WT and ΔslpA B. thuringiensis were cultured for 18 hours as mentioned above, then centrifuged at 3000g for 15 minutes. Supernatants were then filter sterilized (0.22 μm; Millex-GP, Merck Millipore Ltd., Cork, Ireland) and serially diluted 1:2 in PBS (pH 7.4). Diluted supernatants were incubated 1:1 with 4% (vol/vol) sheep erythrocytes (Rockland Immunochemicals, Pottstown, PA, USA) in a 96-well round bottom plate for 30 minutes at 37°C. The plate was centrifuged at 1892g for 10 minutes to remove the unlysed erythrocytes.
The supernatants were carefully transferred into a 96-well, flat-bottom plate and hemoglobin release was measured spectrophotometrically at 490 nm by using a FLUOstar Omega microplate spectrophotometer (BMG Labtech, Cary, NC, USA). Values are expressed as the percent hemolysis relative to a 100% lysis control in which 5% rabbit erythrocytes were lysed in double-distilled H2O.23 (link),27 (link),48 (link),76 ,77 (link) Values represent the mean results ± SEM of two independent experiments. These strains were also incubated on tryptic soy agar (TSA) supplemented with 5% sheep blood (Hardy Diagnostics, Santa Maria, CA, USA) for 18 hours at 37°C, and colony morphology and hemolytic phenotypes were compared.
The supernatants were carefully transferred into a 96-well, flat-bottom plate and hemoglobin release was measured spectrophotometrically at 490 nm by using a FLUOstar Omega microplate spectrophotometer (BMG Labtech, Cary, NC, USA). Values are expressed as the percent hemolysis relative to a 100% lysis control in which 5% rabbit erythrocytes were lysed in double-distilled H2O.23 (link),27 (link),48 (link),76 ,77 (link) Values represent the mean results ± SEM of two independent experiments. These strains were also incubated on tryptic soy agar (TSA) supplemented with 5% sheep blood (Hardy Diagnostics, Santa Maria, CA, USA) for 18 hours at 37°C, and colony morphology and hemolytic phenotypes were compared.
