Background

The discovery of biofilms dates back to the Dutch microscopist Antonie van Leeuwenhoek, who first observed them on tooth surfaces [1]. They were later studied by J.W. Costerton, who elucidated their role in persistent infections and antibiotic resistance [2]. These microbial biofilms are organized communities of microorganisms that adhere to surfaces and are encased in an extracellular matrix (ECM). The biofilm life cycle involves attachment, irreversible adhesion, maturation into microcolonies, and dispersion. The ECM, composed of polysaccharides, proteins, DNA, and lipids, acts as a protective shield, making biofilms highly resistant to antimicrobial treatments and immune responses [3].

Understanding biofilms is crucial for developing effective detection methods and treatments. Biofilm detection is crucial in clinical diagnosis, as biofilms contribute to antibiotic resistance and can lead to chronic conditions [4]. In the food industry, detecting biofilms on processing equipment is vital for preventing contamination and spoilage [5]. Monitoring biofilm growth in industrial water systems, cooling towers, and pipelines is essential to maintain operational efficiency [6]. Biofilm detection is important for studying microbial communities in natural environments, such as streams, soil, and marine ecosystems [7]. Moreover, pharmaceutical companies rely on biofilm detection methods to develop and assess the effectiveness of antibiofilm treatments [8].

Advanced microscopy techniques, such as scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), and fluorescence microscopy, are commonly used to visualize and differentiate biofilms. However, these methods are not feasible in all laboratories because of their high cost and complexity [9]. In contrast, simpler methods like crystal violet and Congo red staining fail to distinguish bacterial cells from the biofilm matrix [10].

This study introduces a novel dual-staining method combining Maneval’s stain and Congo red stain to visualize and differentiate microbial biofilms. Maneval’s stain, initially developed by Edmond Maneval in 1941 for visualizing bacterial capsules, has been adapted in this study for biofilm detection [11]. This simple, cost-effective method requires only basic equipment and minimal reagents, making it suitable for routine laboratory use. The dual-staining method effectively differentiates bacterial cells and biofilm matrix, displaying a distinctive blue polysaccharide layer surrounding the magenta-red bacterial cells. Congo red interacts with the hydrophobic regions of polysaccharides through hydrogen bonds [12], initially staining them red. Upon adding Maneval’s stain, the pH becomes acidic, causing a shift to blue due to the protonation of the azo group [13]. Simultaneously, acid fuchsin binds to the negatively charged bacterial surfaces, creating a magenta-red coloration.

We applied this technique to various microbial species, including Gram-positive bacteria with a thick peptidoglycan layer (Staphylococcus aureus and Enterococcus faecalis), Gram-negative bacteria distinguished by their outer membrane (Escherichia coli and Pseudomonas aeruginosa), and fungi (Candida albicans), which differ significantly in the presence of chitin, glucan, and mannoproteins. While this method does not provide the detailed structural resolution of high-magnification techniques like SEM, it offers a practical and accessible alternative for distinguishing biofilm matrix and bacterial cells.