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.

Oxygen and Carbon Dioxide Availability

Most clinically relevant bacteria are aerobic, facultatively anaerobic, or strictly anaerobic. Aerobic bacteria use oxygen as a terminal electron acceptor and grow well in room air. Most clinically significant aerobic organisms are actually facultatively anaerobic, being able to grow in the presence (i.e., aerobically) or absence (i.e., anaerobically) of oxygen. However, some bacteria, such as Pseudomonas spp., members of the Neisseriaceae family, Brucella spp., Bordetella spp., and Francisella spp., are strictly aerobic and cannot grow in the absence of oxygen. Other aerobic bacteria require only low levels of oxygen (approximately 20%) and are referred to as being microaerophilic, or microaerobic. Anaerobic bacteria are unable to use oxygen as an electron acceptor, but some aerotolerant strains will still grow slowly and poorly in the presence of oxygen. Oxygen is inhibitory or lethal for strictly anaerobic bacteria.

In addition to oxygen, the availability of CO₂ is important for growth of certain bacteria. Organisms that grow best with higher CO₂ concentrations (i.e., 5% to 10% CO₂) than is provided in room air are referred to as being capnophilic. For some bacteria, a 5% to 10% CO₂ concentration is essential for successful cultivation from patient specimens.

Temperature

Bacterial pathogens generally multiply best at temperatures similar to those of internal human host tissues and organs (i.e., 37° C). Therefore, cultivation of most medically relevant bacteria is done using incubators with temperatures maintained in the 35° C to 37° C range. For others, an incubation temperature of 30° C (i.e., the approximate temperature of the body’s surface) may be preferable, but such bacteria are encountered relatively infrequently so that use of this incubation temperature occurs only when dictated by special circumstances.

Recovery of certain organisms can be enhanced by incubation at other temperatures. For example, the gastrointestinal pathogen Campylobacter jejuni is able to grow at 42° C. Therefore, incubation at this temperature can be used as an enrichment procedure. Other bacteria, such as Listeria monocytogenes and Yersinia enterocolitica, can grow at 4° C to 43° C but grow best at temperatures between 20° and 40° C. Cold enrichment has been used to enhance the recovery of these organisms in the laboratory.

pH

The pH scale is a measure of the hydrogen ion concentration in the environment, with a pH value of 7 being neutral. Values less than 7 indicate the environment is acidic; values greater than 7 indicate alkaline conditions. Most clinically relevant bacteria prefer a near neutral pH range, from 6.5 to 7.5. Commercially prepared media are buffered in this range so that checking their pH is rarely necessary.

Moisture

Water is provided as a major constituent of both agar and broth media. However, when media are incubated at the temperatures used for bacterial cultivation, a large portion of water content can be lost by evaporation. Loss of water from media can be deleterious to bacterial growth in two ways: (1) less water is available for essential bacterial metabolic pathways and (2) with a loss of water, there is a relative increase in the solute concentration of the media. An increased solute concentration can osmotically shock the bacterial cell and cause lysis. In addition, increased atmospheric humidity enhances the growth of certain bacterial species. For these reasons, measures such as sealing agar plates to trap moisture or using humidified incubators are utilized to ensure appropriate moisture levels are maintained throughout the incubation period.

Methods for Providing Optimum Incubation Conditions

Although heating blocks and temperature-controlled water baths may be used occasionally, incubators are the primary laboratory devices used to provide the environmental conditions required for cultivating microorganisms. The conditions of incubators can be altered to accommodate the type of organisms to be grown. This section focuses on the incubation of routine bacteriology cultures. Conditions for growing anaerobic bacteria (Section 13), mycobacteria (Section 14), fungi (Chapter 59), and viruses (Chapter 65) are covered in other areas of the text.

Once inoculated with patient specimens, most media are placed in incubators with temperatures maintained between 35° and 37° C and humidified atmospheres that contain 3% to 5% CO₂. It is important to note that some media that contain pH indicators may not be placed in CO₂ incubators. The presence of CO₂ will acidify the media, causing the pH indicator to change color and thereby disrupt the differential properties of the media (e.g., Hektoen-Enteric agar and MacConkey agar). Incubators containing room air may be used for some media, but the lack of increased CO₂ may hinder the growth of certain bacteria.

