Evaluation of a microfluorometer in immunofluorescence assays of individual spores of Bacillus anthracis and Bacillus cereus.

A microfluorometer was constructed by modifying a standard fluorescent microscope with a fibre optic eyepiece and a simple photometric system. It was evaluated in direct immunofluorescence assays of Bacillus anthracis and Bacillus cereus spores immobilised on multispot microscope slides. From measurements of stable fluorescent crystals comparable in size to the spores, it was inferred that the fluorescence intensity of a stained bacterium could be measured with good precision. Fluctuation of a exciting light from a mercury vapour lamp did not contribute significantly to the distribution of fluorescence measurements obtained when samples of 20 spores were assessed. Attempts to correlate spore size with fluorescence intensity suggest that spore fluorescence does not increase in a 1 : 1 ratio with surface area; it is therefore possible that the density of antigenic sites on the surface decreases with increasing spore size. It is concluded that differences in the observed fluorescence of individual spores truly reflect differences in fluorescent antibody binding, but the relative contribution of antigenic variability and of artefacts of the staining procedure remain unknown.