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Inconsistent Findings Between Crystal Violet and Congo Red Methods on Biofilms with Comparative Sugar
Nihan Unubol1,2, Meltem Ayaş1,2, Neval Yurttutan Uyar2,3
1Department of Medical Laboratory Techniques, Vocational School of Health Services, Acibadem Mehmet Ali Aydinlar University, Istanbul 34752, Türkiye.
Microorganisms
|January 28, 2026
Summary
Crystal violet and Congo Red biofilm assays yield inconsistent results, especially in sugar-free conditions. Glucose as a carbon source improved agreement between these common biofilm detection methods.
Area of Science:
- Microbiology
- Molecular Biology
- Biotechnology
Background:
- Biofilm-derived multidrug-resistant bacteria pose a significant global health and environmental threat.
- Crystal violet and Congo Red assays are widely used for biofilm detection but yield inconsistent results.
- Standardized methods are needed for reliable biofilm quantification and characterization.
Purpose of the Study:
- To compare the efficacy of crystal violet and Congo Red methods for biofilm detection.
- To investigate the impact of different sugar sources on biofilm formation.
- To identify factors contributing to inconsistencies in biofilm assay results.
Main Methods:
- Biofilm assays were performed using clinically relevant bacterial strains (ATCC or NCTC).
- Crystal violet and Congo Red methods were employed for biofilm quantification.
- Experiments were conducted with various sugar substrates and in a sugar-free environment.
Main Results:
- Significant differences in biofilm detection were observed between the two methods, influenced by sugar content.
- Seventy-seven percent of strains showed differing results in a sugar-free environment.
- Glucose as a carbon source yielded the highest agreement (68%) between crystal violet and Congo Red assays.
Conclusions:
- The choice of carbon source critically affects biofilm detection consistency between crystal violet and Congo Red methods.
- Glucose appears to be a more reliable carbon source for standardizing biofilm assays.
- Further research is needed to optimize biofilm detection protocols for multidrug-resistant bacteria.
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