Anaerobic Bacillus thuringiensis Growth and Mannan Assay

Bt was cultured anaerobically at 37 °C in minimal media containing an appropriate carbon source. or in TYG (tryptone yeast extract glucose medium) as described previously21 (link). Growth curves presented in the paper are averages of six biological replicates. Bt was grown in 5 mL minimal media on 0.5% w/v Saccharomyces cerevisiae mannan (Sigma) or glucose as the sole source to mid exponential phase (OD_600nm 0.6-0.8). Cells were harvested by centrifugation, 5000 x g 10min at room temperature and washed in 5 ml Phosphate buffered Saline pH 7.1 (PBS) before being resuspended in 500 µL PBS. Cells (50 µL) were assayed against yeast mannan (10 mg ml^-1) at 37 °C for 16 h. Assays were analysed by thin layer chromatography, 5 µL of each sample was spotted onto silica plates and resolved in butanol/acetic acid/water buffer. The plates were dried and sugars visualised by orcinol/sulphuric acid heated to 70°C.