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 (ClO), instead of traditional hazardous fixatives for the cross-linking of cellular proteins to improve immunofluorescence staining of bacteria. The concentration of ClOneeded for 100% fixation is 50g ml, which is much lower than that of traditional fixatives (1000-10000g ml). The ClOmediated 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 usingO157: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 ClOfixation on the staining. The results demonstrated that ClOfixation 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, ClOhas potential practical applications in immunofluorescence staining and fluorescencehybridization for single bacteria/cell analysis.