Non-covalent carbohydrate and mAGP extraction

The method described by Besra et al., (Besra et al., 1998) was followed. Biomass was harvested from three replicate planktonic or biofilm M. tuberculosis samples, inactivated by autoclaving at 126 °C for 30 min, and evaporated to dryness using a Genevac EZ-2 plus evaporator. Planktonic or biofilm biomass was quantified by dry weight to ensure equivalent quantities of planktonic and biofilm biomass were used in the extractions. A quantity of 2 g of biomass was used in each extraction, consisting of pooled cell pastes from replicate cultures. Samples were heated under reflux with 10 mL ethanol–water (1:1) at 75 °C for 4 h. Samples were then left to cool to room temperature, spun at 3000 g for 15 min, and the supernatant was removed into fresh tubes. The pellet was topped up to 10 mL with ethanol–water (1:1) and the heating step and centrifugation were repeated. The supernatants were vacuum-dried, and the pellets were recombined in phenol saturated with PBS. The samples were heated at 75 °C for 30 min and left to cool to room temperature. The phenol and aqueous layers were separated by centrifugation at 3000 g. The aqueous layer was removed into a semi-permeable dialysis membrane (MWCO 3500). The samples were dialysed overnight with running tap water and left in distilled water for 1 h to remove salts from the tap water. Samples were transferred into clean pre-weighed glass tubes and were subsequently dried. The remaining pellet from the ethanol reflux was used for mAGP extraction. 2% SDS (w/v in PBS) was added to planktonic and biofilm pellets that were remaining from the ethanol reflux step of the carbohydrate and lipoglycan extraction. Samples were heated under reflux at 95 °C overnight. Following this, the samples were washed with water, pelleted, washed with 80% acetone (v/v in water), pelleted, washed with 100% acetone, and dried.