A method for the detection and quantification of bacteria in human carious dentine using fluorescent in situ hybridisation

Abstract

Objectives: Previous studies have evaluated bacterial numbers in carious dentine using conventional culturing methods, capable of detecting only a proportion of the total bacteria present within lesions. The aim of this study was to detect and enumerate the total bacterial population present in carious human dentine by means of fluorescent in situ hybridisation.

Method: Five freshly extracted carious molars were sequentially hand excavated under sterile conditions through four levels in the lesions. Replicate samples were probed with a rhodamine-tagged, 16S rRNA-directed probe (EUB338), specific for the bacterial domain. Two of the five original samples were examined using fluorescence microscopy and by using a systematic visual counting strategy; direct enumeration of the bacterial population in carious dentine was performed.

Results: In the superficial, middle and deep/excavation front zones, a mean of 7.34 x 10(6) (standard error of the mean, SEM +/- 0.44), 5.23 x 10(6) (SEM +/- 0.18), and 1.69 x 10(6) (SEM +/- 0.15) total bacteria/mg dentine were found, respectively. In the advancing front zone (beyond the conventional clinical excavation boundary) a mean of 0.34 x 10(6) (SEM +/- 0.05) total bacteria/mg dentine was recorded.

Conclusion: A bacterial enumeration strategy was developed and detected greater numbers of bacteria through the depth of carious lesions that had been reported previously. The technique could be further developed using species-specific probes to determine the distribution, abundance and viability of all bacteria in carious dentine. This new information in turn will lead to a better understanding of the pathological process of caries and ultimately, its clinical treatment.