Growth and identification of the infecting agent in vitro is usually the most sensitive and specific means of diagnosis and is, thus, the method most commonly used. Theoretically, the presence of a single live organism in the specimen can yield a positive result. Most bacteria and fungi can be grown in a variety of artificial media, but strictly intracellular microorganisms (eg, Chlamydia, Rickettsia, and viruses) can be isolated only in cultures of living eukaryotic cells. The culture of some parasites is possible, but used only in highly specialized laboratories.
Isolation and Identification of Bacteria and Fungi
Almost all medically important bacteria can be cultivated outside the host in artificial culture media. A single bacterium placed in the proper culture conditions multiplies to quantities sufficient to be seen by the naked eye. Bacteriologic media are soup-like recipes prepared from digests of animal or vegetable protein supplemented with nutrients such as glucose, yeast extract, serum, or blood to meet the metabolic requirements of the organism. Their chemical composition is complex, and their success depends on matching the nutritional requirements of most heterotrophic living things. The same approaches as well as some of the same culture media used for bacteria are also used for fungi.
Bacteria grow in soup-like media
Growth in media prepared in the fluid state (broths) is apparent when bacterial numbers are sufficient to produce turbidity or macroscopic clumps. Turbidity results from reflection of transmitted light by the bacteria; depending on the size of the organism, more than 106 bacteria per milliliter of broth are required. The addition of a gelling agent to a broth medium allows its preparation in solid form as plates in Petri dishes. The universal gelling agent for diagnostic bacteriology is agar—a polysaccharide extracted from seaweed. Agar has the convenient property of becoming liquid at approximately 95°C but not returning to the solid gel state until cooled to less than 50°C. This allows the addition of a heat-labile substance such as blood to the medium before it sets. At temperatures used in the diagnostic laboratory (37°C or lower), broth–agar exists as a smooth, solid, nutrient gel. This medium, usually termed “agar,” may be qualified with a description of any supplement (eg, blood agar).
Large numbers of bacteria in broth produce turbidity
Agar is a convenient gelling agent for solid media
A useful feature of agar plates is that the bacteria can be separated by spreading a small sample of the specimen over the surface. Bacterial cells that are well separated from others grow as isolated colonies, often reaching 2 to 3 mm in diameter after overnight incubation. This allows isolation of bacteria in pure culture because the colony is assumed to arise from a single organism (Figure 4–7). Colonies vary greatly in size, shape, texture, color, and other features called colonial morphology. Colonies from different species or genera often differ substantially, whereas those derived from the same strain are usually consistent. Differences in colonial morphology are very useful for separating bacteria in mixtures and as clues to their identity. Some examples of colonial morphology are shown in Figure 4–8.
Bacteria may be separated in isolated colonies on agar plates
Colonies may have consistent and characteristic features
Bacteriologic plate streaking. Plate streaking is essentially a dilution procedure. A. (1) The specimen is placed on the plate with a swab, loop, or pipette and evenly spread over approximately part of plate surface with a sterilized bacteriologic loop (2-5). The loop is flamed to remove residual bacteria, and a series of overlapping streaks are made flaming the loop between each one. B. After overnight incubation, heavy growth is seen in the primary areas followed by isolated colonies. More than one organism is present because both a red and a clear colony are seen. (Reproduced with permission from Willey JM: Prescott, Harley, & Klein’s Microbiology, 7th edition. McGraw-Hill, 2008.)
Bacterial colonial morphology. The colonies formed on agar plates by three different gram-negative bacilli are shown at the same magnification. Each is typical for its species, but variations are common. A. Escherichia coli colonies are flat with an irregular scalloped edge. B. Klebsiella pneumoniae colonies with a smooth entire edge and a raised glistening surface. C. Pseudomonas aeruginosa colonies with an irregular reflective surface, suggesting hammered metal.
New methods that do not depend on visual changes in the growth medium or colony formation are also used to detect bacterial growth in culture. These techniques include optical, chemical, and electrical changes in the medium, produced by the growing numbers of bacterial cells or their metabolic products. Many of these methods are more sensitive than classic techniques and, thus, can detect growth hours, or even days, earlier than traditional methods. Some have also been engineered for instrumentation and automation. For example, one fully automated system that detects bacterial metabolism fluorometrically can complete a bacterial identification and antimicrobial susceptibility test in 2 to 4 hours.
