Epifluorescence Microscopy with Image Analysis as a Promising Method for Multispecies Biofilm Quantification

Area of Science:

• Microbiology
• Microscopy
• Biofilm research

Background:

• Biofilm quantification is crucial for understanding microbial communities.
• Crystal violet (CV) staining is a common but potentially limited method.
• Developing alternative, rapid quantification techniques is needed.

Purpose of the Study:

• To evaluate epifluorescence microscopy with image analysis as a biofilm quantification method.
• To compare its performance against crystal violet (CV) staining.
• To assess its accuracy across different biofilm densities and compositions.

Main Methods:

• Generation of multispecies biofilms using varied bacterial assemblages and inoculation sequences.
• Application of epifluorescence microscopy with image analysis for biofilm quantification.
• Comparison with crystal violet (CV) staining for quantitative assessment and variation analysis.

Main Results:

• Both methods showed comparable quantitative variation in biofilms with strong positive relationships (R² ≥ 0.7) for higher colonization levels.
• Microscopy and CV staining exhibited similar coefficients of variation.
• Microscopy was less accurate for biofilms with low surface colonization (≤ 3.7%).

Conclusions:

• Epifluorescence microscopy with image analysis is a promising, rapid method for estimating multispecies biofilm quantity.
• The method is comparable to CV staining for biofilms with significant surface area coverage.
• Microscopy has limitations in quantifying biofilms below a certain colonization threshold.