As discussed in Chapter 2, bacteria have numerous nutritional needs that include different gases, water, various ions, nitrogen, sources for carbon, and energy. The latter two requirements are most commonly provided by carbohydrates (e.g., sugars and their derivatives) and proteins.
In the laboratory, nutrients are incorporated into culture media on or in which bacteria are grown. If a culture medium meets a bacterial cell's growth requirements, then that cell will multiply to sufficient numbers to allow visualization by the unaided eye. Of course, bacterial growth after inoculation also requires that the medium be placed in optimal environmental conditions.
Because different pathogenic bacteria have different nutritional needs, various types of culture media have been developed for use in diagnostic microbiology. For certain bacteria the needs are relatively complex, and exceptional media components must be used for growth. Bacteria with such requirements are said to be fastidious. Alternatively, the nutritional needs of most clinically important bacteria are relatively basic and straightforward. These bacteria are considered nonfastidious.
Growth media are used in either of two phases: liquid (broth) or solid (agar), hi some instances (e.g., certain blood culture methods), a biphasic medium that contains both a liquid and a solid phase may be used.
In broth media, nutrients are dissolved in water, and bacterial growth is indicated by a change in the broth's appearance from clear to turbid (i.e., cloudy). The turbidity, or cloudiness, of the broth is due to light deflected by bacteria present in the culture (Figure 7-1). The more bacteria growth, the greater the turbidity. At least 106 bacteria per milliliter of broth are needed for turbidity to be detected with the unaided eye.
Solid media are made by adding a solidifying agent to the nutrients and water. Agarose^ the most common solidifying agent, has the unique property of melting at high temperatures (a95° C) but resolidifying only after its temperature falls below 50° C. Addition of agar allows a solid medium to be prepared by heating to an extremely high temperature, which is required for sterilization, and cooling to 55a to 60° C for distribution into petri dishes. On further cooling, the agarose-containing medium forms a stable solid gel referred to as agar. The petri dish containing the agar is referred to as the agar plate. Different agar media usually are identified according to the major nutritive components of the medium (e.g., sheep blood agar, bile esculin agar, xylose-lysine-desoxycholate agar).
With appropriate incubation conditions, each bacterial cell inoculated onto the agar medium surface will proliferate to sufficiently large numbers to be observable with the unaided eye (see Figure 7-1). The resulting bacterial population is considered to be derived from a single bacterial cell and is known as a colony, hi other words, all bacterial cells within a single colony are the same genus and species, having identical genetic and phenotypic characteristics (i.e., are a single clone). Bacterial cultures derived from a single colony or clone are considered "pure." Pure cultures are required for subsequent procedures used to identify and characterize bacteria. The ability to select pure (individual) colonies is one of the first and most important steps required for bacterial identification and characterization.
Media are categorized according to their function and use. In diagnostic bacteriology there are four general categories of media: enrichment, supportive, selective, and differential.
Enrichment media contain specific nutrients required for the growth of particular bacterial pathogens that may be present alone or with other bacterial species in a patient specimen. This media type is used to enhance the growth of a particular bacterial pathogen from a mixture of organisms by using nutrient specificity. One example of such a medium is buffered charcoal-yeast extract agar, which provides l-cysteine and other nutrients required for the growth of Legionella pneumophila, the causative agent of legionnaires' disease (Figure 7-2).
Supportive media contain nutrients that support growth of most nonfastidious organisms without giving any particular organism a growth advantage. Selective media contain one or more agents that are inhibitory to all organisms except those being sought. In other words, these media select for the growth of
certain bacteria to the disadvantage of others. Inhibitory agents used for this purpose include dyes, bile salts, alcohols, acids, and antibiotics. An example of a selective medium is phenylethyl alcohol agar, which inhibits the growth of aerobic and facultatively anaerobic gram-negative rods and allows gram-positive cocci to grow (Figure 7-3).
Differential media employ some factor (or factors) that allows colonies of one bacterial species or type to exhibit certain metabolic or culture characteristics that can be used to distinguish them from other bacteria growing on the same agar plate. One commonly used differential medium is MacConkey agar, which differentiates between gram-negative bacteria that can and cannot ferment the sugar lactose (Figure 7-4).
Of importance, many media used in diagnostic bacteriology provide more than one function. For example, MacConkey agar is both differential and selective because it will not allow most gram-positive bacteria to grow. Another example is sheep blood
agar. This is the most commonly used supportive medium for diagnostic bacteriology because it allows many organisms to grow. However, in many ways this agar is also differential because the appearance of colonies produced by certain bacterial species is readily distinguishable (Figure 7-5).
Summary of Artificial Media for Routine Bacteriology
Various broth and agar media that have enrichment, selective, and/or differential capabilities and are used frequently for routine bacteriology are listed alphabetically in Table 7-1. Anaerobic bacteriology (Section 13), mycobacteriology (Section 14), and mycology (Chapter 50) use similar media strategies; details regarding these media are provided in the appropriate chapters.
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