Monitoring bacterial activity by Fluorescence in situ hybridisation (FISH) (CAT#: STEM-MB-1202-WXH)

Introduction

Bacterial activity is linked to DNA synthesis. The detection of newly synthesised DNA by monitoring the uptake and incorporation of tritiated thymidine or, more recently, bromodioxyuridine BrdU is therefore a way to differentiate active from inactive cells. Combining this technique with FISH consequently allows an assertion of which parts of a bacterial community are metabolically active.

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Principle Applications Procedure Materials Notes Related Products

Principle

FISH uses fluorescent probes with complementary base sequences to locate the presence or absence of specific portions of DNA on chromosomes. The probe and target DNA must be denatured with heat or chemicals to break hydrogen bonds in the DNA and to allow hybridisation to occur once the two samples are mixed. The fluorescent probes form new hydrogen bonds with their complementary base pairs on the DNA, and these can then be detected via microscopy.

Applications

Detect and localize the presence or absence of specific DNA sequences on chromosomes.
Detect and localize specific RNA targets (mRNA, lncRNA and miRNA) in cells, circulating tumor cells, and tissue samples.

Procedure

1. Sample preparation
2. Co-denaturation and hybridization
3. Probe detection
4. Wash off of unbound probe
5. Analysis by flow cytometer/fluorescence microscopy

Materials

• Flow cytometer
• Fluorescence microscopy

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