A successful method for quantifying viable oral anaerobic spirochetes

Area of Science:

• Microbiology
• Periodontology
• Bacteriology

Background:

• Spirochetes are prevalent in periodontal disease but difficult to quantify.
• Current methods do not allow for viable counts of oral spirochetes.
• Accurate enumeration is crucial for understanding their role in disease.

Purpose of the Study:

• To develop a reliable method for enumerating viable oral spirochetes.
• To quantify spirochetes as colony-forming units (CFU) in an agarose-based medium.
• To facilitate the study of spirochetes in periodontal disease.

Main Methods:

• Developed an agarose-based medium for enumerating viable oral spirochetes.
• Used enzyme-free, low-temperature-gelling agarose (37°C) in a specialized broth.
• Incubated seeded tissue-culture flasks anaerobically in a glove-box.
• Evaluated the method using pure cultures of Treponema denticola, Treponema vincentii, and Treponema socranskii.

Main Results:

• Achieved reliable, consistent, and reproducible viable counts of pure spirochete cultures.
• Averted temperature-sensitivity issues by using agarose at 37°C.
• Observed distinctive colony morphologies for different spirochete species.
• Confirmed no decrease in CFU count with the addition of rifampin.

Conclusions:

• The developed method successfully enumerates viable oral spirochetes.
• This technique is valuable for studying spirochetes in periodontal pockets.
• It enables selection of mutants and detection of biochemical activity.
• The method supports the inclusion of spirochetes as predominant cultivable flora in periodontal disease research.