Methods Of Cultivation

The microbial world consists of aerobic and anaerobic organisms, defined by the ability to tolerate molecular oxygen. Sonnewirth (63) traced the evolution of anaerobic methodology and recorded that Leeuwenhoek demonstrated that some life forms could exist in the presence of gases other than oxygen and that Pasteur discovered an-aerobiosis in 1861, coining the terms aerobes and anaerobes. The separation between these groups is not clearly defined. A convenient way to categorize these organisms is by observing their ability to grow at different oxidation-reduction potentials (Eh). Eh can be measured and expressed + or — millivolts (mV). Thus, aerobes can grow between + 300 and — 50 mV; facultative anaerobes, + 300 to —420 mV; aerotolerant anaerobes, +180 to —350 mV; and obligate anaerobes, — 150 to —420 mV (28).

The microbiological procedures for studying obligate anaerobes are similar to those used for the more familiar aerobic organisms, with the exception of the need to exclude oxygen from the working environment during manipulation, inoculation, and incubation of cultures and samples. As a class of organisms, anaerobes are very diversified in their behavior in terms of oxygen tolerance and nutritional requirements for growth. There is no single set of procedures for isolation of all anaerobes. Clinically important anaerobes constitute the most studied class. Specific methods were developed for isolating anaerobes from various sites in patients (57).

A detailed treatment of clinically important anaerobic organisms is recorded in the Manual for the Determination of the Clinical Role of Anaerobic Microbiology by Gall and Riely (43). This manual contains a definition of clinically important anaerobic bacteria methods for the collection and transportation of anaerobic specimens, culturing of anaerobic specimens, and identification of anaerobes.

The book Isolation of Anaerobes, by Shapton and Board (56), contains chapters describing the isolation of clostridia from animal tissues, feces, soil, and foods. Some chapters deal with the isolation of anaerobes from rumen as well as with sulfate-reducing bacteria and photosynthetic bacteria. Zeikus (62) describes the biology of methanogenic bacteria and methods of studying these organisms. In the area of food microbiology, very little attention has been given to the anaerobic microbiology. The Compendium ofMethods for the Microbiological Examination of Foods (59) contains some chapters on anaerobes, mainly on the spore formers and the detection of toxins in foods. Anderson and Fung (30) reviewed the status of anaerobic methods, techniques, and principles for food bacteriology in detail. The history of cultivation of anaerobes was detailed by Hall (44) and more recently summarized by Fung (1).

The general conclusion on isolation procedures is that samples must be free from contact with air during collection time. The best method of collecting samples is by aspiration with needle and syringe. Sample should be placed in media that are designed to handle anaerobes. A variety of liquid and solid media are marketed for anaerobic cultivation by such companies as DIFCO, BBL, UNIPATH/ OXOID, and others (69).

Currently, anaerobic cultivation systems include anaerobic jars, anaerobic glove boxes, roll tubes, anaerobic tube systems, and biological membrane systems.

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