Introduction
An anaerobic jar (such as the GasPak system) is an indispensable tool in microbiology designed to create an oxygen-free environment for culturing obligate anaerobes. When oxygen is present, strict anaerobes cannot survive due to the toxic effects of reactive oxygen species. To solve this, the sealed chamber removes atmospheric oxygen and replaces it with an environment rich in hydrogen and carbon dioxide, allowing fastidious anaerobic bacteria to proliferate safely.
Mechanism of Anaerobiosis Generation
The transformation of the internal atmosphere relies on a chemical gas-generating envelope activated by water, which produces hydrogen (H_2) and carbon dioxide (CO_2). A palladium catalyst attached to the underside of the lid rapidly facilitates the reaction between residual oxygen inside the jar and the newly generated hydrogen gas to yield water vapor (2H_2 + O_2 \rightarrow 2H_2O). Within an hour, oxygen levels fall below 1%, while carbon dioxide accumulates to roughly 5 to 10 percent, establishing the optimal microaerophilic or anaerobic microenvironment.
Function of Chemical Indicators
To verify that complete anaerobiosis has been established and maintained throughout the incubation cycle, chemical redox indicator strips are placed inside the jar. These indicators rely on oxidation-reduction state changes to signal the presence or absence of free oxygen. The two most common chemical indicators are methylene blue and resazurin. In an aerobic state, methylene blue remains distinctively blue, but upon complete reduction in an oxygen-free jar, it transitions to a completely colorless form known as leucomethylene blue. Similarly, resazurin appears bright pink in an oxidized environment and turns colorless once reduced.
Function of Biological Indicator
In addition to chemical strips, biological indicators serve as absolute quality control checks to confirm functional anaerobiosis. Known control strains of bacteria are inoculated onto plates and placed directly into the jar alongside the test specimens. Laboratories routinely use Pseudomonas aeruginosa, a strict obligate aerobe, as a negative growth control; the complete absence of Pseudomonas growth confirms that no residual oxygen remained in the jar. Simultaneously, an obligate anaerobe such as Clostridium sporogenes is included as a positive control, where robust colony growth confirms that the environmental conditions were thoroughly reduced and supportive of anaerobic life.
