Novel Method for Quantitative Estimation of Biofilms.

Area of Science:

• Microbiology
• Biotechnology

Background:

• Biofilms protect bacteria but are sensitive to traditional quantification methods like the crystal violet (CV) assay.
• The CV assay's washing and staining steps can damage delicate biofilms, particularly pellicles formed at the liquid-air interface, common in mycobacteria.

Purpose of the Study:

• To develop a modified protocol for accurate biofilm quantification, preventing the loss of sensitive biofilm layers.
• To introduce a new method, the Syal method, for estimating early-phase and aerobic biofilms formed at the liquid-air interface.

Main Methods:

• A novel protocol involves removing media with a syringe, drying the biofilm, and dissolving it with tetrahydrofuran (THF) for absorbance measurement at 595 nm.
• This method avoids traditional washing and staining steps, crucial for preserving pellicle biofilms.
• The protocol was validated using *E. coli*, *B. subtilis*, and *M. smegmatis*, with results compared to the standard CV assay.

Main Results:

• The Syal method precisely quantified biofilms in *E. coli*, *B. subtilis*, and *M. smegmatis*.
• The protocol effectively estimated early-phase and aerobic pellicle biofilms, which are often lost in conventional assays.
• The inhibitory effects of isoniazid on *M. smegmatis* biofilm formation were accurately quantified using the Syal method.

Conclusions:

• The Syal method offers a more robust and accurate approach for quantifying bacterial biofilms, especially those formed at the liquid-air interface.
• This technique minimizes sample loss and preserves biofilm integrity, making it suitable for studying early-stage biofilms and the effects of antimicrobial agents.