When handling, transferring, or incubating anaerobic cultures, it is important to avoid unnecessary exposure to oxygen as it will cause the viability of cells to decrease rapidly. Anaerobic conditions for handling or transferring cells may be obtained by the use of an anaerobic gas chamber or placement of test tubes under a gassing cannula system connected to anaerobic gas.

For the incubation of anaerobic cultures, the appropriate conditions may be obtained by any of the following:

• Loose screw caps on test tubes in an anaerobic chamber
• Loose screw caps on test tubes in an activated anaerobic gas pack jar
• Use of sterile butyl rubber stoppers on test tubes so that an anaerobic gas headspace is retained

Unless specifically mentioned, the standard anaerobic gas mixture is 80% N2, 10% CO2, and 10% H2. Also, it is recommended that you always use freshly prepared pre-reduced media or pre-reduced media that has been previously prepared but stored under anaerobic conditions.