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

Abstract 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 2 ), instead of traditional hazardous fixatives for the cross-linking of cellular proteins to improve immunofluorescence staining of bacteria. The concentration of ClO 2 needed for 100% fixation is 50 μ g ml −1 , which is much lower than that of traditional fixatives (1000–10000 μ g ml −1 ). The ClO 2 mediated 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 using E. coli O157: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 ClO 2 fixation on the staining. The results demonstrated that ClO 2 fixation 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, ClO 2 has potential practical applications in immunofluorescence staining and fluorescence in situ hybridization for single bacteria/cell analysis.