Isolation and culture of hydrogen-producing and fermentative bacteria is an important foundation on biohydrogen production process. There are complicated operation and composition in present anaerobic techniques and culture media. Hungate technique was improved and plate of culture bottle was established by comparing anaerobic methods and bacterium growth. Isolation and enrichment culture media were confirmed by the test of different composition and the species and amount of hydrogen-producing and fermentative bacterium. 550 bacterium strains were isolated by the anaerobic operation.