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Most experts agree that incubation of tubes and plates for mycobacteria should include at least 8-10% CO2. Specimens on Middlebrook agar require CO2 for growth enhancement, while those on egg-based media are stimulated by CO2. The length of incubation in CO2 is less definable. Some recommend 1-2 weeks others a little longer. Candle-jars are inappropriate as they generate only 3% CO2. Overlong incubation with caps slightly uncrewed to allow CO2 penetration may dry out the agar, thus a compromise, as stated in Cumitech 16A, would be to incubate tubes with loosened caps for no less than 1 week. Caps can then be tightened.
Reference: Cernoch PL, Enns RK, Saubolle MA, and Wallace, Jr., RJ. 1994. Cumitech 16A, Laboratory diagnosis of mycobacteriosis. Coordinating Ed., Alice Weissfeld. American Society for Microbiology, Washington, D.C.
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