Limit of resolution of flow cytometry for the detection of selected bacterial species

The enumeration of bacteria in dental plaque samples is a vital but time-consuming procedure that uses standard cultural methods. Flow cytometry has proven to be a useful tool for the analysis of eukaryotic cells. In the present investigation, the utility of this technology for the enumeration of bacteria in mixtures was explored. Rabbit antisera were produced against the putative periodontal pathogens A. actinomycetemcomitans, B. intermedius, B. gingivalis, E. corrodens, W. recta, B. forsythus, as well as the frequently isolated supragingival species S. sanguis. Cross-reactive antibodies were removed by absorption, and the specificity of each antiserum was confirmed by being tested against a panel of 235 oral microbial strains (79 genera; 94 species) by means of ELISA. Conditions were established for the indirect immunofluorescent labeling of cells without agglutination with use of a goat anti-rabbit Ig-FITC second antibody. When an internal bead standard was used, it was found that unstained bacteria were enumerated by light-scattering parameters with poor efficiency (less than 3%). However, cells exposed to FITC either in the presence of specific or non-specific first antibody were enumerated with high efficiency (102.6 +/- 29.3%), indicating that a small amount of non-specific binding of fluorochrome facilitates bacterial detection. Clear discrimination between specifically- and non-specifically-stained bacteria was achieved with all six rabbit antisera. Mixtures of known composition were made (1) with pure cultures or (2) with a known species and supragingival plaque devoid of that species by culture. The results from both approaches with various species combinations revealed that the limit of resolution for accurate quantitation of a selected species was approximately 5%, although specific organisms could be detected qualitatively when present at approximately 1%.