Optimizing the environmental conditions to support the most robust growth of clinically relevant bacteria is as important as meeting the organism's nutritional needs for in vitro cultivation. The four most critical environmental factors to consider include oxygen and carbon dioxide (C02) availability, temperature, pH, and moisture content of medium and atmosphere.
Most clinically relevant bacteria are either aerobic, facultatively anaerobic, or strictly anaerobic. Aerobic bacteria use oxygen as a terminal electron acceptor and grow well in room aif, Most clinically significant aerobic organisms are actually facultatively anaerobic, being able to grow in the presence (i.e., aerobically) or absence (i.e., anaerobically) of oxygen. However, some bacteria, such as Pseudomonas spp., members of the Neisseriaceae family, Brucella spp., Bordetella spp., and Francisella spp., are strictly aerobic and cannot grow in the absence of oxygen. Other aerobic bacteria require only low levels of oxygen and are referred to as being microaerophilic, or microaerobic. Anaerobic bacteria are unable to use oxygen as an electron acceptor, but some aerotolerant strains will still grow slowly and poorly in the presence of oxygen. Oxygen is inhibitory or lethal for strictly anaerobic bacteria.
In addition to oxygen, the availability of C02 is important for growth of certain bacteria. Organisms that grow best with higher C02 concentrations <i.e., 5% to 10% C02) than is provided in room air are referred to as being capnophilic. For some bacteria, a 5% to 10% C02 concentration is essential for successful cultivation from patient specimens.
Bacterial pathogens generally multiply best at temperatures similar to those of internal human host tissues and organs (i.e., 37° C). Therefore, cultivation of most medically important bacteria is done using incubators with temperatures maintained in the 35" to 37° C range. For others, an incubation temperature of 30° C (i.e., the approximate temperature of the body's surface) may be preferable, but such bacteria are encountered relatively infrequentiy so that use of this incubation temperature occurs only when dictated by special circumstances.
Recovery of certain organisms can be enhanced by incubation at other temperatures. For example, the gastrointestinal pathogen Campylobacter jejuni grows at 42° C, whereas many other pathogens and nonpatho-gens cannot. Therefore, incubation at this temperature can be used as an enrichment procedure. Other bacteria, such as Listeria monocytogenes and Yersinia enterocolitica, can grow at 0° C, but grow best at temperatures between 20° and 40° C. Cold enrichment has been used to enhance the recovery of these organisms in the laboratory.
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