Application of stereological principles for quantification of bacteria in intact dental biofilms

Quantative confocal laser scanning microscopy (CLSM) in combination with fluorescent in situ hybridization (FISH) may help to increase our knowledge about biofilm formation. The purpose of this study was to develop and evaluate a stereological method for quantification of bacteria in intact biofilm. The method was applied in a quantitative study of the proportion of streptococci relative to other bacteria in initial in-situ-grown dental biofilms as a function of time. Biofilms were collected on standardized glass slabs mounted in intra-oral appliances and worn by 10 individuals for 6, 12, 24, and 48 h. Biofilms were analysed using CLSM. Quantification of bacteria labelled with 16S ribosomal RNA oligonucleotide probes was performed with stereological tools: the unbiased counting frame and the two-dimensional fractionator. Results showed a notable increase in the total number of bacteria and streptococci over time, with a considerable inter-individual variation at each time-point. After 48 h there was a 12.5-fold difference between individuals in the total number of bacteria and a 12.6-fold difference in the number of streptococci. The number of streptococci exceeded that of other bacteria and over the examination period there was a relatively constant relationship between the number of streptococci and other bacteria (streptococci vs. non-streptococci: median 15.2; minimum 1.0; maximum 89.3). The study demonstrates that the combined use of FISH and stereology is a relevant and reliable tool for obtaining unbiased information about the numerical contributions of specific bacterial populations during early biofilm formation.