Rapid LED-based fluorescence microscopy distinguishes between live and dead bacteria in oral clinical samples.

Despite the existence of several methods for the diagnosis of oral infectious diseases, few rapid and quantitative methods exist for discriminating between live and dead bacterial cells in oral clinical samples. In this study, we characterized a light-emitting diode (LED) fluorescence microscopic technique for quantifying live and dead oral bacterial cells stained with 4',6'-deamidino-2-phenyllindole and propidium iodide. Four bacterial strains representative of the human oral microflora were used in this study. In addition, saliva and subgingival fluid specimens were collected from healthy volunteers. Saliva was obtained from the donors without stimulation, whereas subgingival fluid was obtained by inserting a sterile endodontic paper point into the subgingival sites of the first molar. The samples were cultured on agar plates and subjected to LED microscopy. The correlations between both methods were analyzed. The number of live bacterial cells as determined by LED-based fluorescence microscopy and standard colony counts on agar plates correlated well for the known oral bacterial strains and bacterial cells in the clinical specimens. The LED illumination method characterized in this study can be used for the rapid enumeration of living and dead cells. However, to show specificity, this method requires further innovations.

The identification of genes specific to Prevotella intermedia and Prevotella nigrescens using genomic subtractive hybridization.

Prevotella intermedia and Prevotella nigrescens, which are often isolated from periodontal sites, were once considered two different genotypes of P. intermedia. Although the genomic sequence of P. intermedia was determined recently, little is known about the genetic differences between P. intermedia and P. nigrescens. The subtractive hybridization technique is a powerful method for generating a set of DNA fragments differing between two closely related bacterial strains or species. We used subtractive hybridization to identify the DNA regions specific to P. intermedia ATCC 25611 and P. nigrescens ATCC 25261. Using this method, four P. intermedia ATCC 25611-specific and three P. nigrescens ATCC 25261-specific regions were determined. From the species-specific regions, insertion sequence (IS) elements were isolated for P. intermedia. IS elements play an important role in the pathogenicity of bacteria. For the P. intermedia-specific regions, the genes adenine-specific DNA-methyltransferase and 8-amino-7-oxononanoate synthase were isolated. The P. nigrescens-specific region contained a Flavobacterium psychrophilum SprA homologue, a cell-surface protein involved in gliding motility, Prevotella melaninogenica ATCC 25845 glutathione peroxide, and Porphyromonas gingivalis ATCC 33277 leucyl-tRNA synthetase. The results demonstrate that the subtractive hybridization technique was useful for distinguishing between the two closely related species. Furthermore, this technique will contribute to our understanding of the virulence of these species.