Comparison of different assays for the aggregation of oral bacteria by human whole saliva.

For comparison, human whole saliva-induced aggregation was studied by phase-contrast microscopy, spectrophotometry combined with macroscopic observations, and in microtiterplate assay under identical experimental conditions for Actinomyces viscosus HG 85 (T14-V) and HG 380 (T14-AV), Bacteroides gingivalis HG 66 (W 83), Streptococcus rattus HG 59 (BHT), and Streptococcus sanguis I HG 169. The entire process of formation, extension, and sedimentation of aggregates could merely be observed by the combination of these assays. The very first stages of aggregation could only be detected and quantitated by phase-contrast microscopy. Within 2 1/2 min, 50% of the A. viscosus, S. rattus, and S. sanguis cells were aggregated, denoted as T50. In microtiterplates, however, aggregates were observed in general only after sedimentation at 30-45 min of incubation, expressed as TA. For interpretation of the spectrophotometric curves, additional microscopic and macroscopic data were a prerequisite. The small decline in absorbance during the first 30-45 min (phase 1) corresponded to the formation and extension of nonsedimenting aggregates, whereas the subsequent pronounced fall in absorbance (phase 2) was caused by the massive sedimentation of aggregates. The moment of inflexion between both phases, TI, marked the onset of sedimentation of aggregates and corresponded very well with TA, at which time already 92-98% of the cells were aggregated as quantitated by microscopy. In conclusion, only by microscopy the formation and extension of aggregates could be observed within a few minutes and quantitated in terms of aggregation rate. From 30-45 min, merely the sedimentation of aggregates was visualized in microtiterplates, whereas the time course of the overall process was recorded indirectly by spectrophotometry.