A staining procedure was developed for screeningClostridium botulinumtype A (tox+) cells fixed (smear) on glass slides. the method is based on reaction between cells and an immunoenzyme‐conjugate consisting of antibody against type A toxin linked to horseradish peroxidase (HRPO) enzyme. Fixed smears were stained for perioxidase activity (30 min), using one drop of 3,3′‐diaminobenzidine‐hydrogen peroxide solution (pH 7.6) as oxidizable substrate, followed by washing, drying and microscopic examination (1000 X). the cell‐walls of vegetativeC. botulinumtype A appeared reddish brown and periphery of spores (if present), stained dark brown. However,C. botulinumtype B (tox+) cross‐reacted (faint brown) with the same antibody‐HRPO‐conjugate. Specificity and sensitivity of this procedure compared well with the immunodiffusion method of Ferreiraet al.(1981, 1983). Factors affecting cell‐staining were: fixation time and type used, incubation time of antibody‐HRPO‐conjugate with cells, growth medium, culture age, and crowding of cells in smears. Ethanol fixation (5 min) of logarithmic and/or stationary phase cells and 30 min incubation with antibody‐HRPO‐conjugate were optimal and yielded the most appropriate procedure for detecting cells containi
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