Various atmosphere-generating systems are commercially available and are used instead of CO₂-generating incubators. For example, a self-contained culture medium and a compact CO₂-generating system can be used for culturing fastidious organisms such as Neisseria gonorrhoeae. A tablet of sodium bicarbonate is dissolved by the moisture created within an airtight plastic bag and releases sufficient CO₂ to support growth of the pathogen. As an alternative to commercial systems, a candle jar can also generate a CO₂ concentration of approximately 3% and has historically been used as a common method for cultivating certain fastidious bacteria. The burning candle, which is placed in a container of inoculated agar plates that is subsequently sealed, uses just enough oxygen before it goes out (from lack of oxygen) to lower the oxygen tension and produce CO₂ and water by combustion. Other atmosphere-generating systems are available to create conditions optimal for cultivating specific bacterial pathogens (e.g., Campylobacter spp. and anaerobic bacteria).

Finally, the duration of incubation required for obtaining good bacterial growth depends on the organisms being cultured. Most bacteria encountered in routine bacteriology will grow within 24 to 48 hours. Certain anaerobic bacteria may require longer incubation, and mycobacteria frequently take weeks before detectable growth occurs.

Evaluation of Colony Morphologies

The initial evaluation of colony morphologies on the primary plating media is extremely important. Laboratorians can provide physicians with early preliminary information regarding the patient’s culture results. This information is also important for deciding how to proceed for definitive organism identification and characterization.

Colony Characteristics.

Noting key features of a bacterial colony is important for any bacterial identification; success or failure of subsequent identification procedures often depends on the accuracy of these observations. Criteria frequently used to characterize bacterial growth include the following:

• Colony size (usually measured in millimeters or described in relative terms such as pinpoint, small, medium, large)

• Colony pigmentation

• Colony shape (includes form, elevation, and margin of the colony [Figure 7-11])

• Colony surface appearance (e.g., glistening, opaque, dull, dry, transparent)

• Changes in agar media resulting from bacterial growth (e.g., hemolytic pattern on blood agar, changes in color of pH indicators, pitting of the agar surface; for examples, see Figures 7-3 through 7-8)

• Odor (certain bacteria produce distinct odors that can be helpful in preliminary identification)

Many of these criteria are somewhat subjective, and the adjectives and descriptive terms used may vary among different laboratories. Regardless of the terminology used, nearly every laboratory’s protocol for bacterial identification begins with some agreed-upon colony description of the commonly encountered pathogens.

Although careful determination of colony appearance is important, it is unwise to place total confidence on colony morphology for preliminary identification. Bacteria of one species often exhibit colony characteristics that are nearly indistinguishable from those of many other species. Additionally, bacteria of the same species exhibit morphologic diversity. For example, certain colony characteristics may be typical of a given species, but different strains of that species may have different morphologies.

Gram Stain and Subcultures.

Isolation of individual colonies during cultivation not only is important for examining morphologies and characteristics but also is necessary for timely performance of Gram stains and subcultures.

The Gram stain and microscopic evaluation of cultured bacteria are used with colony morphology to decide which identification steps are needed. To avoid confusion, organisms from a single colony are stained. In many instances, staining must be performed on all different colony morphologies observed on the primary plate. In other cases, staining may not be necessary because growth on a particular selective agar provides dependable evidence of the organism’s Gram stain morphology (e.g., gram-negative bacilli essentially are the only clinically relevant bacteria that grow well on MacConkey agar).

Following characterization of growth on primary plating media, all subsequent procedures for definitive identification require the use of pure cultures (i.e., cultures containing one strain of a single species). If sufficient inocula for testing can be obtained from the primary media, then a subculture is not necessary, except as a precaution to obtain more of the etiologic agent if needed and to ensure that a pure inoculum has been used for subsequent tests (i.e., a “purity” check). However, frequently the primary media do not yield sufficient amounts of bacteria in pure culture and a subculture step is required (Figure 7-12).

Using a sterile loop, a portion of an isolated colony is taken and transferred to the surface of a suitable enrichment medium that is then incubated under conditions optimal for the organism. When making transfers for subculture, it is beneficial to flame the inoculating loop between streaks to each area on the agar surface. This avoids over inoculation of the subculture media and ensures individual colonies will be obtained. Once a pure culture is available in a sufficient amount, an inoculum for subsequent identification procedures can be prepared.

Microscopic Morphology and Staining Characteristics

Microscopic evaluation of bacterial cellular morphology, as facilitated by the Gram stain or other enhancing methods discussed in Chapter 6, provides the most basic and important information on which final identification strategies are based. Based on these findings, most clinically relevant bacteria can be divided into four distinct groups: gram-positive cocci, gram-negative cocci, gram-positive bacilli, and gram-negative bacilli (Figure 7-13). Some bacterial species are morphologically indistinct and are described as “gram-negative coccobacilli,” “gram-variable bacilli,” or pleomorphic (i.e., exhibiting various shapes). Still other morphologies include curved or rods and spirals.

