Automated immunofluorescence for enumeration of selected taxa in supragingival dental plaque

The present study investigated a recently developed automated image analysi s technique for its applicability to the enumeration of selected bacteria i n supragingival dental plaque. Following initial calibration, the system is capable to count fluorescence-labeled target cells in up to 48 samples wit hout user interference. Test samples contained a characteristic mixture of planktonic bacteria, small almost planar bacterial aggregates, and large, v irtually indisruptable clumps with cells from multiple species. Due to thei r complex composition, these samples provided a challenging validation step for the image analysis system. Automated enumeration of target bacteria wa s compared with visual counting of the fluorescence-labeled bacteria. Resul ts are shown for six taxa (Actinomyces naeslundii, Fusobacterium nucleatum, Prevotella intermedia/Prev. nigrescens, Streptococcus gordonii/Strep. oral is/Strep, sanguis, Strep. sobrinus, and Veillonella dispar/V. parvula) with characteristic differences in abundance, cell morphology and aggregation b ehavior. Results revealed good correspondence between the two enumeration t echniques (correlation coefficients ranging from 0.77 to 0.92) provided tha t the portion of target bacteria exceeded 0.05% of the total bacterial cell number. This work demonstrates the applicability and usefulness of fully a utomated immunofluorescence to analyze such complex ecosystems as supraging ival dental plaque.