FLUORESCEIN-LABELED BETA-GLUCOSIDASE AS A BACTERIAL STAIN

• Main Authors: Pital, Abe, Janowitz, Sheldon L, Hudak, Charles E, Lewis, Evelyn E
• Format: Report
• Language: English
• Subjects: BACTERIA; BIOASSAY; BIOLOGICAL STAINS; CELL WALL; CELLS(BIOLOGY); Coatings, Colorants and Finishes; ELECTRON MICROSCOPY; ENZYMES; FLUORESCENCE; GLYCOSIDES; LABELED SUBSTANCES; MICROORGANISMS; MICROSCOPY; PREPARATION; SPORES; TISSUES(BIOLOGY); VISIBILITY

Beta-glucosidase labeled with fluorescein isothiocyanate was used as a simple staining reagent with selected gram-positive and gram-negative organisms. Staining appeared to be dependent on the presence of accessible glycosidic-type linkages as well as the partial inhibition of enzyme activity by high enzyme concentrations. Labeled beta-glucosidase separated into three major fractions upon Sephadex column passage to remove excess dye. These fractions differed in protein content and enzyme activity, but produced similar staining reactions. Extensive wall damage or lysis did not occur when stained cells were suspended in washing and mounting solutions. The apparent specificity of labeled enzyme for wall substance was determined by one-step and Alican Blue blocking reactions as well as by formation of cell wall lesions on prolonged staining with the enzyme. Spores and a majority of the gram-negative organisms tested were effectively stained after prior exposure to thioglycollic acid at 70 C. The present studies suggest that disulfide bonds may play an important role in maintaining the cellular integrity of some gram-negative species. Fluorescein- labeled beta-glucosidase appears to be potentially useful staining reagent for demonstrating in situ glycosidic-type linkages in the bacterial cell wall. A potential for staining tissues and cell lines may also exist.