Optical, chemical, and electrical methods can detect growth
Over the last 100 years, countless media have been developed by microbiologists to aid in the isolation and identification of medically important bacteria and fungi. Only a few have found their way into routine use in clinical laboratories. These may be classified as nutrient, selective, or indicator media.
The nutrient component of a medium is designed to satisfy the growth requirements of the organism to permit isolation and propagation. For medical purposes, the ideal medium would allow rapid growth of all agents. No such medium exists; however, several suffice for good growth of most medically important bacteria and fungi. These media are prepared with enzymatic or acid digests of animal or plant products such as muscle, milk, or soybeans. The digest reduces the native protein to a mixture of polypeptides and amino acids that also includes trace metals, coenzymes, and various undefined growth factors. For example, one common broth contains a digest of casein (milk curd) and a digest of soybean meal. To this nutrient base, salts, vitamins, or body fluids such as serum may be added to provide pathogens with the conditions needed for optimum growth.
Media are prepared from animal or plant products
Selective media are used when specific pathogenic organisms are sought in sites with an extensive microbiota (eg, Campylobacter species in fecal specimens). In these cases, other bacteria may overgrow the suspected etiologic species in simple nutrient media, either because the pathogen grows more slowly or because it is present in much smaller numbers. Selective media usually contain dyes, other chemical additives, or antimicrobial agents at concentrations designed to inhibit contaminating flora but not the suspected pathogen.
Unwanted organisms are inhibited with chemicals or antimicrobials
Indicator media contain substances designed to demonstrate biochemical or other features characteristic of specific pathogens or organism groups. The addition to the medium of one or more carbohydrates and a pH indicator is frequently used. A color change in a colony indicates the presence of acid products and thus of fermentation or oxidation of the carbohydrate by the organism. The addition of red blood cells (RBCs) to plates allows the hemolysis produced by some organisms to be used as a differential feature. In practice, nutrient, selective, and indicator properties are often combined to various degrees in the same medium. It is possible to include an indicator system in a highly nutrient medium and also make it selective by adding appropriate antimicrobials. Some examples of culture media commonly used in diagnostic microbiology are listed in Appendix 4–1, and more details of their constitution and application are provided in Appendix 4–2.
Metabolic properties of bacteria are demonstrated by substrate and indicator systems
After inoculation, cultures of most aerobic bacteria are placed in an incubator with temperature maintained at 35°C to 37°C. Slightly higher or lower temperatures are used occasionally to selectively favor a certain organism or organism group. Most bacteria that are not obligate anaerobes grow in air; however, CO2 is required by some and enhances the growth of others. Incubators that maintain a 2% to 5% concentration of CO2 in air are frequently used for primary isolation, because this level is not harmful to any bacteria and improves isolation of some. A simpler method is the candle jar, in which a lighted candle is allowed to burn to extinction in a sealed jar containing plates. This method adds 1% to 2% CO2 to the atmosphere. Some bacteria (eg, Campylobacter) require a microaerophilic atmosphere with reduced oxygen (5%) and increased CO2 (10%) levels to grow. This can be achieved by using a commercially available packet that is placed in a jar which is then sealed similar to the anaerobic system described further.
Incubation temperature and atmosphere vary with organism
Strictly anaerobic bacteria do not grow under the conditions just described, and many die when exposed to atmospheric oxygen or high oxidation–reduction potentials. Most medically important anaerobes grow in the depths of liquid or semisolid media containing any of a variety of reducing agents, such as cysteine, thioglycollate, ascorbic acid, or even iron filings. An anaerobic environment for incubation of plates can be achieved by replacing air with a gas mixture containing hydrogen, CO2, and nitrogen and allowing the hydrogen to react with residual oxygen on a catalyst to form water. A convenient commercial system accomplishes this chemically in a packet that is added before the jar is sealed. Specimens suspected to contain significant anaerobes should be processed under conditions designed to minimize exposure to atmospheric oxygen at all stages.
Anaerobes require reducing conditions and protection from oxygen
Clinical Microbiology Procedures
Routine laboratory procedures for processing specimens from various sites are needed because no single medium or atmosphere is ideal for all bacteria. Combinations of broth and solid-plated media and aerobic, CO2, and anaerobic incubation must be matched to the organisms expected at any particular site or clinical circumstance. Examples of such routines are shown in Table 4–1. In general, it is not practical to routinely include specialized media for isolation of rare organisms such as Corynebacterium diphtheriae or Legionella pneumophila. For detection of these and other uncommon organisms, the laboratory must be specifically informed of their possible presence by the physician. Appropriate media and special procedures can then be included.
