Detection of bacterial and fungal pathogens by culture
Culture on manufactured medium is the most commonly used technique for detecting bacteria and fungi in clinical specimens. Although not as rapid as direct examination, it is more sensitive and much more specific. For the majority of human pathogens, culture requires only 1 to 2 days of incubation. For particularly slow-growing organisms, such as M. tuberculosis and some fungi, the incubation period may last for weeks. By growing the organism, it is available for further phenotypic and genotypic analysis, such as antimicrobial susceptibility testing, serotyping, virulence factor detection, and molecular epidemiology studies.
Environmental and nutritional aspect of bacterial and fungal culture
Certain basic strategies are used to recover bacterial and fungal pathogens. These strategies are dependent on the phenotypic characteristics of the organisms to be isolated and the presence of competing microbiota in a patient’s clinical specimen. Most human pathogens grow best at 37°C, human body temperature. Most bacterial and fungal cultures are performed, at least initially, at this temperature. Certain skin pathogens, such as dermatophytes and some Mycobacterium spp., grow better at 30°C. When seeking these organisms, cultures may be done at this lower temperature. A few clinically significant microorganisms will grow at low temperatures (4°C), while others prefer higher temperatures (42°C). These incubation temperatures may be used when attempting to recover a specific organism from specimens with a resident microbiota, such as feces, as few organisms other than the target organism can grow at these temperature extremes.
Another important characteristic of human bacterial and fungal pathogens is the impact of the presence of oxygen on the growth of these organisms. Microbes can be divided into three major groups based on their ability to grow in the presence of oxygen. Organisms that can only grow in the presence of oxygen are called aerobes. Fungi and many bacteria are aerobic organisms. Organisms that can only grow in the absence of oxygen are called anaerobes. The majority of bacteria that make up the resident microbiota of the gastrointestinal and female genital tracts are anaerobic organisms. Some bacteria can grow either in the presence or in the absence of oxygen. These organisms are called facultative organisms. A subgroup of facultative organisms is called microaerophiles. These organisms grow best in an atmosphere with reduced levels of oxygen. Campylobacter spp. and Helicobacter spp. are examples of microaerophiles.
Besides temperature and oxygen, nutrients are an important third factor in the growth of microbes. Many bacteria have very simple growth requirements. They require an energy and carbon source, such as glucose; a nitrogen source, which may be ammonium salts or amino acids; and trace amounts of salts and minerals, especially iron. Some human pathogens have much more complex growth requirements, needing certain vitamins or less well-defined nutrients such as animal serum. Organisms with highly complex growth requirements are often referred to as being fastidious. A fastidious bacterium that is frequently encountered clinically is Haemophilus influenzae. This bacterium requires both hemin, an iron-containing molecule, and NAD for growth.
Media
The selection of media to be used in isolation of pathogens from clinical specimens is dependent on several factors. First, the nutritional requirements of the specific pathogen must be met. For example, fastidious organisms require a medium that is enriched with specific nutrients, such as animal blood, serum, or other growth factors. If the clinical specimen is obtained from a site that has a resident microbiota, certain strategies will be necessary to isolate a specific pathogen from the accompanying resident microbiota. Often in this setting, a special type of medium called selective medium is used to recover these pathogens. This medium selects for the growth of a specific group of organisms. This is done by adding substances, such as dyes, antibiotics, or bile salts, that inhibit the growth of one group of organisms while permitting the growth of another. For example, MacConkey agar is a selective medium that contains bile salts and the dye crystal violet. These two substances are inhibitory for Gram-positive organisms as well as some Gram-negative ones. A wide variety of Gram-negative rods grow on this medium. Some selective media are also differential. MacConkey agar is an example of a selective and differential medium. The Gram-negative bacilli that grow on this agar can be differentiated from one another on the basis of the organism’s ability to ferment the carbohydrate lactose. Organisms that ferment lactose are called lactose positive, and organisms that are unable to ferment lactose are called lactose negative (Fig. 10). When selecting media for culturing clinical specimens from sites with a resident microbiota, typically both enriched and selective media are used. If Gram-negative bacilli are a component of this microbiota, than a selective-differential medium might be used as well.
Certain organisms will not grow on media commonly used to culture clinical specimens, because the media may not be enriched enough or may contain inhibitory substances. When these organisms are sought, the laboratory must be notified so that special isolation medium can be used. Two important respiratory tract pathogens, B. pertussis and L. pneumophila, are examples of organisms that do not grow on standard laboratory media and require special media for their isolation.
Organism identification and susceptibility testing
Once organisms are isolated, they may be identified, and in some cases susceptibility testing needs to be performed. Bacteria and fungi grow as colonies on agar plates. The appearance of these colonies is often useful in determining the identity of the organism. Colonies may appear flat or raised, smooth or rough; may pit the agar; or may hemolyze red blood cells in blood-containing agar. Molds, for example, have very characteristic “fuzzy” growth on agar. Colonies of organisms such as S. aureus may be pigmented or may secrete a diffusible pigment, as seen with Pseudomonas aeruginosa. Skilled microbiologists often have a very good idea of the identification of a microorganism based solely on its colonial appearance.
In specimens that come from an area of the body with a resident microbiota, it is important to separate the colonies of organisms that may represent the resident microbiota from the colonies of organisms that may be pathogens. Much of the time, this can be done on the basis of colonial appearance. However, some potential pathogens, such as S. pneumoniae, a common cause of bacterial pneumonia, cannot be readily differentiated from viridans group streptococci, a member of the resident oropharyngeal microbiota. In patients with suspected bacterial pneumonia, a sputum specimen may be obtained. Sputum consists of secretions coughed up from the lower airways that are expectorated through the oropharynx and submitted for culture. Because they pass through the oropharynx, sputum specimens almost always contain viridans group streptococci. The appearance of colonies produced by viridans group streptococci is very similar to that produced by S. pneumoniae. To determine whether or not these colonies are S. pneumoniae, one must do tests based on the phenotypic characteristics of the organism; these are referred to as biochemical tests. The biochemical test that is done most often to distinguish between these two organisms is the disk diffusion test, in which the organism’s susceptibility to the compound optochin is examined. S. pneumoniae (Fig. 11) is susceptible to optochin, while the viridans group streptococci are not. On the basis of this easily performed test, the identity of S. pneumoniae can be determined from a sputum specimen.
