The pH scale is a measure of the hydrogen ion concentration of an organism's 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.
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 taken to ensure appropriate moisture levels are maintained throughout the incubation period.
Although heating blocks and temperature-controlled water baths may be used occasionally, incubators are the primary laboratory devices used to provide the environmental 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 (Section 13), mycobacteria (Section 14), fungi (Chapter 50), and viruses (Chapter 51) 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% C02. Incubators containing room air may be used for some media, but the lack of increased C02 may hinder the growth of certain bacteria.
Various atmosphere-generating systems are commercially available and are used instead of C02-generating incubators. For example, a self-contained culture medium and a compact C02-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 C02 to support growth of the pathogen. As an alternative to commercial systems, a candle jar can also generate a C02 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 up just enough oxygen before it goes out (from lack of oxygen) to lower the oxygen tension and produce C02 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, if not sooner. Certain anaerobic bacteria may require longer incubation, and mycobacteria frequently take weeks before detectable growth occurs.
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