General Concepts of Culture Media

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.

Phases of Growth Media

Growth media are used in either of two phases: liquid (broth) or solid (agar). In 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). More growth indicates a higher cell density and greater turbidity. At least 10⁶ bacteria per milliliter of broth are needed for turbidity to be detected with the unaided eye.

In addition to amount of growth present, the location of growth within thioglycollate broth indicates the type of organism present based on oxygen requirements. Strict anaerobes will grow at the bottom of the broth tube, whereas aerobes will grow near the surface. Microaerophilic organisms will glow slightly below the surface where oxygen concentrations are lower than atmospheric concentrations. In addition, facultative anaerobes and aerotolerant organisms will grow throughout the medium, as they are unaffected by the variation in oxygen content.

A solid medium is a combination of a solidifying agent added to the nutrients and water. Agarose, the most common solidifying agent, has the unique property of melting at high temperatures (≥95° C) but re-solidifying only after its temperature falls below 50° C. The 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 55° C 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 pure colony. In other words, all bacterial cells within a single colony are the same genus and species, having identical genetic and phenotypic characteristics (i.e., are derived from a single clone). 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 Classifications and Functions

Media are categorized according to their function and use. In diagnostic bacteriology there are four general categories of media: enrichment, nutritive, 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 providing specific nutrients for the organism’s growth. 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).

Enrichment media may also contain specialized enrichment broths used to enhance the growth of organisms present in low numbers. Broths may be used to ensure growth of an organism when no organisms grow on solid media following initial specimen inoculation. Enrichment broths used in the clinical laboratory often include thioglycollate for the isolation of anaerobes, LIM broth for selective enrichment of group B streptococci, and gram-negative (GN) broth for the selective enrichment of enteric gram-negative organisms.

Nutritive media or supportive media contain nutrients that support growth of most nonfastidious organisms without giving any particular organism a growth advantage. Nutrient media include tryptic soy agar, or nutrient agar plates for bacteria or Sabouraud’s dextrose agar for fungi. Selective media contain one or more agents that are inhibitory to all organisms except those “selected” by the specific growth condition or chemical. 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 (PEA) agar, which inhibits the growth of aerobic and facultatively anaerobic gram-negative rods and allows gram-positive cocci to grow (Figure 7-3). Selective and inhibitory chemicals included within nutritive media prevent the overgrowth of normal flora or contaminating organisms that would prevent the identification of pathogenic organisms. However, it is important to note that the use of selective media does not ensure that the inhibited organisms are not present in small quantity and may simply be too small to see.

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 it 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 or combination media because it will not allow most gram-positive bacteria to grow. Another example is sheep blood agar. This is the most commonly used nutritive 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, as indicated in Figure 5-2. Figure 7-5 shows differential hemolytic patterns by various organisms.

Summary of Artificial Media for Routine Bacteriology

Various broth and agar media that have enrichment, selective, 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 60) use similar media strategies; details regarding these media are provided in the appropriate chapters.

Of the dozens of available media, only those most commonly used for routine diagnostic bacteriology are summarized in this discussion. Part VII discusses which media should be used to culture bacteria from various clinical specimens. Similarly, other chapters throughout Part III discuss media used to identify and characterize specific organisms.

Hektoen Enteric (HE) Agar.

Hektoen enteric (HE) agar contains bile salts and dyes (bromthymol blue and acid fuchsin) to selectively slow the growth of most nonpathogenic gram-negative bacilli found in the gastrointestinal tract and allow Salmonella spp. and Shigella spp. to grow. The medium is also differential because many non-enteric pathogens that do grow will appear as orange to salmon-colored colonies. This colony appearance results from the organism’s ability to ferment the lactose in the medium, resulting in the production of acid, which lowers the medium’s pH and causes a change in the pH indicator bromthymol blue. Salmonella spp. and Shigella spp. do not ferment lactose, so no color change occurs and their colonies maintain the original blue-green color of the medium. As an additional differential characteristic, the medium contains ferric ammonium citrate, an indicator for the detection of H₂S, so that H₂S-producing organisms, such as Salmonella spp., can be visualized as colonies exhibiting a black precipitate (Figure 7-6).

Thioglycollate Broth.

Thioglycollate broth is the enrichment broth most frequently used in diagnostic bacteriology. The broth contains many nutrient factors, including casein, yeast and beef extracts, and vitamins, to enhance the growth of most medically important bacteria. Other nutrient supplements, an oxidation-reduction indicator (resazurin), dextrose, vitamin K1, and hemin have been used to modify the basic thioglycollate formula. In addition, this medium contains 0.075% agar to prevent convection currents from carrying atmospheric oxygen throughout the broth. This agar supplement and the presence of thioglycolic acid, which acts as a reducing agent to create an anaerobic environment deeper in the tube, allow anaerobic bacteria to grow.

Gram-negative, facultatively anaerobic bacilli (i.e., those that can grow in the presence or absence of oxygen) generally produce diffuse, even growth throughout the broth, whereas gram-positive cocci demonstrate flocculation or clumps. Strict aerobic bacteria (i.e., require oxygen for growth), such as Pseudomonas spp., tend to grow toward the surface of the broth, whereas strict anaerobic bacteria (i.e., those that cannot grow in the presence of oxygen) grow at the bottom of the broth (Figure 7-7).

Xylose-Lysine-Desoxycholate (XLD) Agar.

As with HE agar, xylose-lysine-desoxycholate (XLD) agar is selective and differential for Shigella spp. and Salmonella spp. The salt, sodium desoxycholate, inhibits many gram-negative bacilli that are not enteric pathogens and inhibits gram-positive organisms. A phenol red indicator in the medium detects increased acidity from carbohydrate (i.e., lactose, xylose, and sucrose) fermentation. Enteric pathogens, such as Shigella spp., do not ferment these carbohydrates, so their colonies remain colorless (i.e., the same approximate pink to red color of the un-inoculated medium). Even though they often ferment xylose, colonies of Salmonella spp. are also colorless on XLD, because of the decarboxylation of lysine, which results in a pH increase that causes the pH indicator to turn red. These colonies often exhibit a black center that results from Salmonella spp. producing H₂S. Several of the nonpathogenic organisms ferment one or more of the sugars and produce yellow colonies (Figure 7-8).

Preparation of Artificial Media

Nearly all media are commercially available as ready-to-use agar plates or tubes of broth. If media are not purchased, laboratory personnel can prepare agars and broths using dehydrated powders that are reconstituted in water (distilled or deionized) according to manufacturer’s recommendations. Generally, media are reconstituted by dissolving a specified amount of media powder, which usually contains all necessary components, in water. Boiling is often required to dissolve the powder, but specific manufacturer’s instructions printed in media package inserts should be followed exactly. Most media require sterilization so that only bacteria from patient specimens will grow and not contaminants from water or the powdered media. Broth media are distributed to individual tubes before sterilization. Agar media are usually sterilized in large flasks or bottles capped with either plastic screw caps or plugs before being placed in an autoclave.