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Bacterial biofilms are difficult to eradicate from surfaces using conventional antimicrobial interventions. High-throughput 96-well microplate methods are frequently used to cultivate bacterial biofilms for rapid antimicrobial susceptibility testing to calculate minimal biofilm eradication concentration (MBEC) values. Standard biofilm devices consist of polystyrene pegged-lids fitted to 96-well microplates and are ideal for measuring biofilm biomass and MBEC values, but these devices are limited by available peg surface area for biomass accumulation and cost. Here, we outline a protocol to use self-assembled polypropylene 96-well deep well PCR-plate pegged-lid device to grow Escherichia coli BW25113 and Pseudomonas aeruginosa PAO1 biofilms. A comparison of 24-hour biofilms formed on standard and deep well devices by each species using crystal violet biomass staining and MBEC determination assays are described. The larger surface area of deep well devices expectedly increased overall biofilm formation by both species 2-4-fold. P. aeruginosa formed significantly greater biomass/mm 2 on deep well pegs as compared to the standard device. E. coli had greater biomass/mm 2 on standard polystyrene devices as compared the deep well device. Biofilm eradication assays with disinfectants such as sodium hypochlorite (bleach) or benzalkonium chloride (BZK) showed that both compounds could eliminate E. coli and P. aeruginosa biofilms from both devices but at different MBEC values. BZK biofilm eradication resulted in variable E. coli MBEC values between devices, however, bleach demonstrated reproducible MBEC values for both species and devices. This study provides a high throughput deep well method for growing larger quantities of biofilms on polypropylene devices for downstream studies requiring higher amounts of static biofilm.

Generation of Greater Bacterial Biofilm Biomass using PCR-Plate Deep Well Microplate Devices