Routine procedures are designed to detect the most common organisms
TABLE 4–1Routine Use of Gram Smear and Isolation Systems for Selected Clinical Specimensa ||Download (.pdf) TABLE 4–1 Routine Use of Gram Smear and Isolation Systems for Selected Clinical Specimensa
|MEDIUM (INCUBATION) ||BLOOD ||CEREBROSPINAL FLUID ||WOUND, PUS ||GENITAL, CERVIX ||THROAT ||SPUTUM ||URINE ||STOOL |
|Gram smear || ||× ||× ||× || ||× || || |
|Soybean–casein digest broth (CO2) ||× || || || || || || || |
|Selenite F broth (air) || || || || || || || ||× |
|Blood agar (CO2) || ||× ||× || || ||× ||× || |
|Blood agar (anaerobic) || || ||× || ||×b || || || |
|MacConkey agar (air) || || ||× || || ||× ||× ||× |
|Chocolate agar (CO2) || ||× ||× ||× PCR preferred || ||× || || |
|Martin–Lewis agar (CO2) || || || ||× PCR preferred || || || || |
|Hektoen agar (air) || || || || || || || ||× |
|Campylobacter agar (CO2, 42°C)c || || || || || || || ||× |
When growth is detected in any medium, the process of identification begins. Identification involves methods for obtaining pure cultures from single colonies, followed by tests designed to characterize and identify the isolate. The exact tests and their sequences vary with different groups of organisms, and the taxonomic level (genus, species, subspecies, etc) of identification needed varies according to the medical usefulness of the information. In some cases, only a general description or the exclusion of particular organisms is important. For example, a report of “mixed oral flora” in a sputum specimen or “No Salmonella, Shigella, or Campylobacter isolated” in a fecal specimen may provide all the information needed. MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) mass spectroscopy has become major new tool used for the rapid identification of microorganisms already isolated in pure culture; it complements but does not replace fully the need for traditional methods.
Extent of identification is linked to medical relevance
Features Used to Classify Bacteria and Fungi
Cultural characteristics include the demonstration of properties such as unique nutritional requirements, pigment production, and the ability to grow in the presence of certain substances (sodium chloride, bile) or on certain media (MacConkey, nutrient agar). Demonstration of the ability to grow at a particular temperature or to cause hemolysis on blood agar plates is also used. For fungi, growth as a yeast colony or a mold is the primary separator. For molds, the morphology of the mold structures (hyphae, conidia, etc) are the primary means of identification.
Growth under various conditions has differential value
The ability to attack various substrates or to produce particular metabolic products has broad application to the identification of bacteria and yeast. The most common properties examined are listed in Appendix 4–3. Biochemical and cultural tests for bacterial identification are analyzed by reference to tables that show the reaction patterns characteristic of individual species. In fact, advances in computer analysis have now been applied to identification of many bacterial and fungal groups. These systems use the same biochemical principles together with computerized databases to determine the most probable identification from the observed test pattern.
Biochemical reactions analyzed by tables and computers give identification probability
Toxin Production and Pathogenicity
Direct evidence of virulence in laboratory animals is rarely needed to confirm a clinical diagnosis. In some diseases caused by production of a specific toxin, the toxin may be detected in vitro through cell cultures or immunologic methods. Neutralization of the toxic effect with specific antitoxin is the usual approach to identify the toxin. Toxin testing is conducted only in specialized laboratories. Molecular assays have been developed for some toxins.
Detection of specific toxin may define disease
Viruses, bacteria, fungi, and parasites possess many antigens, such as capsular polysaccharides, surface proteins, and cell wall components. Serology involves the use of antibodies of known specificity to detect antigens present on whole organisms or free in extracts (soluble antigens). The methods used for demonstrating antigen-antibody reactions are discussed in “Antibody Detection (Serology).”
Antigenic structures of organism demonstrated with antisera
Nucleic acid–sequence relatedness as determined by homology and direct sequence comparisons have become a primary determinant of taxonomic decisions. They are discussed later in the section on nucleic acid methods.
Isolation and Identification of Viruses
Living cell cultures that can support their replication are the primary means of isolating pathogenic viruses. The cells are derived from a tissue source by outgrowth of cells from a tissue fragment (explant) or by dispersal with proteolytic agents such as trypsin. They are allowed to grow in nutrient media on a glass or plastic surface until a confluent layer one cell thick (monolayer) is achieved. In some circumstances, a tissue fragment with a specialized function (eg, fetal trachea with ciliated epithelial cells) is cultivated in vitro and used for viral detection. This procedure is known as organ culture.
