Chan E C, Siboo R, Touyz L Z, Qui Y S, Klitorinos A
Department of Oral Biology, Department of Microbiology and Immunology, Faculty of Medicine, McGill University, Montreal, Quebec, Canada.
Oral Microbiol Immunol. 1993 Apr;8(2):80-3. doi: 10.1111/j.1399-302x.1993.tb00549.x.
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 for 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 spirochetes were seeded and diluted. The flasks were anaerobically incubated in a glove-box. Reliable, consistent and reproducible viable counts of pure spirochete cultures were obtained. The injurious effects of spirochete temperature-sensitivity were averted by using molten agarose at 37 degrees C. Distinctive colony morphologies of spirochete species could be compared from pure cultures. Addition of rifampin into the medium showed no decrease in spirochete CFU count. The method as described allows for selection of mutants and detection of biochemical activity and is potentially useful for enumeration of spirochetes from periodontal pockets as members of the predominant cultivable flora.
螺旋体在牙周疾病中显著流行，但由于无法通过活菌计数对其进行量化，因此未被列为主要的可培养微生物。已开发出一种成功的方法，用于在基于琼脂糖的培养基中以菌落形成单位（CFU）来计数活的口腔螺旋体。处于对数生长期的齿垢密螺旋体、文森特密螺旋体和索氏密螺旋体在新的口腔螺旋体（NOS）肉汤中用于该方法的评估。该方法的关键组成部分包括在小组织培养瓶中的NOS培养基中使用无酶低温凝胶化（37℃）琼脂糖，将螺旋体接种并稀释到其中。这些培养瓶在手套箱中进行厌氧培养。获得了可靠、一致且可重复的纯螺旋体培养物活菌计数。通过使用37℃的融化琼脂糖避免了螺旋体温度敏感性的有害影响。可以从纯培养物中比较螺旋体物种独特的菌落形态。向培养基中添加利福平并未使螺旋体CFU计数降低。所描述的方法允许选择突变体并检测生化活性，并且可能有助于从牙周袋中计数螺旋体，作为主要可培养菌群的成员。
