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 (ClO2), instead of traditional hazardous fixatives for the cross-linking of cellular proteins to improve immunofluorescence staining of bacteria. The concentration of ClO2 needed for 100% fixation is 50 μg ml−1, which is much lower than that of traditional fixatives (1000–10000 μg ml−1). The ClO2 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 ClO2 fixation on the staining. The results demonstrated that ClO2 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, ClO2 has potential practical applications in immunofluorescence staining and fluorescence in situ hybridization for single bacteria/cell analysis.

Rapid and Sensitive Quantification of Bacterial Viability Using Ratiometric Fluorescence Sensing

“…E. coli O157:H7 was cultured in LB broth as described by Sun et al The bacteria were harvested at an optical density (OD 600 ) of 1–1.2 by centrifugation at 5000 rpm for 5 min. The cells were washed three times with drink water and resuspended to an OD 600 of 10 (containing approximately 1 × 10 10 cfu/mL).…”

Bridge-DNA synthesis triggered by an allosteric aptamer for the colorimetric detection of pathogenic bacteria

“…For comparison of the DNA synthesis efficiency between different lysis agents, 50 mL bacterial suspension (10 7 cfu mL −1 ) was mixed with 50 mL lysis agent, and the mixture was incubated for 10 min. The concentrations of the lysis agents were 1% for Triton-X100, 100 mg mL −1 for lysozyme, and 0.2 M for NaOH, as described by Sun et al 23…”

On-bead DNA synthesis triggered by allosteric probe for detection of carcinoembryonic antigen

“…The suspension was incubated for 20 min at room temperature. After magnetic separation, the magnetic beads were resuspended with 500 μL TAE buffer and subjected to flow cytometry analysis as described by Sun et al [ 16 ]. The magnetic beads with DNA on its surface were further verified via laser-scanning confocal microscopy and transmission electron microscopy (FEI Tecnai G2 F30).…”