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Bacterial aggregating activity in human saliva: simultaneous determination of free and bound cells

Two new assays for saliva-mediated aggregation of oral bacteria have been developed, based on the use of [3H]thymidine-labeled cells. One assay separates free cells from aggregated cells by centrifugation through sucrose, whereas the other utilizes membrane filters (8 micrometers, Nuclepore) to effect the separation. Comparison of these assays with the turbidity method reveals that they are faster (X20 to 40) and require 10 times less saliva and bacteria. The aggregation of Streptococcus sanguis M5, as determined with these assays, is complete in 5 min and is dose dependent on added cells and saliva. The reaction exhibits a temperature optimum of 42 degrees C with no reaction at 0 degrees C. If the pH is reduced to below 5, saliva-dependent aggregation is inhibited. The salivary factor(s) are heat labile, losing 100% of their activity after 100 degrees C, 10 min or 70 degrees C, 30 min.