FLUORESCENT IN SITU HYBRIDIZATION FISH

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

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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.
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• 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.

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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
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rRNA features

• Single strand
• Ancient molecules
• Constant functions
• Universally distributed
• Well conserved phylogenetically

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Eubacteria DAPI