To detect with whole-cell fluorescence in situ hybridization (FISH), Staphylococcus aureus is typically permeabilized with lysozyme and lysostaphin. We tested whether it was feasible to detect S. aureus and differentiate it from Staphylococcus epidermidis with lysozyme-only permeabilization. We compared lysozyme permeabilization to S. aureus permeabilized with lysozyme in combination with lysostaphin. It was determined that S. aureus treated with agarose, methanol, and lysozyme could be detected with FISH. The 1 hr protocol is a useful alternative to conventional FISH.