Immunofluorescence analysis of bacillus spores and vegetative cells by flow cytometry

A commercially available flow cytometer (Cytofluorograf) was used for the immunofluorescence (IF) analysis of spores of Bacillus anthracis, Bacillus cereus, and Bacillus subtilis, using fluorescein-labelled antispore conjugates. The cytometer was modified to allow analysis of known numbers of bacteria. In attempting to identify the region of the cytometer fluorescence histogram associated with the presence of stained spores, evidence was produced for signal components due to antibody bound to extracellular antigens. Under some reaction conditions these components were large enough partially or completely to obscure the fluorescence distribution imputed to the spores. The results support the hypothesis that the fluorescence histogram for a bacterial suspension can be modified by subtracting the histogram of the cell-free centrifugation supernatant to provide a fluorescence distribution more representative of the bacteria themselves. Spore and vegetative forms of B. anthracis could be differentiated in the flow IF assay by comparing the peak and area (integral) values of the photomultiplier output. The 90 degrees scatter histograms of the stained spores and their cell-free supernatants were so alike in shape that it was not possible to ascribe a unique peak to the spores themselves. Overall, these results confirm the considerable potential of flow cytometry for the rapid and quantitative IF assay of bacterial populations.