Heparin enhances cell retention within a biofilm but does not promote attachment. Heparin enhances cell retention within a biofilm but does not promote attachment. (A) The fraction of planktonic cells compared to biofilm cells was determined by suspending either the entire contents of a microtiter well (total) or just the biofilm after removal of spent medium (biofilm). When heparin was present, the number of planktonic cells was negligible compared to the number of cells in the biofilm. (B) The wells of a polystyrene or glass-bottom microtiter plate were incubated with 100 U/ml of heparin in PBS for 24 h [indicated by “(c)” (for coated)], washed to remove free heparin, and inoculated with strain LAC. After 18 h of incubation, biofilm biomass was assessed. As a negative control, wells were preincubated with PBS, and as a positive control, heparin was added at the time of inoculation with the LAC strain. (C) To assess the kinetics of heparin enhancement of biofilm formation, heparin was added at the time of inoculation (0 h) or after 18 h of growth in the absence of heparin (18 h). Biofilm biomass was determined after one additional hour of incubation. (D) Effect of washing on heparin-dependent biofilm formation. Strain LAC was grown for 18 h in the absence of heparin, at which time the cells were washed to remove secreted proteins and then transferred to the wells of a microtiter plate. Heparin was added, and biofilm biomass was assessed after 1 h. For comparison, the assay was also performed using the planktonic cells directly (unwashed). A glucose-only control (glc) received no heparin. Values in each panel are the averages ± standard deviations from three independent experiments. Statistical significance (*, P < 0.05; **, P < 0.005) was determined by a Student t test and is based on comparison to the value for the untreated control for each condition. Surabhi Mishra, and Alexander R. Horswill mSphere 2017; doi:10.1128/mSphere.00135-17