Laboratory of Veterinary Bacteriology and Mycology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium.
J Microbiol Methods. 2010 Dec;83(3):335-40. doi: 10.1016/j.mimet.2010.09.001. Epub 2010 Sep 17.
Limited reports are available on the growth response of Mycoplasma hyopneumoniae in Friis medium and the routinely used color changing units (CCU) assay has not yet been profoundly compared with other titration methods. Firstly, growth kinetics of 7 diverse M. hyopneumoniae isolates were followed by ATP luminometry in five Friis medium batches. Secondly, results of the CCU and ATP assays were compared hereby evaluating the methods. Growth curves of all isolates had log, stationary and senescence phases, and reached similar maximal titres when cultured in the same batch of Friis medium. Doubling times (Tds) of the isolates grown in slowly shaken cultures varied between 4.8 and 7.8 h. Maximal titres, Tds, growth phase in which the phenol red indicator turned from red to yellow due to acidification by mycoplasmal metabolism, and the length of the stationary phase varied depending on the Friis medium batch. The effect of static vs. shaking culture conditions on the Td depended on the isolate. ATP and CCU assays obtained similar growth curves, but when maximal levels were reached the CCU titre dropped earlier than the ATP titre. During log phase, CCU and ATP titres were strongly linearly linked. We developed a model enabling transformation of ATP into CCU titres or vice versa. The calculated amount of ATP per CCU (1.77 amol ATP/ml) indicated that the CCU assay likely underestimates the actual cell concentration. When titres were determined as means of 3 measurements, the ATP assay was 7 times more accurate and had 11-fold lower outliers than the CCU assay. Unlike the CCU assay, luminometry only requires one measurement to obtain sufficient accuracy. It was concluded that the ATP assay constitutes a valuable robust alternative for reproducible real-time titre assessment of freshly grown M. hyopneumoniae cultures. It is faster, more accurate and time, work and cost efficient compared to the CCU assay. The assay is preferred to better standardise and describe M. hyopneumoniae cultures used in various experiments.
关于猪肺炎支原体在 Friis 培养基中的生长反应，已有有限的报道，且常规使用的变色单位（CCU）检测法尚未与其他滴定方法进行深入比较。首先，通过 5 批 Friis 培养基中的 ATP 发光法对 7 株不同的猪肺炎支原体分离株的生长动力学进行了跟踪。其次，通过比较 CCU 和 ATP 检测法的结果，对这些方法进行了评估。所有分离株的生长曲线均具有对数、静止和衰老阶段，并且在相同批次的 Friis 培养基中培养时达到相似的最大滴度。在缓慢摇动培养物中生长的分离株的倍增时间（Td）在 4.8 和 7.8 小时之间变化。最大滴度、Td、由于支原体代谢酸化而使酚红指示剂从红色变为黄色的生长阶段以及静止阶段的长度取决于 Friis 培养基批次。静态与摇动培养条件对 Td 的影响取决于分离株。ATP 和 CCU 检测法获得了相似的生长曲线，但当达到最大水平时，CCU 滴度比 ATP 滴度更早下降。在对数阶段，CCU 和 ATP 滴度具有很强的线性关系。我们开发了一种模型，能够将 ATP 转化为 CCU 滴度或反之亦然。每个 CCU 的 ATP 量（1.77 amol ATP/ml）表明，CCU 检测法可能低估了实际细胞浓度。当通过 3 次测量确定滴度时，ATP 检测法比 CCU 检测法更准确 7 倍，且异常值少 11 倍。与 CCU 检测法不同，发光法仅需一次测量即可获得足够的准确性。因此，ATP 检测法是一种有价值的可靠替代方法，可用于重现性实时评估新鲜生长的猪肺炎支原体培养物的滴度。与 CCU 检测法相比，它更快、更准确，且更节省时间、工作和成本。该检测法优于更好地标准化和描述用于各种实验的猪肺炎支原体培养物。
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