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.

Antimicrobial Resistance, Genetic Lineages, and Biofilm Formation in Pseudomonas aeruginosa Isolated from Human Infections: An Emerging One Health Concern

“…In this study, all clinical isolates of P. aeruginosa were capable of biofilm formation, and the microtiter assay proved to be a reliable and consistent method for screening and detecting biofilm-producing bacteria. This conclusion is supported by studies conducted by other authors who utilized the same methods for biofilm evaluation [ 52 , 53 , 54 ]. These studies also affirm the accuracy and quantitative capabilities of this technique, making it a recommended approach for assessing biofilm formation in clinical isolates of P. aeruginosa [ 22 , 55 ].…”

Influence of the Environment on Biofilm Formation Candida albicans of Vulvovaginal Candidiasis Isolate Patient

“…Then, growth media is needed to study the properties of a microorganism. 15,23 In addition to incubation time considerations, environmental parameters affecting biofilm formation include incubation temperature, isolate amount, and crystal violet solvent. Compared to biomass 10 8 CFU/mL, biofilm formation was higher on biomass 10 7 CFU/mL, resulting in a high OD value.…”

Comparative <i>in vitro</i> activity of various antibiotic against planktonic and biofilm and the gene expression profile in <i>Pseudomonas aeruginosa</i>

“…Bacterial inhibition increases with antibiotic concentration and incubation time. A biofilm is an aggregation of one or more species of microorganisms adhered to a surface, as compared to planktonic bacteria, which exist as individual organisms [20] , [51] . Biofilm-forming bacteria are reported to be 100–1000 times more resistant to antibiotics than planktonic bacteria [13] , [14] .…”