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

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Abstract

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.

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