The medium consists of a base containing a protein source (e.g., tryptones), soybean protein digest (containing a slight amount of natural carbohydrate), sodium chloride, agar, and 5% sheep blood.
Certain bacteria produce extracellular enzymes that lyse red blood cells in the agar (hemolysis). This activity can result in complete clearing of the red blood cells around the bacterial colony (beta hemolysis) or in only partial lysis of the cells to produce a greenish discoloration around the colony (alpha hemolysis). Other bacteria have no effect on the red blood cells, and no halo is produced around the colony (gamma or nonhemolysis). Microbiologists often use colony morphology and the degree or absence of hemolysis as criteria for determining what additional steps will be necessary for identification of a bacterial isolate. To read the hemolytic reaction on a blood agar plate accurately, the technologist must hold the plate up to the light and observe the plate with the light coming from behind (i.e., transmitted light).
Thayer-Martin Agar. Thayer-Martin agar is an enrichment and selective medium for the isolation of Neisseria gonorrhoeae, the causative agent of gonorrhea, and Neisseria meningitidis, a life-threatening cause of meningitis. The enrichment portion of the medium is the basal components and the chocolatized blood, while the addition of antibiotics provides a selective capacity. The antibiotics include colistin to inhibit other gram-negative bacteria, vancomycin to inhibit grampositive bacteria, and nystatin to inhibit yeast. The antimicrobial trimethoprim is also added to inhibit Proteus spp., which tend to swarm over the agar surface and mask the detection of individual colonies of the two pathogenic Neisseria spp. A further modification, Martin-Lewis agar, substitutes ansa-mycin for nystatin and has a higher concentration of vancomycin.
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 Kl, 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
Figure 7-7 Growth characteristics of various bacteria in thioglycollate broth. A, Facultatively anaerobic gram-negative bacilli (i.e., those that grow in the presence or absence of oxygen) grow throughout broth. B, Gram-positive cocci grow as "puff balls." C, Strictly aerobic organisms (i.e., those that require oxygen for growth), such as Pseudomonas aeruginosa, grow toward the top of the broth. D, Strictly anaerobic organisms (i.e., those that do not grow in the presence of oxygen) grow in the bottom of the broth.
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, allows 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 frequently grow as discrete "puffballs." 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 uninoculated medium). Colonies of Salmonella spp. are also colorless on XID, 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 nonpathogens ferment one or more of the sugars and produce yellow colonies (Figure 7-8).
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 those that are 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.
Media Sterilization. The timing of autoclave sterilization should start from the moment the temperature reaches 121° C and usually requires a minimum of 15 minutes. Once the sterilization cycle is completed, molten agar is allowed to cool to approximately 50° C before being distributed to individual petri
plates (usually 25 mL of molten agar per plate). If other ingredients are to be added (e.g., supplements such as sheep blood or specific vitamins, nutrients, or antibiotics), they should be incorporated when the molten agar has cooled, just before distribution to plates.
Delicate media components that cannot withstand steam sterilization by autoclaving (e.g., serum, certain carbohydrate solutions, certain antibiotics, and other heat-labile substances) can be sterilized by membrane filtration. Passage of solutions through membrane filters with pores ranging in size from 0.2 to 0.45 fim in diameter will not remove viruses but does effectively remove most bacterial and fungal contaminants. Finally, all media, whether purchased or prepared, must be subjected to stringent quality control before being used in the diagnostic setting (for more information regarding quality control see Chapter 63).
Cell Cultures. Although most bacteria grow readily on artificial media, certain pathogens require factors provided only by living cells. These bacteria are obligate intracellular parasites that require viable host cells for propagation. Although all viruses are obligate intracellular parasites, chlamydiae, rickettsiae, and rickettsiae-like organisms are bacterial pathogens that require living cells for cultivation.
The cultures for growth of these bacteria comprise layers of living cells growing on the surface of a solid matrix such as the inside of a glass tube or the bottom of a plastic flask. The presence of bacterial pathogens within the cultured cells is detected by specific changes in the cells' morphology. Alternatively, specific stains, composed of antibody conjugates, may be used to detect bacterial antigens within the cells. Cell cultures may also detect certain bacterial toxins (e.g., Clostridium difficile cytotoxin). Cell cultures are most commonly used in diagnostic virology. Cell culture maintenance and inoculation is addressed in Chapter 51.
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