General Characteristics

The organisms described in this chapter and in Chapter 42 usually do not grow in the presence of oxygen (O₂); they are obligate, or strict, anaerobes (0% O₂). Obligate anaerobes are killed upon brief exposure (less than a few minutes) to atmospheric oxygen. Obligate anaerobes include Prevotella sp., Fusobacterium sp., and Bacteroides spp. These chapters also include some aerotolerant organisms (5% O₂), such as Actinomyces spp., Bifidobacterium spp., and Clostridium spp., which are capable of growth in the presence of either reduced or atmospheric oxygen but grow best under anaerobic conditions. Finally, facultative anaerobes do not require atmospheric oxygen but are capable of growth in oxygen and anaerobic environments.

Anaerobic organisms lack superoxide dismutase and catalase, the enzymes required to breakdown reactive oxygen species produced during respiration or aerobic metabolism. In addition, oxygen has a high affinity for organic compounds containing nitrogen, hydrogen, carbon, and sulfur, which interferes with normal biologic activity. Because they are unable to protect themselves against the action of oxygen, anaerobes require an environment free of oxygen to survive and grow.

Specimen Collection and Transport

The importance of proper collection and transport of specimens for anaerobic culture cannot be overemphasized. Because indigenous anaerobes are often present in large numbers as normal flora on mucosal surfaces, even minimal contamination of a specimen can produce misleading results. Box 41-1 shows the specimens acceptable for anaerobic culture; Box 41-2 presents specimens that are likely to be contaminated and therefore are unacceptable for anaerobic culture. In general, material for anaerobic culture is best obtained by tissue biopsy or by aspiration using a needle and syringe. Use of swabs is a poor alternative because of excessive exposure of the specimen to the deleterious effects of drying, the possibility of contamination during collection, and the easy retention of microorganisms in the fibers of the swab. If a swab must be used, it should be from an oxygen-free transport system.

A crucial factor in obtaining valid results with anaerobic cultures is the transport of the specimen; the lethal effect of atmospheric oxygen must be nullified until the specimen can be processed in the laboratory. Recapping a syringe and transporting the needle and syringe to the laboratory is no longer acceptable because of safety concerns involving needle stick injuries. Therefore, even aspirates must be injected into an oxygen-free transport tube or vial.

Three kinds of anaerobic transport systems are shown in Figures 41-1 to 41-3. Figure 41-1 shows is a rubber-stoppered collection vial containing an agar indicator system. The vial is gassed out with oxygen-free carbon dioxide (CO₂) or nitrogen. The specimen (pus, body fluid, or other liquid material) is injected through the rubber stopper after all air has been expelled from the syringe and needle. If only a swab specimen can be obtained, a special collection device with an oxygen-free atmosphere is required (see Figure 41-2). When the swab is reinserted, care must be taken not to tip the container, which would cause the oxygen-free CO₂ or nitrogen to spill out and be displaced by ambient air. A tissue specimen can be immersed in a small amount of liquid to prevent it from drying and then placed in an anaerobic pouch (see Figure 41-3). All specimens should be held at room temperature pending processing in the laboratory, because refrigeration can oxygenate the specimen.

Macroscopic Examination of Specimens

Upon receipt in the laboratory, specimens should be inspected for characteristics that strongly indicate the presence of anaerobes: (1) foul odor; (2) sulfur granules (associated with Actinomyces spp., Propionibacterium spp., or Eubacterium nodatum); and (3) brick red fluorescence under long wavelength ultraviolet (UV) light (associated with pigmented Prevotella or Porphyromonas spp).

The cytotoxin (toxin B) of Clostridium difficile can be detected using a tissue culture assay. This assay, performed in various cell lines, is based on the neutralization of cytopathic effect when the cell-free fecal extract is adsorbed using either Clostridium sordellii or C. difficile antitoxins. Latex particle agglutination tests or enzyme-linked immunosorbent assays (ELISA) to detect toxin A or toxin B (or both) are also available.

Screening of stool samples for the production of glutamate dehydrogenase (GDH) using enzyme immunoassays is commonly used. GDH-positive samples should be examined for production of toxin A and B. Commercially available DNA-based methods are available. However, C. difficile colonization occurs, and therefore molecular testing does not indicate C. difficile enteric disease.

Specimen Processing

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 41-4) 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.

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 41-3. Besides specimen transport, the pouch also can be used to incubate one or two agar plates.

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 41-5). 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.

Anaerobic Media

Initial processing of anaerobic specimens involves inoculation of appropriate media. Table 41-2 lists commonly used anaerobic media. Primary plates should be freshly prepared or used within 2 weeks of preparation. Plates stored for longer periods accumulate peroxides and become dehydrated; this results in growth inhibition. Reduction of media in an anaerobic environment eliminates dissolved oxygen but has no effect on the peroxides. Prereduced, anaerobically sterilized (PRAS) media are produced, packaged, shipped, and stored under anaerobic conditions. They are commercially available from Anaerobe Systems (Morgan Hill, California) (Figure 41-6) and have an extended shelf life of up to 6 months.