Standardizing biofilm quantification: harmonizing crystal violet absorbance measurements through extinction...

Area of Science:

• Microbiology
• Biotechnology
• Analytical Chemistry

Background:

• Biofilm quantification is critical across various fields, yet inconsistencies arise from varying measurement wavelengths.
• The microtiter plate assay with crystal violet (CV) is widely used but susceptible to wavelength-dependent discrepancies.
• Differences in CV extinction coefficients at different wavelengths lead to varied biofilm categorization.

Purpose of the Study:

• To establish standardized wavelengths for accurate CV-based biofilm quantification.
• To develop a method for harmonizing biofilm categorization across studies using different wavelengths.
• To enable consistent comparison of biofilm research data for advancing therapeutic strategies.

Main Methods:

• Determined the absorbance maximum (λmax) for crystal violet resolubilized in 33% acetic acid (585 nm) and 94-100% ethanol (580 nm).
• Developed a method to adjust biofilm categorization thresholds based on CV extinction coefficient ratios.
• Validated the adjusted thresholds using E. coli and S. aureus biofilms.

Main Results:

• Identified specific λmax values for CV in common resolubilizing agents, crucial for accurate absorbance measurements.
• The developed adjustment method ensures consistent biofilm categorization regardless of the wavelength used.
• Validation confirmed that adjusted thresholds yield results comparable to those obtained at λmax.

Conclusions:

• Standardized biofilm quantification using adjusted CV thresholds enhances data consistency and comparability.
• This approach facilitates the integration of existing biofilm research data for developing new treatments.
• Accurate and reproducible biofilm quantification is essential for combating biofilm-related infections and advancing research.