Cell cultures derived from human or animal tissues are used to isolate viruses
Three basic types of cell culture monolayers are used in diagnostic virology. The primary cell culture, in which all cells have a normal chromosome count (diploid), is derived from the initial growth of cells from a tissue source. Redispersal and regrowth produce a secondary cell culture, which usually retains characteristics similar to those of the primary culture (diploid chromosome count and virus susceptibility). Monkey and human embryonic kidney cell cultures are examples of commonly used primary and secondary cell cultures.
Monkey kidney is used in primary and secondary culture
Further dispersal and regrowth of secondary cell cultures usually lead to one of two outcomes: the cells eventually die, or they undergo spontaneous transformation, in which the growth characteristics change, the chromosome count varies (haploid or heteroploid), and the susceptibility to virus infection differs from that of the original. These cell cultures have characteristics of “immortality”; that is, they can be redispersed and regrown many times (serial cell culture passage). They can also be derived from cancerous tissue cells or produced by exposure to mutagenic agents in vitro. Such cultures are commonly called cell lines. A common cell line in diagnostic use is the Hep-2, derived from a human epithelial carcinoma. A third type of culture is often termed a cell strain. This culture consists of diploid cells, commonly fibroblastic, that can be redispersed and regrown a finite number of times; usually, 30 to 40 cell culture passages can be made before the strain dies out or spontaneously transforms. Human embryonic tonsil and lung fibroblasts are common cell strains used in clinical virology laboratories that are continuing to perform cultures. Molecular assays that are faster, more sensitive, and more cost-effective are replacing the standard viral culture techniques in many laboratories.
Cell strains regrow a limited number of times
Shell vial techniques using coverslips with monolayers of cell lines have been developed for some viruses (eg, cytomegalovirus, respiratory viruses) to provide a more rapid culture method. Virus is amplified in the cell culture vials after low-speed centrifugation. Then fluorescent staining techniques using monoclonal antibodies for the specific virus are used to detect early viral antigens prior to the development of cytopathic effect (CPE).
Primary cultures either die out or transform
Cells from Cancerous Tissue May Grow Continuously
Detection of Viral Growth
Viral growth in susceptible cell cultures can be detected in several ways. The most common effect is seen with lytic or cytopathic viruses; as they replicate in cells, they produce alterations in cellular morphology (or cell death), which can be observed directly by light microscopy under low magnification (×30 or ×100). This CPE varies with different viruses in different cell cultures. For example, enteroviruses often produce cell rounding, pleomorphism, and eventual cell death in various culture systems, whereas measles and respiratory syncytial viruses cause fusion of cells to produce multinucleated giant cells (syncytia). The microscopic appearance of some normal cell cultures and the CPE produced in them by different viruses are illustrated in Figure 4–9.
Viral CPE is due to morphologic changes or cell death
CPE is characteristic for some viruses
Viral cytopathic effect (CPE). A. Normal human diploid fibroblast cell monolayer. B. CPE caused by infection with adenovirus. C. CPE caused by infection with herpes simplex virus. (Reproduced with permission from Willey JM: Prescott, Harley, & Klein’s Microbiology, 7th edition. McGraw-Hill, 2008.)
Other viruses may be detected in cell culture by their ability to produce hemagglutinins. These hemagglutinins may be present on the infected cell membranes, as well as in the culture media, as a result of release of free, hemagglutinating virions from the cells. Addition of erythrocytes to the infected cell culture results in their adherence to the cell surfaces—a phenomenon known as hemadsorption. Another method of viral detection in cell culture is by interference. In this situation, the virus that infects the susceptible cell culture produces no CPE or hemagglutinin, but can be detected by “challenging” the cell culture with a different virus that normally produces a characteristic CPE. The second, or challenge, virus fails to infect the cell culture because of interference by the first virus, which is thus detected. This method is obviously cumbersome, but has been applied to the detection of rubella virus in certain cell cultures.
