analysis– Physical association of cells

• Community composition…?• Cheap and quick!

Thank you

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