FLUORESCENT IN SITU HYBRIDIZATION FISH - PowerPoint PPT Presentation

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FLUORESCENT IN SITU HYBRIDIZATION FISH

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1 Identifying/quantifying uncultivated and/or cultivated ... Add anti-bleaching mountant. View under epifluorescence microscope. rRNA features. Single strand ... – PowerPoint PPT presentation

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Title: FLUORESCENT IN SITU HYBRIDIZATION FISH


1
FLUORESCENT IN SITU HYBRIDIZATION (FISH)
2
  • Fluorescent in situ Hybridization detects nucleic
    acid sequences by a fluorescent probe that
    hybridizes specifically to its complementary
    target sequence within the intact cell

3
Applications 1 Identifying/quantifying
uncultivated and/or cultivated organisms in
natural environmental samples, within organisms
like endosymbionts, or enrichments. 2 Monitoring
population dynamics in environments and/or
enrichments. 3 Visualizing spatial relationships
among microbes in communities like biofilms,
sludge, etc. 4 Identifying morphology or
relationships among types of organisms.
4
  • Limitations
  • 1. Decreased amounts of rRNA target low or no
    ribosomes low or no signal due to metabolically
    inactive or quiescent cells
  • 2. Background autofluorescence, including
    cyanobacteria, plant tissues, etc
  • 3. Permeability issues or difficulty getting
    probes into tough cells like Gram, Archaea, etc.

5
Main stepsFix SamplesImmobilize on
slidesHybridize with fluorescently-labeled
oligosWash slides (optimize stringency)Counter
stain with non-specific dye (like DAPI)Add
anti-bleaching mountantView under
epifluorescence microscope
6
rRNA features
  • Single strand
  • Ancient molecules
  • Constant functions
  • Universally distributed
  • Well conserved phylogenetically

7
Eubacteria DAPI
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