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It is important to remember that fermentation can occur with or without the presence of oxygen, and during prolonged incubation periods, many bacteria will begin growing oxidatively on the peptone after exhausting the supplied carbohydrate. This results in neutralization of the indicator, turning it black to red due to the alkaline conditions created by ammonia production. Consequently, fermentation tubes should not be incubated over 48 hours after inoculation for this reason.

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Chad Rath

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Q: Why are fermentation tubes evaluated at 24 and 48 hours?
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