Department of Medical Microbiology and Infectious Diseases, University of Manitoba.
Department of Medical Microbiology and Infectious Diseases, University of Manitoba; National Microbiology Laboratory, Public Health Agency of Canada.
J Vis Exp. 2022 Apr 22(182). doi: 10.3791/63069.
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 on deep well pegs as compared to the standard device. E. coli had greater biomass/mm 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.
细菌生物膜很难用常规抗菌干预从表面消除。高通量 96 孔微孔板方法常用于培养细菌生物膜，以便快速进行抗菌药敏试验，计算最小生物膜清除浓度 (MBEC) 值。标准生物膜装置由带有销钉的聚苯乙烯盖子组成，适用于 96 孔微孔板，非常适合测量生物膜生物量和 MBEC 值，但这些装置受到用于生物量积累的可用销钉表面积和成本的限制。在这里，我们概述了一种使用自组装聚丙烯 96 孔深孔 PCR 板带钉盖装置培养大肠杆菌 BW25113 和铜绿假单胞菌 PAO1 生物膜的方案。描述了每种生物在标准和深孔装置上形成的 24 小时生物膜通过结晶紫生物量染色和 MBEC 测定分析进行比较。深孔装置的较大表面积预计会使两种生物的总生物膜形成增加 2-4 倍。与标准设备相比，深孔钉上的铜绿假单胞菌形成的生物量/mm 明显更大。与深孔设备相比，大肠杆菌在标准聚苯乙烯设备上具有更大的生物量/mm。消毒剂（如次氯酸钠（漂白剂）或苯扎氯铵（BZK））的生物膜清除试验表明，这两种化合物都可以从两种设备中消除大肠杆菌和铜绿假单胞菌生物膜，但所需的 MBEC 值不同。BZK 生物膜清除导致两种设备之间大肠杆菌的 MBEC 值变化，但漂白剂可提供两种生物和设备的可重复的 MBEC 值。本研究提供了一种高通量深孔方法，用于在聚丙烯装置上培养更多数量的生物膜，用于需要更高量静态生物膜的下游研究。
