Evaluation of the effect of different growth media and incubation time on the suitability of biofilm formation by Pseudomonas aeruginosa and Streptococcus pyogenes

Abstract

Biofilm formation has different stages and can be classified based on the bacterial strain, culture vessel, and the method employed. Biofilm formation is carried out in culture vessels to represent mode of infection in humans. Microbial concentration, growth medium, supplement, and incubation time are key factors to successfully form biofilm in a culture vessel. This study aimed to identify the optimum conditions for biofilm formation in a 96-well plate by culturing Pseudomonas aeruginosa and Streptococcus pyogenes. We utilized the infectious and pathogenic bacteria, P. aeruginosa and S. pyogenes strains. These bacteria were cultured in Mueller–Hinton Broth (MHB) and Tryptic Soy Broth (TSB) at two different optical densities OD₆₀₀ (0.05) and OD₆₀₀ (0.1). After a certain incubation time, the formed biofilm was stained by using 0.1% crystal violet. The stained bacteria were disaggregated and measured using a microplate reader. Biofilm was then classified based on bacterial adherence to the plate. Our results showed that P. aeruginosa and S. pyogenes biofilms were strongly formed on days 3 and 5 in MHB and TSB, respectively. However, the strongest biofilm formation was seen on day 3 after P. aeruginosa being incubated in MHB at OD₆₀₀ (0.1) and after S. pyogenes being incubated in MHB at OD₆₀₀ (0.05). Biofilm formation is ranged between weak, moderate, and strong in accordance with the density of bacterial adhesion. P. aeruginosa and S. pyogenes biofilms were optimized at specific OD₆₀₀ (0.1 and 0.05, respectively) for 3 days’ cultivation in MHB.