fish technique

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FLUORESCENT IN SITU HYBRIDIZATION (FISH) : 

FLUORESCENT IN SITU HYBRIDIZATION (FISH)

Slide 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

Slide 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.

Slide 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.

Main stepsFix SamplesImmobilize on slidesHybridize with fluorescently-labeled oligosWash slides (optimize stringency)Counterstain with non-specific dye (like DAPI)Add anti-bleaching mountantView under epifluorescence microscope : 

Main stepsFix SamplesImmobilize on slidesHybridize with fluorescently-labeled oligosWash slides (optimize stringency)Counterstain with non-specific dye (like DAPI)Add anti-bleaching mountantView under epifluorescence microscope

rRNA features : 

rRNA features Single strand Ancient molecules Constant functions Universally distributed Well conserved phylogenetically

Eubacteria DAPI : 

Eubacteria DAPI