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Chapter: Microbiology and Immunology: Culture Methods

Anaerobic Culture: Specimen, Culture Media, Methods

Obligate anaerobes are bacteria that can live only in the absence of oxygen. These anaerobes are killed when exposed to the atmosphere for as briefly as 10 minutes.

Anaerobic Culture

Obligate anaerobes are bacteria that can live only in the absence of oxygen. These anaerobes are killed when exposed to the atmosphere for as briefly as 10 minutes. Some anaer-obes are tolerant to small amounts of oxygen. Facultative anaerobes are those anaerobes that grow with or without oxygen.

Anaerobic bacterial culture is a method used to grow anaer-obes from a clinical specimen. Culture and identification of anaerobes is essential for initiating appropriate treatment.

The failure to do so may have serious consequences, such as amputation, organ failure, sepsis, meningitis, and even death.

Specimen Collection

Specimens frequently used for anaerobic culture include:

·           Blood, bile, bone marrow, cerebrospinal fluid, direct lung aspirate, and tissue biopsy from a normally sterile site;

·           Fluid aspirated from a normally sterile site, such as a joint;

·           Pus specimens from dental abscess, burn wound, abdominal or  pelvic abscess; and

·           Specimens from knife, gunshot, or surgical wounds.

Collection of a contamination-free specimen and protecting it from oxygen exposure during collection form the mainstay of anaerobic culture. The specimens need to be obtained from an appropriate site without contaminating the sample with bacteria from the adjacent skin, mucous membrane, or tissue.

Abscesses or fluids are usually collected by using a sterile syringe and is then tightly capped to prevent entry of air. Tissue samples are placed into a degassed bag and sealed, or into a gassed out screw top vial that may contain oxygen-free prereduced culture medium and tightly capped. The specimens need to be plated as rapidly as possible onto culture media for isolation of bacteria.

Culture Media

The commonly used media for anaerobic culture include Robertson cooked meat broth, thioglycollate broth, Willis and Hobbs’ media, and neomycin blood agar. Robertson cooked meat (RCM) broth is the most widely used medium in an anaer-obic culture. It consists of nutrient broth and pieces of fat-free minced cooked meat of ox heart with a layer of sterile liquid paraffin over it. Unsaturated fatty acids and even glutathione and cysteine present in the meat extract utilize oxygen for auto-oxidation. The medium before inoculation is usually boiled at 80°C in a water bath to make the medium free of oxygen. The media after inoculation and incubation allows the growth of even strict anaerobes and also indicates their saccharolytic or proteolytic activities as meat is turned red or black, respectively.

Methods of Anaerobic Culture

Anaerobic cultures are carried out in an environment that is free of oxygen, followed by incubation at 95°F (35°C) for at least 48 hours before the plates are examined for growth. The cultures of anaerobic bacteria are carried out as follows:

1. McIntosh–Fildes anaerobic jar: It is the most widely usedand dependable method of anaerobiosis (Color Photo 3). It consists of a glass or metal jar with a metal lid that can be clamped air tight with the help of a screw. The lid has one inlet tube and another outlet tube. The outlet tube is connected to a vacuum pump by which the air is evacuated out of the jar. The inlet tube is connected to a source of hydrogen supply. The lid has two electric terminals also that can be connected to an electric supply. The underside of the lid contains a catalyst (e.g., alumina pellets coated with palladium) that catalyzes the combination of hydro-gen with residual oxygen present in the air. This method ensures complete anaerobiosis.

      The culture media are inoculated with the specimens suspected to contain anaerobic bacteria. The inoculated media are then kept inside the jar, and the lid is closed air tight. The anaerobiosis in the jar is carried out by first evacuating the air from the jar through outlet tube with the help of a vacuum pump. The outlet tube is closed, then the sealed jar containing the culture plates is replaced with hydrogen gas passed through inlet tube till reduced atmospheric pressure is brought to normal atmospheric pressure, which is monitored on the vacuum gauge as zero. The electrical terminals are then switched on to heat the catalyst that catalyzes combination of hydrogen with residual oxygen and ensures complete anaerobiosis in the jar.

Reduced methylene blue is used as the indicator of anaerobiosis in the jar. If anaerobiosis is complete, it remains colorless; if anaerobiosis is not complete, it turns blue on exposure to oxygen.

Gas pack system is a simple and effective method ofproduction of hydrogen gas for anaerobiosis. It does not require the cumbersome method of evacuation and filing up of gases after evacuation. Carbon dioxide, which is also generated, is required for growth by some anaerobes. Water activates the gas pack system, resulting in the production of hydrogen and carbon dioxide. Hydrogen combines with oxygen in the air in the presence of catalyst and maintains anaerobiosis. In this method, the inoculated plates are kept along with the gas pack envelope with water added in the air tight jar.

2. Anaerobic glove box: The anaerobic glove box is anotherinnovation developed for isolating anaerobic bacteria. It is essentially a large clear-vinyl chamber with attached gloves, containing a mixture of 80% nitrogen, 10% hydrogen, and 10% carbon dioxide. A lock at one end of the chamber is fit-ted with two hatches, one leading to outside and the other to the inside of the chamber. Specimens are placed in the lock, the outside hatch is closed, and the air in the lock is evacu-ated and replaced with the gas mixture. The inside hatch is then opened to introduce the specimen into the chamber.


3. Anoxomat: This is a fully automated system that evacu-ates a portion of the jar contents and refills the jar with an anaerobic gas mixture. During this procedure, the oxygen concentration in the air is rarefied. For anaero-bic atmosphere, this procedure is repeated three times, after which the oxygen concentration is rarefied to 0.16%. A small catalyst removes this very small percentage of oxygen content. Anoxomat is capable of producing micro-aerophilic conditions also. The method is being increas-ingly used for processing clinical specimens for isolation of anaerobic bacteria.

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