Studies show that species-specific immunofluorescent antibody labeling of bacteria provides an alternative method to complex anaerobic culturing for the quantitative identification specific periodontopathogenic bacteria. Analysis of bacterial fluorescent antibody labeling with fluorescent microscopy is laborious and yields subjective data on fluorescence intensity for only several hundred cells in each sample. Flow cytometers measure individual cell fluorescence as cells flow past a laser with sensitive optics and electronics at a rate of thousands of cells per second. Recent reports suggest that flow cytometry provides an alternative way to measure bacterial cell fluorescence of large numbers of bacteria. The use of lasers to excite fluorescence and sensitive photomultiplier tubes to measure fluorescence by flow cytometers permits use of lower titers of labeling antibody which results in a reduction of non-specific labeling. This study will use monoclonal antibodies that label all tested strains of Actinobacillus actinomycetemcomitans (Aa) to define an immunofluorescent labeling protocol which optimizes flow cytometric detection of Aa cells within bacterial cell mixtures. Indirect and direct immunofluorescent labeling techniques will be compared using FITC conjugated antibodies. The sensitivity and specificity of flow cytometric analyses using optimized flow cytometric technique for detecting Aa cells within mixtures will be compared to analysis of the same mixtures using fluorescence microscopy. Effects that controlled variables in flow cytometric analysis techniques or in bacteria cells have on measuring Aa cells within mixtures will be defined. Bacteria cell variables to be studied include cell preservation techniques, cell concentration or size, and cell growth cycle. Use of nucleic acid staining and addition of an internal bead standard will be tested to see if either improves analyses. Finally, flow cytometric analyses will be used to assess alterations in Aa cell morphology, DNA or protein content produced by in-vitro antibiotic exposure. These cell alterations will be compared to changes in numbers of colony forming units. The techniques defined by this study will provide new investigative tools to measure Aa infection in larger populations.
