FIG. 2.

Biofilm assays and slot blot analysis of PNAG extracts indicate that PNAG levels do not correspond with maximal biofilm levels in S. aureus. (A) Biofilm formation by S. aureus Newman, 8325-4, and the Newman ΔsigB::erm mutant. (B) PNAG production by strains Newman and 8325-4. Bacteria were grown in CRPMI overnight and diluted to an OD₅₉₅ of 0.1 in CRPMI (Fe−) or CRPMI plus 50 μM Fe₂(SO₄)₃ (Fe+) with (NaCl+) or without the addition of 3% (vol/vol) NaCl and inoculated in quadruplicate into wells of a polystyrene microtiter plate. After 24 h the wells were washed with phosphate-buffered saline, adherent bacteria were stained with safranin, and biofilm formation was assessed by OD₄₉₀ measurements. Results represent the averages of at least eight independent experiments and the standard error of the mean. For measurement of PNAG production, cells were harvested by centrifugation from 24-h static cultures. PNAG was extracted by boiling cell suspensions in EDTA; cell debris was removed by centrifugation, and 50-μl samples of serial dilutions (1:10 and 1:100) were applied to nitrocellulose membrane for probing with PNAG-specific rabbit antiserum. Each experiment was repeated at least three times using PNAG extracts obtained from cultures grown on different days with similar results in each case.