Using a safe and effective fixative to improve the immunofluorescence staining of bacteria

The emerging and development of green chemistry has once again drawn the researchers' attention to eliminating the use and generation of hazardous materials. Here we report the use of a safe and effective fixative, chlorine dioxide (ClO₂), instead of traditional hazardous fixatives for the cross-linking of cellular proteins to improve immunofluorescence staining of bacteria. The concentration of ClO₂needed for 100% fixation is 50μg ml^-1, which is much lower than that of traditional fixatives (1000-10000μg ml^-1). The ClO₂mediated cross-linking can preserve the integrity of bacterial cells and prevent cell loss through lysis. Meanwhile, lysozyme can permeabilize the bacterial cells, allowing the labelled antibodies to diffuse to their intracellular target molecules. By usingE. coliO157:H7/RP4 as a gram-negative bacteria model, immunofluorescence staining assays for both intracellular protein and surface polysaccharide were carried out to investigate the effect of ClO₂fixation on the staining. The results demonstrated that ClO₂fixation could prevent the target antigens from cracking off the bacteria without damage on the interaction between the antibodies and antigens (either for polysaccharide or protein). As a safe and effective fixative, ClO₂has potential practical applications in immunofluorescence staining and fluorescencein situhybridization for single bacteria/cell analysis.