Standardization and Comparative Evaluation of in vitro Methods for Bacterial Biofilm Formation

Abstract

Biofilms are communities of microorganisms embedded in an extracellular matrix, which enhance their survival, virulence, and resistance to antimicrobial treatments. Due to their importance in clinical and environmental settings, effective in vitro biofilm models are crucial for studying biofilm biology and assessing antibiofilm strategies.

This study is focussed on standardizing and comparing various in vitro methods for biofilm formation using six bacterial strains: Escherichia coli MTCC 1687, Klebsiella pneumoniae MTCC 432, Staphylococcus aureus MTCC 3160, Staphylococcus epidermidis MTCC 3615, Pseudomonas aeruginosa MTCC 2453, and Bacillus subtilis MTCC 2423. Biofilm formation was evaluated through qualitative, semi-quantitative, and quantitative methods, including the Congo red agar assay, tube method with optimized inoculum density, biofilm formation on glass slides and coverslips using tube, plate, and 6 well-plate methods, and the 96-well microtiter plate assay. The Congo red assay detected exopolysaccharide production in E. coli, K. pneumoniae, S. aureus, and P. aeruginosa, while S. epidermidis and B. subtilis did not exhibit the characteristic blackening. The optimal inoculum density for biofilm formation differed among species, highlighting the need for culture specific standardization. Plate-based methods consistently facilitated stronger and more uniform biofilm formation compared to tube-based systems. Among the methods tested, the 6-well plate coverslip method and the 96-well microtiter plate assay generated dense, reproducible biofilms within 24–48 hours, with P. aeruginosa showing the highest biofilm-forming capability. Statistical analyses using one-way and two-way ANOVA revealed significant differences between methods and among bacterial strains (p < 0.0001). In summary, this study offers standardized and reproducible in vitro biofilm models that can be effectively used for comparative biofilm research and for testing antibiofilm and antimicrobial agents.