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 environ mental factors to consider include oxygen and carbon dioxide (CO2) availability, temperature, pH, and mois ture content of medium and atmosphere.
Oxygen and Carbon Dioxide Availability
Most clinically relevant bacteria are aerobic, facultatively anaerobic, or strictly anaerobic. Aerobic bacteria use oxygen as a terminal electron acceptor and grow well in room air. 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 (approximately 20%) and are referred to as being micro aerophilic, 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 CO2 is important for growth of certain bacteria. Organisms that grow best with higher CO2 concentrations (i.e., 5% to 10% CO2) than is provided in room air are referred to as being capnophilic. For some bacteria, a 5% to 10% CO2 concentration is essential for successful cultivation from patient specimens.
Temperature
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 relevant bacteria is done using incubators with temperatures maintained in the 35° C 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 infrequently 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 is able to grow at 42° C. Therefore, incubation at this temperature can be used as an enrichment procedure. Other bacteria, such as Listeria monocytogenes and Yersinia enterocolitica, can grow at 4° C to 43° 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.
pH
The pH scale is a measure of the hydrogen ion concentration in the environment, with a pH value of 7 being neutral. Values less than 7 indicate the environment is acidic; values greater than 7 indicate alkaline conditions. Most clinically relevant bacteria prefer a near neutral pH range, from 6.5 to 7.5. Commercially prepared media are buffered in this range so that checking their pH is rarely necessary.
Moisture
Water is provided as a major constituent of both agar and broth media. However, when media are incubated at the temperatures used for bacterial cultivation, a large portion of water content can be lost by evaporation. Loss of water from media can be deleterious to bacterial growth in two ways: (1) less water is available for essential bacterial metabolic pathways and (2) with a loss of water, there is a relative increase in the solute concentration of the media. An increased solute concentration can osmotically shock the bacterial cell and cause lysis. In addition, increased atmospheric humidity enhances the growth of certain bacterial species. For these reasons, measures such as sealing agar plates to trap moisture or using humidified incubators are utilized to ensure appropriate moisture levels are maintained throughout the incubation period.
Methods for Providing Optimum Incubation Conditions
Although heating blocks and temperature-controlled water baths may be used occasionally, incubators are the primary laboratory devices used to provide the environ mental conditions required for cultivating microorganisms. The conditions of incubators can be altered to accommodate the type of organisms to be grown. This section focuses on the incubation of routine bacteriology cultures. Conditions for growing anaerobic bacteria, mycobacteria, fungi, and viruses are covered in other areas of the text.
Once inoculated with patient specimens, most media are placed in incubators with temperatures maintained between 35° and 37° C and humidified atmospheres that contain 3% to 5% CO2. It is important to note that some media that contain pH indicators may not be placed in CO2 incubators. The presence of CO2 will acidify the media, causing the pH indicator to change color and thereby disrupt the differential properties of the media (e.g., Hektoen-Enteric agar and MacConkey agar). Incubators containing room air may be used for some media, but the lack of increased CO2 may hinder the growth of certain bacteria.
Various atmosphere-generating systems are commercially available and are used instead of CO2-generating incubators. For example, a self-contained culture medium and a compact CO2-generating system can be used for culturing fastidious organisms such as Neisseria gonorrhoeae. A tablet of sodium bicarbonate is dissolved by the moisture created within an airtight plastic bag and releases sufficient CO2 to support growth of the pathogen. As an alternative to commercial systems, a candle jar can also generate a CO2 concentration of approximately 3% and has historically been used as a common method for cultivating certain fastidious bacteria. The burning candle, which is placed in a container of inoculated agar plates that is subsequently sealed, uses just enough oxygen before it goes out (from lack of oxygen) to lower the oxygen tension and produce CO2 and water by combustion. Other atmosphere-generating systems are available to create conditions optimal for cultivating specific bacterial pathogens (e.g., Campylobacter spp. and anaerobic bacteria).
Finally, the duration of incubation required for obtaining good bacterial growth depends on the organisms being cultured. Most bacteria encountered in routine bacteriology will grow within 24 to 48 hours. Certain anaerobic bacteria may require longer incubation, and mycobacteria frequently take weeks before detectable growth occurs.
