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 hybridizationincubation 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 1hr. 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.