A novel dual-staining method for cost-effective visualization and differentiation of microbial biofilms.

Microbial biofilms are intricate communities that pose significant challenges in clinical and microbiological settings due to their resistance to antibiotics and immune responses. Advanced microscopy techniques, such as scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), and fluorescence microscopy, are often employed to visualize and differentiate between these biofilms. However, these methods are not feasible in all laboratories because of their high cost and complexity. In contrast, simpler techniques like crystal violet and Congo red staining fail to differentiate bacterial cells from the biofilm matrix. This study introduces a novel dual-staining method using Maneval's stain for microbial biofilm detection and differentiation. This simple, cost-effective method requires only basic equipment and minimal reagents, making it suitable for routine use across various settings. We applied the dual-staining method to various microbial species, including Staphylococcus aureus, Enterococcus faecalis, Candida albicans, Escherichia coli, and Pseudomonas aeruginosa. When compared with the microtiter plate assay, results showed strong agreement, with the dual-staining method effectively differentiating between bacterial cells and the surrounding biofilm matrix, displaying a distinctive blue polysaccharide layer surrounding the magenta‒red bacterial cells. This technique offers a viable alternative to more expensive and complex biofilm detection methods, with potential applications in clinical diagnostics and biofilm research.