Inconsistent Findings Between Crystal Violet and Congo Red Methods on Biofilms with Comparative Sugar Supplementation

In recent years, the World Health Organization has highlighted biofilm-derived multidrug-resistant bacteria as a critical threat to both global health and the environment. Although various testing methods are available, crystal violet and Congo Red methods are among the most frequently used methods for biofilm detection in the literature. However, inconsistent findings across studies have raised concerns. To address these issues and offer valuable insights for researchers in the field, this study used clinically relevant standard bacterial strains (ATCC or NCTC strains) to perform biofilm assays with both methods and compare the results. To investigate the effect of different sugar sources on biofilm formation, various sugar substrates were also examined using both biofilm methods under controlled culture conditions in this study. When the results were evaluated, significant differences were found between the two methods closely related to sugar content. Of the 22 strains tested, 17 (77%) showed different biofilm results in a sugar-free environment. Similar inconsistencies were also observed with glucose (32% of strains) and sucrose (50% of strains). With fructose, some strains (<i>P. aeruginosa</i> strains, <i>E. faecalis</i> ATCC 29212, <i>K. pneumoniae</i> High Level ESBL, <i>K. pneumoniae</i> BAA 1706, <i>A. baumannii</i> BAA 747) were negative with Congo Red and positive with crystal violet, while others (<i>S. mutans</i> ATCC 25175, <i>E. coli</i> NCTC 13846, <i>E. coli</i> ATCC 25922) gave the opposite results. Fructose caused inconsistencies in approximately 45% of strains. The highest agreement between the two methods (approximately 68%) was observed when glucose was used as the carbon source.