Attempts to increase viable count recovery of human supragingival dental plaque.

Abstract

Studies of the microbiota of dental plaque have been hampered by an inability to cultivate all or even a majority of microorganisms present in this site. Failure to recover the microorganisms could be attributed to at least 3 types of losses; inadequate dispersion, adhesion to glassware used in dilution and spreading, and an inability of any single cultural environment to recover all of the resident microorganisms. Clumps of microorganisms could be more effectively dispersed by sonic oscillation than tissue grinders. Anaerobic sonication proved to be significantly more effective in maintaining viability of plaque isolates than aerobic sonication while dispersion in dilute salt solutions permitted recovery of greater numbers of organisms than dispersion in broth.About 5% of the organisms were lost due to adsorption to glassware as determined by pouring molten agar media over the used apparatus and counting the resultant colonies.Optimum recovery of plaque organisms occurred in this study when samples were dispersed by anaerobic sonic oscillation in pre‐reduced anaerobically sterilized 1/4 strength Ringer's solution supplemented with 1% sodium metaphosphate, 0.05% L cysteine, and 0.0001% resazurin. When the resulting suspension was anaerobically serially diluted, and plated on trypticase soy 5% sheep blood agar plates which were incubated in Brewer jars containing 80% N2, 10% H2. and 10% CO2, 60% of the total microscopic cell count could be recovered.The use of additional primary isolation environments including blood agar plates incubated aerobically, as well as trypticase soy and reduced benzyl viologen roll tubes increased the recovery an additional 15%. Thus an average of 75% of the microscopic count could be cultivated. An additional 5% was lost due to adsorption to glassware and an average of 10% of the microbiota remained undispersed in clumps.