Demonstration of bacterial cells and glycocalyx in biofilms on human tonsils.

OBJECTIVES To demonstrate mucosal biofilms in human tissue by direct visualization of bacteria and glycocalyx using confocal laser scanning microscopy with double fluorescent staining on tonsils and to compare the findings with the results of scanning electron microscopy analysis. DESIGN Prospective study. SETTING Tertiary university-based referral center. PATIENTS Twenty-four tonsils were obtained from children with chronic or recurrent tonsillitis. INTERVENTIONS Tonsils were prepared for analysis by scanning electronic microscopy and by confocal laser scanning microscopy. MAIN OUTCOME MEASURES Double fluorescent staining for confocal laser scanning microscopy consisted of propidium iodide staining to detect bacterial cells and fluorescein isothiocyanate concanavalin A staining to detect the glycocalyx matrix. Images were analyzed for characteristic biofilm morphologic features by 3 investigators who evaluated the images independently in a blinded retrospective manner. Consensus of all observers was required to demonstrate the presence of a biofilm in a specimen. RESULTS Findings from analyses using scanning electronic microscopy suggested the presence of biofilm formations on tonsils by showing bacterial cells in microcolonies. Double-staining technique using confocal laser scanning microscopy showed bacterial cells and the glycocalyx matrix, providing visual evidence for the presence of biofilms on tonsils. CONCLUSION Using a novel visualization approach in single sections of human mucosal tissue, the presence of biofilms was demonstrated on tonsils in most (17/24 [70.8%]) patients with tonsillitis.