Anaerobic culturing

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Anaerobes demand special methods for their culture. Since the growth of anaerobes is inhibited by oxygen, they should be protected against its deleterious activity.

For successful culturing of anaerobes it is mandatory to inoculate the large amounts of clinical specimen into the nutrient medium. The specimen is commonly taken by tissue puncture into closed syringe and immediately transferred into appropriate anaerobic medium. The nutrient medium should maintain a proper viscosity by balancing certain agar concentrations. The dissolved air is eliminated by boiling prior to inoculation, and subsequent contact with air is prevented by covering of the medium with mineral oil (liquid vaseline) with a layer of 0.5-1 cm thick. Anaerobiosis can be created by the adsorption of oxygen with reducing chemicals or by adding of porous absorbing substances (like the charcoal).

For anaerobic culture transportation the thioglycolate broth is widely used. It contains sodium thioglycolate (the salt of mercaptoacetic acid) that is a strong reducing agent for elimination of dissolved oxygen. After primary inoculation the medium-containing test tubes with specimens should be tightly closed.

Another medium for anaerobic cultivation is iron sulphite agar, that contains MPA, glucose, iron citrate or chloride, and NaHSO3. It is prepared as a high butt of agar covered with liquid vaseline. Microbial inoculation is made like deep agar culture. The growth of anaerobes (e.g., Clostridium perfringens) is followed by the blackening of the medium from the production of hydrogen sulfide.

The growth of the anaerobes can be also produced within a Kitt-Tarozzi medium composed of broth, 0.5% of glucose and pieces of animal organs (as liver) or minced meat for oxygen absorption. The top of the medium is covered with liquid vaseline.

Deep agar cultures are performed by stab inoculation of the specimen into long tubes filled with solidified anaerobic nutrient medium, covered with mineral oil.

For plate inoculation anaerobe Shaedler agar is commonly used. It includes protein enzymatic digest, yeast extract, D-glucose (dextrose), hemin, L-cystin, and agar. For anaerobic culture Shaedler agar should be put into an anaerobic jar.

The most advanced and effective are the methods of apparatus-based anaerobic culturing. They use special closed cylinders – anaerobic jars (or anaerostats), where the air oxygen has been pumped out. A number of Petri dishes with inoculated anaerobic media are placed inside the jars for incubation.

In addition, the air from the anaerobic jar might be substituted with certain inert gas (nitrogen, argone, etc.)

Another common mode of anaerobic jar culture employs the chemical reagent placed into the jar as a disposable sachet (gas-pack or anaeropack). This reagent removes oxygen from closed jar volume. For instance, in some techniques the chemical reagent produces hydrogen that (under the action of catalyst) binds to any free oxygen inside the jar to form water.

Sometimes biological method of culture can be used, where Petri dish with appropriate media like blood agar is inoculated. At first, aerobic culture is plated upon the one half of Petri dish and the material, containing anaerobes, is inoculated onto opposite side of the medium. Primarily aerobes grow and consume the oxygen inside tightly sealed Petri dish; subsequently, anaerobic bacteria begin to propagate.

Now this method is mostly of historical interest, as the maintenance of proper anaerobic conditions poses serious difficulties in such “hand-made” system.