A SUCCESSFUL METHOD FOR QUANTIFYING VIABLE ORAL ANAEROBIC SPIROCHETES

Spirochetes are markedly prevalent in periodontal disease but are not included as predominant cultivable organisms because of the inability to quantify them by viable count. A successful method was developed fo r enumerating viable oral spirochetes as colony-forming units (CFU) in an agarose-based medium. Treponema denticola, Treponema vincentii and Treponema socranskii in log-phase growth in new oral spirochete (NOS) broth were used for evaluation of the method. Critical components of the method include enzyme-free low temperature-gelling (37-degrees-C) agarose in NOS medium in small tissue-culture flasks into which the sp irochetes were seeded and diluted. The flasks were anaerobically incub ated in a glove-box. Reliable, consistent and reproducible viable coun ts of pure spirochete cultures were obtained. The injurious effects of spirochete temperature-sensitivity were averted by using molten agaro se at 37-degrees-C. Distinctive colony morphologies of spirochete spec ies could be compared from pure cultures. Addition of rifampin into th e medium showed no decrease in spirochete CFU count. The method as des cribed allows for selection of mutants and detection of biochemical ac tivity and is potentially useful for enumeration of spirochetes from p eriodontal pockets as members of the predominant cultivable flora.