Detection of Biofilm Formation: Evaluation of Congo Red Agar and Crystal Violet Staining Method

Abstract

This study focuses on the detection of biofilm formation using Congo Red Agar (CRA) and Crystal Violet Staining (CVS) methods. Biofilms are complex microbial communities formed by bacteria adhering to surfaces, surrounded by extracellular polymeric substances, which provide protection against external stressors. Biofilm formation plays a critical role in infection control and combating antibiotic resistance. In this research, the biofilm production capacities of 24 standard bacterial strains, 11 multidrug-resistant (MDR) strains, 9 foodborne isolates, and 24 clinical isolates were evaluated. The CRA method qualitatively identifies biofilm formation through colony morphology, while the CVS assay quantitatively measures biofilm production. The results demonstrated that 66.18% of the 68 microorganisms tested using CRA were biofilm positive, with Gram-positive bacteria generally exhibiting stronger biofilm production. The CVS assay further provided more precise measurements of biofilm production, identifying strong biofilm producers such as Enterococcus faecalis ATCC 29212, Pseudomonas fluorescens P1, and Staphylococcus aureus ATCC 25923. The study found that while CRA is effective in detecting strong biofilm producers, it has limitations in identifying weaker biofilm producers. In contrast, the CVS assay proved to be more sensitive and reliable. The findings also highlight that variations in biofilm production capacity emphasize the significance of biofilm management in clinical infections and the challenges encountered in treatment.

Keywords

• Biofilm
• Congo Red Agar
• Crystal Violet Staining
• Clinical Isolates
• Pathogen bacteria

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