Enumeration of viable oral spirochetes from periodontal pockets

We recently developed a successful method for quantifying oral anaerobic spirochetes in pure culture by a viable count. New oral spirochete medium was used with low temperature-gelling agarose in polystyrene tissue-culture flasks. We have extended the use of this method to determine the viable count of spirochetes from periodontal pockets. Sixteen subgingival plaque samples were obtained by insertion of sterile paper points into deep periodontal pockets. The points were placed into reduced transport medium at chairside, vortexed in the microbiology laboratory and aliquots of the medium inoculated into molten new oral spirochete-agarose medium (37°C) containing rifampin (20 μg/ml) in a flask. Subsequent dilutions were made from this initial flask to other flasks containing selective medium in sequence. All flasks were incubated anaerobically. Most other subgingival bacteria were selectively inhibited by rifampin. Spirochete colonies were typically spherical and were either dense or cottony. Their identities were checked by darkfield examination. Counts of colony-forming units of cultivable spirochetes ranged from 12.5% to 28.2% of the total cultivable anaerobic flora by the method described.