Objectives

Methanogenic Archaea are considered as extremely oxygen-sensitive organisms, and their culture is fastidious, requiring specific equipment. We report here conditions allowing the cultivation of Methanobrevibacter smithii in an anaerobic chamber without the addition of hydrogen.

Methods

We first enriched the stool sample in an anaerobic liquid medium. To cultivate M. smithii with Bacteroides thetaiotaomicron and other hydrogen-producing bacteria on solid medium in an anaerobic chamber, we divided the agar plates into two compartments and seeded each strain on each compartment. Methane production was assessed by gas chromatography, and the growing colonies were authenticated by MALDI-TOF MS.

Results

We successfully cultured M. smithii from a liquid culture medium inoculated with stool collected from a healthy donor in an anaerobic chamber. The isolation in pure culture permitted successful culture on agar medium by our performing a co-culture with B. thetaiotaomicron. We also successfully tested the co-cultivation of M. smithii with other known hydrogen-producing bacteria. Gas chromatographic tests showed that these strains produced hydrogen in different amounts. Agar colonies of methanogens were obtained by co-culture with these bacteria, and methane production was detected.

Conclusions

We propose a new approach to isolate and cultivate new strains of M. smithii by using a co-culture–based technique that can facilitate and make available the isolation of new methanogenic Archaea strains in clinical microbiology laboratories.

中文翻译：

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在琼脂平板上与产氢细菌共培养分离和分离史密斯甲烷杆菌

目标

产甲烷的古生菌被认为是对氧气非常敏感的生物，其养殖是挑剔的，需要特定的设备。我们在这里报告的条件允许在不添加氢气的情况下在厌氧室内培养史密斯甲烷杆菌。

方法

我们首先在厌氧液体培养基中富集了粪便样品。要在厌氧室的固体培养基上用拟杆菌（Bacteroides thetaiotaomicron）和其他产氢细菌培养史密斯氏菌，我们将琼脂平板分成两个隔室，并将每个菌株接种到每个隔室中。通过气相色谱法评估甲烷的产生，并通过MALDI-TOF MS对生长的菌落进行鉴定。

结果

我们成功地从接种了健康供体的粪便中接种的液体培养基中，在厌氧室中培养了史密斯氏菌。通过与B. thetaiotaomicron进行共培养，纯净培养物中的分离允许在琼脂培养基上成功培养。我们还成功地测试了史密斯氏菌与其他已知的产氢细菌的共培养。气相色谱测试表明，这些菌株产生的氢气量不同。通过与这些细菌共培养获得产甲烷菌的琼脂菌落，并检测到甲烷的产生。

结论

我们提出了一种通过使用基于共培养的技术来分离和培养史密斯氏梭菌新菌株的新方法，该技术可以促进临床微生物实验室中产甲烷的新产甲烷菌菌株的分离。