Culturing is routine for most bacterial and fungal infections but is rarely used to identify helminths or protozoa. Culturing of many pathogens is straightforward, for example, streaking a throat swab onto a blood agar plate in search of group A β-hemolytic streptococcus. However, certain pathogens are very slow growing (for example, M. tuberculosis) or are cultured only with difficulty (for example, Bartonella henselae). Microorganisms isolated in culture are identified using such characteris tics as colony size, shape, color, Gram stain, hemolytic reactions on solid media, odor, and metabolic properties. In addition, pure cultures provide samples for antimicrobial susceptibility testing . The success of culturing depends on appropriate collection and transport techniques and on selection of appropriate culture media, because some organisms may require special nutrients. Also, some media are used to suppress the growth of certain organisms in the process of identifying others

 A. Specimen collection

 Many organisms are fragile and must be transported to the laboratory with minimal delay. For example, gonococci and pneumococci are very sensitive to heating and drying. Samples must be cultured promptly, or, if this is not possible, transport media must be used to extend the viability of the organism to be cultured. When anaerobic organisms are suspected, the patient’s specimen must be protected from the toxic effect of oxygen (Figure 1).

Fig1. With anaerobic transport media containing a nonnutritive medium that retards diffusion of oxygen after addition of the specimen, microorganisms may remain viable for up to 72 hours. It is important to use an airless syringe for liquid specimens, such as pus, rather than a swab in a tube— especially when transport medium is not available. Also, if promptly transported in a syringe, the relative proportions of each morphology in mixed infections is visible.

 B. Growth requirements

 All clinically important bacteria are heterotrophs (that is, they require organic carbon for growth). Heterotrophs may have complex or simple requirements for organic molecules. [Note: Organisms that can reduce carbon dioxide and, therefore, do not require organic compounds for cell growth, are called autotrophs.] Most bacteria require varying numbers of growth factors, which are organic com pounds required by the cell to grow, but which the organism cannot itself synthesize (for example, vitamins). Organisms that require either a large number of growth factors or must be supplied with very specific ones are referred to as fastidious.

 C. Oxygen requirements

 Bacteria can be categorized according to their growth responses in the presence and absence of oxygen. Strict aerobes cannot survive in the absence of oxygen and produce energy only by oxidative phosphorylation. Strict anaerobes generate energy by fermentation or by anaerobic respiration and are killed in the presence of oxygen. Facultative anaerobes can grow in the absence of oxygen but grow better in its presence. Aerotolerant anaerobes have mechanisms to protect themselves from oxygen (therefore, being able to grow in its presence or absence) but do not use oxygen in their metabolism. Finally, microaerophiles require oxygen for their metabolism but cannot survive at atmospheric levels of oxygen. Microaerophiles are found in lakes and wet soil where the oxygen concentration is within an acceptable range.

D. Media

 Two general strategies are used to isolate pathogenic bacteria, depending on the nature of the clinical sample. The first method uses enriched media to promote the nonselective growth of any bacteria that may be present. The second approach employs selective media that only allow growth of specific bacterial species from specimens that normally contain large numbers of bacteria (for example, stool, genital tract secretions, and sputum). Isolation of a bacterium is usually performed on solid medium. Liquid medium is used to grow larger quantities of a culture of bacteria that have already been isolated as a pure culture.

 1. Enriched media: Media fortified with blood, yeast extracts, or brain or heart infusions are useful in growing fastidious organisms. For example, sheep blood agar contains protein sources, sodium chloride, and 5 percent sheep blood and supports the growth of most gram-positive and gram-negative bacteria isolated from human sources. However, Haemophilus influenzae and Neisseria gonorrhoeae, among others, are highly fastidious organisms. They require chocolate agar, which contains red blood cells (RBCs) that have been lysed . This releases intracellular nutrients, such as hemoglobin, hemin (“X” factor), and nicotinamide adenine dinucleotide (“V” factor), required by these organisms. Enriched media are useful for culturing normally sterile body fluids, such as blood and CSF, in which the finding of any organisms provides reasonable evidence for infection by that organism. Failure to culture an organism may indicate that the culture medium is inadequate or that the incubation conditions do not support bacterial growth.

2. Selective media: The most commonly used selective medium is MacConkey agar , which supports the growth of most gram-negative rods, especially the Enterobacteriaceae, but inhibits growth of gram-positive organisms and some fastidious gram-negative bacteria, such as Haemophilus and Neisseria species. Growth on blood agar and chocolate agar but not MacConkey agar suggests a gram-positive isolate or a fastidious gram-negative species. In contrast, most gram-negative rods often form distinctive colonies on MacConkey agar. This agar is also used to detect organisms able to metabolize lactose (Figure 2). Clinical samples are routinely plated on blood agar, chocolate agar, and MacConkey agar. Hektoen enteric agar is also a selective medium that differentiates lactose/sucrose fermenters and nonfermenters as well as H₂S producers and nonproducers. It is often used to culture Salmonella and Shigella species. Thayer Martin agar is another selective medium composed of chocolate agar supplemented with several antibiotics that suppress the growth of nonpathogenic Neisseria and other normal and abnormal flora. This medium is normally used to isolate gonococci. When submitting samples for culture, the physician must alert the laboratory to likely pathogens whenever possible, especially when unusual organisms are suspected. This allows inclusion of selective media that might not be used routinely. It also alerts the laboratory to hold specimens longer if a slow-growing organism, such as Nocardia, is suspected.

Fig2. Lactose-fermenting, gram-negative rods produce pink colonies on MacConkey agar.

مواضيع ذات صلة

Immunologic Detection of Microorganisms

Identification of Bacteria

Cultivation of Listeria, Corynebacterium, and Similar Organisms

Pathogenesis and Spectrum of Disease for Listeria, Corynebacterium, and Similar Organisms

The Growth Curve in Batch Culture

The Measurement of Microbial Concentrations

Bacterial Pathogenesis

Distribution of Normal Flora in The Body

Cultivation of Bacillus and Similar Organisms

Diagnostic Laboratory Tests for Borrelia Burgdorferi

Bacterial Cell wall

Bacillus cereus