Hemadsorption or interference marks cells that may not show CPE
For some agents, such as Epstein-Barr virus (EBV) or human immunodeficiency virus (HIV), even more novel approaches may be applied. Both EBV and HIV can replicate in vitro in suspension cultures of normal human lymphocytes such as those derived from neonatal cord blood. Their presence may be determined in several ways; for example, EBV-infected B lymphocytes and HIV-infected T lymphocytes express virus-specified antigens and viral DNA or RNA, which can be detected with immunologic or genomic probes. In addition, HIV reverse transcriptase can be detected in cell culture by specific assay methods. In addition, immunologic and nucleic acid probes (see further text) can also be used to detect virus in clinical specimens or in situations in which only incomplete, noninfective virus replication has occurred in vivo or in vitro. An example is the use of in-situ cytohybridization, whereby specific labeled nucleic acid probes are used to detect and localize papillomavirus genomes in tissues where neither infectious virus nor its antigens can be detected.
EBV and HIV antigens are expressed on lymphocytes
Immunologic or genomic probes detect incomplete viruses
In-Vivo Isolation Methods
In-vivo methods for isolation, although carried out only in highly specialized laboratories, are also sometimes necessary. The embryonated hen’s egg is still often used for the initial isolation and propagation of influenza A virus. Virus-containing material is inoculated on the appropriate egg membrane, and the egg is incubated to permit viral replication and recognition. Animal inoculation is now only occasionally used by highly specialized laboratories for detecting some viruses. The usual animal host for viral isolation is the mouse; suckling mice in the first 48 hours of life are especially susceptible to many viruses. Evidence of viral replication is based on the development of illness, manifested by such signs as paralysis, convulsions, poor feeding, or death. The nature of the infecting virus can be further elucidated by histologic and immunofluorescent examination of tissues or by detection of specific antibody responses. Many arboviruses and rabies virus can be detected in this system.
Embryonated eggs and animals are used for isolation of some viruses in highly specialized laboratories
Viral isolation from a suspect case involves a number of steps. First, the viruses that are believed to be most likely involved in the illness are considered, and appropriate specimens are collected. Centrifugation or filtration and addition of antimicrobials are frequently required with respiratory or fecal specimens to remove organic matter, cellular debris, bacteria, and fungi, which can interfere with viral isolation. The specimens are then inoculated into the appropriate cell culture systems. The time between inoculation and initial detection of viral effects varies; however, for most viruses positive cultures are usually apparent within 5 days of collection. With proper collection methods and application of the diagnostic tools, as discussed further, many infections can even be detected within hours. In contrast, some viruses may require culture for a month or more before they can be detected.
Specimen preparation is required
Time to detection varies from days to weeks
On isolation, a virus can usually be tentatively identified to the family or genus level by its cultural characteristics (eg, the type of CPE produced). Confirmation and further identification may require enhancement of viral growth to produce adequate quantities for testing. This result may be achieved by inoculation of the original isolate into fresh culture systems (viral passage) to amplify replication of the virus, as well as improve its adaptation to growth in the in vitro system.
Nature of CPE and cell cultures affected may suggest virus
Neutralization and Serologic Detection
Of the several ways of identifying the isolate, the most common is to neutralize its infectivity by mixing it with specific antibody to known viruses before inoculation into cultures. The inhibition of the expected viral effects on the cell culture such as CPE or hemagglutination is then evidence of that virus. As in bacteriology, demonstration of specific viral antigens is a useful way to identify many agents. Immunofluorescence and enzyme immunoassay (EIA) are the most common methods.
Neutralization of biologic effect with specific antisera confirms identification
In some instances, viruses produce specific cytologic changes in infected host tissues that aid in diagnosis. Examples include specific intranuclear inclusions seen in neuronal infections due to herpes simplex (Cowdry type A bodies) and due to intracytoplasmic inclusions in rabies (Negri bodies), and cell fusion, which results in multinucleated epithelial giant cells (eg, measles and varicella zoster). Although such findings are useful when seen, their overall diagnostic sensitivity and specificity are usually considerably less than those of the other methods discussed.
Inclusions and giant cells suggest viruses
When virions are present in sufficient numbers, they may be further characterized by specific agglutination of viral particles on mixture with type-specific antiserum. This technique, immune electron microscopy, can be used to identify viral antigens specifically or to detect antibody in serum using viral particles of known antigenicity.
Immune electron microscopy shows agglutinated viral particles
Some viruses (eg, human rotaviruses and hepatitis A and B viruses) grow poorly or not at all in the laboratory culture systems currently available. However, they can be efficiently detected by immunologic or molecular methods (described later in this chapter).
Not all viruses grow in culture