Table 1.

Comparison of the Optimized One‐Step or Two‐Step Permeabilization FISH Assays

One‐step (1)	Two‐step (This study)
Preparation: Cultures of clinical isolates were diluted with PBS, spotted, fixed to slides at 80°C, fixed in methanol, and air‐dried
Permeabilization: Slides were spotted with lysis reagent (15 mg/ml lysozyme, 0.1 mg/ml lysostaphin, 20 mM Tris–HCl at pH 7.0 in MQ water) and incubated at 40°C, rinsed with methanol, and air‐dried	Permeabilization: Slides were spotted with 15 mg/ml lysozyme with 20 mM Tris–HCl at pH 7.0 in MQ water and incubated at 38°C, rinsed with PBS, and dried with pressurized air
	Permeabilization: Slides were spotted with 0.1 mg/ml lysostaphin, 20 mM Tris–HCl diluted in PBS and incubated at 47°C, rinsed with methanol, and air‐dried
Hybridization: Slides were spotted with hybridization buffer (30% formamide, 0.9 M NaCl, 20 mM Tris–HCl at pH 8.0, 0.01% SDS, a 1‐μM probe, and MQ water), and incubated at 47°C
Washing: Slides were incubated with washing buffer (0.3–0.9 M NaCl, 20 mM Tris–HCl pH 8.0, 0.01% SDS, 10 mM EDTA, and MQ water) at 47°C, rinsed with PBS
Mounting: Slides were mounted with a cover‐slip while wet	Streptavidin/Mounting: Slides were spotted with streptavidin‐f and incubated at 38°C, rinsed with PBS, and mounted with a cover‐slip while wet