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Updated: May 29, 2026
Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization
Published on: March 25, 2009
Optimization of a two-step permeabilization fluorescence in situ hybridization (FISH) assay for the detection of
Thomas S Lawson1, Russell E Connally, Subramanyam Vemulpad
1Macquarie University, Faculty of Science, Sydney, New South Wales, Australia. tomxlawson@gmail.com
Background:
Aspects of the fluorescence in situ hybridization (FISH) method for the detection of clinically important bacteria, such as Staphylococcus aureus, Staphylococcus epidermidis, and Escherichia coli, were investigated for optimization.
Methods:
Various approaches to optimizing the FISH procedure were taken and different methods were compared. To save time, hybridization and washing buffers were prepared beforehand and stored at -20 °C and mixed to their final formamide and NaCl concentrations just before use. The use of 50-ml tubes for hybridization incubation reduced drying out, reagent wastage, and reaction times.
Results:
A two-step permeabilization FISH assay was developed that used phosphate-buffered saline as a buffer for lysostaphin. It could detect bacteria with DNA probes conjugated to fluorophores with a higher signal intensity and the less expensive biotinylated DNA probes with minimal cell lysis in 1 hr.
Conclusions:
The two-step assay might be used when the FISH signal is weak, bacterial numbers are low or if there is a need to use other reporter molecules.
