Demonstration of biofilm in infections crystalline keratopathy using ruthenium red and electron microscopy

Objective: Bacterial biofilm formation has been implicated in the pathogenesis of infectious crystalline keratopathy. Biofilm cannot be visualized by electron microscopy without the addition of a fixative that stabilizes the polysaccharide-rich bacterial extracellular matrix that surrounds the bacterial colonies in a biofilm. We used ruthenium red as a fixative to evaluate corneal biopsy specimens for the presence of bacterial biofilm in three cases of infectious crystalline keratopathy (ICK) and five cases of chronic microbial keratitis without crystalline changes. Design: Case series with clinicopathologic correlation. Participants: Eight patients underwent corneal biopsy or therapeutic keratoplasty as part of their management for chronic unresponsive microbial keratitis. Methods: The corneal specimens removed were trisected for microbiology, pathology, and transmission electron microscopy (TEM). The TEM specimens were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer with 0.05% ruthenium red. Main Outcome Measures: Demonstration of bacterial biofilm with TEM. Results: TEM demonstrated organisms with a surrounding extracellular matrix consistent with a bacterial biofilm in the three cases of ICK but not in the five other cases of chronic microbial keratitis. Conclusions: The presence of biofilm in ICK can be demonstrated with TEM with appropriate fixation techniques that stabilize the bacterial extracellular matrix. Biofilm stains intensely with periodic acid-Schiff because of the polysaccharide-rich extracellular matrix and weakly with Gram stain because of the high proportion of nonviable organisms. Biofilm formation occurs in ICK but probably not in chronic bacterial keratitis without crystalline changes. Secretion of an extracellular matrix by bacteria to form a biofilm is a response to a nutrient-deprived environment in which growth and replication is depressed. The extracellular matrix of the biofilm may mask bacterial antigens, explaining the relative lack of inflammatory response in these infections. It may also be one of the mechanisms explaining the resistance to in vivo antimicrobial therapy when in vitro sensitivities have been proven. Ophthalmology 2001;108:1088-1092 (C) 2001 by the American Academy of Ophthalmology.