Anaerobic Culture Media, Specimen Collection and Methods of Anaerobic Culture

Anaerobic Culture Media, Specimen Collection and Methods of Anaerobic Culture

Anaerobic Culture Media, Specimen Collection and Methods of 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 anaerobes 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 anaerobes from a clinical specimen.
  • Culture and identification of anaerobes is essential for initiating appropriate treatment.

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.
  • Specimens from knife, gunshot, or surgical wounds.
  • Swabs are always avoided when collecting specimens for anaerobic culture because cotton fibers may be detrimental to anaerobes.
  • Coughed throat discharge (sputum), rectal swab, nasal or throat swab, urethral swab, and voided urine are some of the specimens that are not suitable for processing anaerobic cultures.

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 anaerobic 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.
  • 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.

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 examine.

The cultures of anaerobic bacteria are carried out as follows:

  1. McIntosh-Fildes anaerobic jar
  2. Anaerobic glove box
  3. Anoxomat

McIntosh-Fildes anaerobic jar:

  • It is the most widely used and dependable method of anaerobiosis.
  • 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 hydrogen with residual oxygen present in the air. This method ensures complete anaerobiosis.
  • The inoculated media are then kept inside the jar, and the lid is closed air tight.

McIntosh-Fildes anaerobic jar

  • 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:

  • Gas pack system is a simple and effective method of production of hydrogen gas for anaerobiosis.
  • Carbon dioxide  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.

Anaerobic glove box:

  • The anaerobic glove box is another innovation 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 fitted 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 evacuated and replaced with the gas mixture.
  • The inside hatch is then opened to introduce the specimen into the chamber.

Anoxomat:

  • This is a fully automated system that evacuates 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 anaerobic atmosphere, this procedure is repeated three times, after which the oxygen concentration is rarefied to 0.16%.

Anoxomat

  • A small catalyst removes this very small percentage of oxygen content.
  • Anoxomat is capable of producing microaerophilic conditions also.
  • The method is being increasingly used for processing clinical specimens for isolation of anaerobic bacteria.

Anaerobic Culture Media, Specimen Collection and Methods of Anaerobic Culture

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