Quantifying Biofilm Formation in NTHi

Previously described methods were used (50 (link), 51 (link)). Briefly, an overnight NTHi broth culture was diluted 1:200 in fresh broth and 200 μL was inoculated into the wells of a 96-well CELLSTAR tissue culture plate (Greiner Bio-One, Monroe, NC). The plates were incubated at 37°C on a nutator for 24 h. Before biofilm quantitation, growth was assessed by measuring the OD₄₉₀ in a Bio-Rad plate reader. To quantitate biofilm formation, 20 μL of Difco crystal violet (Becton, Dickinson and Co., Sparks, MD) was added to each well and incubated at room temperature for 15 min. Wells were washed vigorously with distilled water and the plate was air-dried. A 230-μL volume of 95% ethanol was added to each well and the OD₅₇₀ was measured. Each plate included four control wells which contained sterile broth instead of bacteria but were otherwise treated identically. The OD₅₇₀ was standardized against these wells. Strains were tested in quadruplicate wells. The mean and standard deviation were calculated from values for each strain. Comparison of biofilm formation between mutant and parent strains was performed using an unpaired t test.