Methanogen cultures require hydrogen produced by fermentative bacteria such as Bacteroides thetaiotaomicron (biological method). We developed an alternative method for hydrogen production using iron filings and acetic acid with the aim of cultivating methanogens more efficiently and more quickly (chemical method). We developed this new method with a reference strain of Methanobrevibacter oralis, compared the method to the biological reference method with a reference strain of Methanobrevibacter smithii and finally applied the method to 50 saliva samples. Methanogen colonies counted using ImageJ software were identified using epifluorescence optical microscopy, real-time PCR and PCR sequencing. For cultures containing pure strains of M. oralis and M. smithii, colonies appeared three days postinoculation with the chemical method versus nine days with the biological method. The average number of M. smithii colonies was significantly higher with the chemical method than with the biological method. There was no difference in the delay of observation of the first colonies in the saliva samples between the two methods. However, the average number of colonies was significantly higher with the biological method than with the chemical method at six days and nine days postinoculation (Student’s test, p = 0.005 and p = 0.04, respectively). The chemical method made it possible to isolate four strains of M. oralis and three strains of M. smithii from the 50 saliva samples. Establishing the chemical method will ease the routine isolation and culture of methanogens.

产甲烷菌培养物需要发酵细菌（如拟杆菌（Theactotaomicron）的微生物）产生的氢。我们开发了一种使用铁屑和乙酸生产氢气的替代方法，目的是更高效，更快地培养产甲烷菌（化学方法）。我们开发了一种新的方法，将其与口头上的甲烷短杆菌进行了比较，将该方法与生物学上的参考方法与史密斯短杆菌进行了比较，最后将该方法应用于50个唾液样品中。使用落射荧光显微镜，实时PCR和PCR测序鉴定使用ImageJ软件计数的产甲烷菌菌落。用于含有纯口头支原体菌株的培养物和史密斯氏菌，化学方法接种后三天出现菌落，而生物学方法则为九天。化学方法使史密斯氏菌菌落的平均数量显着高于生物方法。两种方法之间唾液样品中最初菌落的观察延迟没有差异。然而，在接种后六天和九天，采用生物学方法的平均菌落数要明显高于采用化学方法的菌落数（Student's test，p = 0.005和p = 0.04）。化学方法使得可以分离出四个口头分支杆菌和三个史密斯氏菌菌株。从50个唾液样本中 建立化学方法将简化产甲烷菌的常规分离和培养。