Determination of optimum incubation time for formation of Pseudomonas aeruginosa and Streptococcus pyogenes biofilms in microtiter plate

Abstract

Abstract Pseudomonas aeruginosa and Streptococcus pyogenes are the most common pathogens to humans and are able to form a biofilm following ineffective precautionary approach. Biofilm is defined as a surface-attached community of bacterium embedded in an extracellular matrix which leads to tremendous problems in the environment, among humans and animals. This study aims to investigate the ability of P. aeruginosa and S. pyogenes to form biofilms in 96-well plate before further study in antibiofilm will be done. Initially, the 96-well plate was added with 100 μl of overnight P. aeruginosa culture with optical density (OD) 0.1 and S. pyogenes culture with OD 0.05. The cultures were incubated for 7 days at 37 °C to justify the formation of biofilm. Subsequently, stained blue biofilm was detached from the plate by using 95% ethanol. Biofilms were finally measured using a micro plate reader at 570 nm and were classified based on the adherence strength formula. P. aeruginosa and S. pyogenes biofilms strongly adhered to the plates on days three, four, five and six. Interestingly on day three, biofilms showed the highest formation. However, moderate biofilm formation onto the plates by both P. aeruginosa and S. pyogenes were observed on day two, but non-adherence was observed on days one and seven. Day three is the optimum cultivation period for P. aeruginosa and S. pyogenes to switch into a strong biofilm in microtiter plate and could be beneficial for antibiofilm experiments.

Keywords

• Biofilm
• Pseudomonas aeruginosa
• Streptococcus pyogenes