Specimen Processing of Anaerobic Bacteriology
المؤلف:
Patricia M. Tille, PhD, MLS(ASCP)
المصدر:
Bailey & Scotts Diagnostic Microbiology
الجزء والصفحة:
13th Edition , p460-464
2025-08-25
607
Specimens for anaerobic culture may be processed in the biologic safety cabinet, after which they are incubated in anaerobic jars or pouches or in an anaerobic chamber. The roll tube method developed at Virginia Polytechnic Institute is no longer widely used and is not discussed here.
ANAEROBE JARS OR POUCHES
The most frequently used system for creating an anaerobic atmosphere is the anaerobe jar. Anaerobe jars are available commercially from several companies. For example, the GasPak (Figure 1) is made by Becton Dickinson (Sparks, Maryland); other companies that produce these devices include EM Diagnostic Systems (Gibbstown, New Jersey) and Oxoid U.S.A. (Columbus, Maryland). All of these systems use a clear, heavy plastic jar with a lid that is clamped down to make it airtight. Anaerobic conditions can be set up by two methods. The easiest method uses a commercially available envelope containing a hydrogen and CO2 generator that is activated either by adding water (GasPak) or by the moisture on the agar plates (EM Diagnostic Systems and Oxoid USA). The production of heat within a few minutes (detected by touching the top of the jar) and subsequent development of moisture on the walls of the jar are indications that the catalyst and generator envelope are functioning properly. Reduced conditions are achieved in 1 to 2 hours, although the methylene blue or resazurin indicators take longer to decolorize. Alternatively, the “evacuation-replacement” method can be used. Air is removed from the sealed jar by drawing a vacuum of 25 inches (62.5 cm) of mercury. This process is repeated two times, with the jar being filled with an oxygen-free gas, such as nitrogen, between evacuations. The final fill of the jar is made with a gas mixture containing 80% to 90% nitrogen, 5% to 10% hydrogen, and 5% to 10% CO2. Many anaerobes require CO2 for maximal growth.
Fig1. GasPak anaerobe jar (BD Diagnostic Systems, Sparks, Md.). Inside the jar are inoculated plates, an activated gas-generating envelope, and an indicator strip. A wire-mesh basket attached to the lid of the jar contains palladium-coated alumina pellets that catalyze the reaction to remove oxygen. Newer models of the GasPak jar use reagent packs that simply require the addition of water to catalyze a reaction.
The atmosphere in the jars is monitored using an indicator to check anaerobiosis. Anaerobe bags or pouches are useful for laboratories processing small numbers of anaerobic specimens. A widely used anaerobic pouch, the GasPak Pouch, is shown in Figure 2. Besides specimen transport, the pouch also can be used to incubate one or two agar plates.
Fig2. Anaerobic transport system for tissue specimens. Tissue is placed in a small amount of saline to keep it moist. It then is inserted into a self-contained, atmosphere-generating anaerobic bag for transportation. This system is called the GasPak Pouch. (Courtesy BD Diagnostic Systems, Sparks, Md.)
HOLDING JARS
If anaerobic jars or pouches are used for incubation, holding jars should be used during specimen processing and examination of cultures. Holding jars are anaerobic jars with loosely fitted lids attached by rubber tubing to nitrogen gas. Uninoculated plates are kept in holding jars pending use for culture setup, and inoculated plates are kept in holding jars pending incubation or examination; this minimizes exposure to oxygen.
ANAEROBE CHAMBER
Anaerobic chambers, or glove boxes, are made of molded or flexible clear plastic. The flexible clear plastic chambers are the most widely used type. Specimens and other materials are placed in the chamber through an air lock. The technologist uses gloves (Forma Scientific, Marietta, Ohio) or sleeves (Sheldon Manufacturing, Cornelius, Oregon), to form airtight seals around the arms (Figure 3). Media stored in the chamber are kept oxygen free, and all work on a specimen, from inoculation through workup, is performed under anaerobic conditions. A gas mixture of 5% CO2, 10% hydrogen, and 85% nitrogen, plus a palladium catalyst, maintain the anaerobic environment inside the chamber.
Fig3. Gloveless anaerobe chamber. Anaerobic chambers were developed more than 40 years ago, and a variety of new models are being used in cutting-edge research. (Courtesy Anaerobe Systems, Morgan Hill, Calif.)
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