Even without staining, examination of a wet preparation of bacterial colonies under oil immersion (1000× magnification) can provide clues as to possible identity. For example, a wet preparation prepared from a translucent, alpha-hemolytic colony on blood agar may reveal cocci in chains, a strong indication that the bacteria are probably streptococci. Also, the presence of yeast, whose colonies can closely mimic bacterial colonies but whose cells are generally much larger, can be determined (Figure 7-14).

In most instances, schemes for final identification are based on the cellular morphologies and staining characteristics of bacteria. To illustrate, an abbreviated identification flowchart for commonly encountered bacteria is shown in Figure 7-13 (more detailed identification schemes are presented throughout Part III); this flowchart simply illustrates how information about microorganisms is integrated into subsequent identification schemes that are based on the organism’s nutritional requirements and metabolic capabilities. In certain cases, staining characteristics alone are used to definitively identify a bacterial species. Examples are mostly restricted to the use of fluorescent-labeled specific antibodies and fluorescent microscopy to identify organisms such as Legionella pneumophila and Bordetella pertussis.

Macroscopic (Colony) Morphology

Evaluation of colony morphology includes considering colony size, shape, odor, color (pigment), surface appearance, and any changes that colony growth produces in the surrounding agar medium (e.g., hemolysis of blood in blood agar plates). A characteristic odor can be utilized in supporting an identification of an organism such as Pseudomonas aeruginosa described as having a fruity or grapelike smell. (Note: Smelling plates in a clinical setting can be dangerous and is strongly discouraged.)

Although these characteristics usually are not sufficient for establishing a final or definitive identification, the information gained provides preliminary information necessary for determining what identification procedures should follow. However, it is unwise to place too much confidence on colony morphology alone for preliminary identification of isolates. Microorganisms often grow as colonies whose appearance is not that different from many other species, especially if the colonies are relatively young (i.e., less than 14 hours old). Therefore, unless colony morphology is distinctive or unless growth occurs on a particular selective medium, other characteristics must be included in the identification scheme.

Environmental Requirements for Growth

Environmental conditions required for growth can be used to supplement other identification criteria. However, as with colony morphologies, this information alone is not sufficient for establishing a final identification. The ability to grow in particular incubation atmospheres most frequently provides insight about the organism’s potential identity. For example, organisms growing only in the bottom of a tube containing thioglycollate broth are not likely to be strictly aerobic bacteria, thus eliminating these types of bacteria from the list of identification possibilities. Similarly, anaerobic bacteria can be discounted in the identification schemes for organisms that grow on blood agar plates incubated in an ambient (room) atmosphere. An organism’s requirement, or preference, for increased carbon dioxide concentrations can provide hints for the identification of other bacteria such as Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria gonorrhoeae.

In addition to atmosphere, the ability to survive or even thrive in temperatures that exceed or are well below the normal body temperature of 37° C may be helpful for organism identification. The growth of Campylobacter jejuni at 42° C and the ability of Yersinia enterocolitica to survive at 0° C are two examples.

Resistance or Susceptibility to Antimicrobial Agents

The ability of an organism to grow in the presence of certain antimicrobial agents or specific toxic substances is widely used to establish preliminary identification information. This is accomplished by using agar media supplemented with inhibitory substances or antibiotics (for examples, see Table 7-1) or by directly measuring an organism’s resistance to antimicrobial agents that may be used to treat infections (for more information regarding antimicrobial susceptibility testing, see Chapter 12).

As discussed earlier in this chapter, most clinical specimens are inoculated to several media, including some selective or differential agars. Therefore, the first clue to identification of an isolated colony is the nature of the media on which the organism is growing. For example, with rare exceptions, only gram-negative bacteria grow well on MacConkey agar. Alternatively, other agar plates, such as Columbia agar with CNA, support the growth of gram-positive organisms to the exclusion of most gram-negative bacilli. Certain agar media can be used to differentiate even more precisely than simply separating gram-negative and gram-positive bacteria. Whereas chocolate agar will support the growth of all aerobic microorganisms including Neisseria spp., the antibiotic-supplemented Thayer-Martin formulation will almost exclusively support the growth of the pathogenic species N. meningitidis and N. gonorrhoeae.

Directly testing a bacterial isolate’s susceptibility to a particular antimicrobial agent may be a very useful part of an identification scheme. Many gram-positive bacteria (with a few exceptions, such as certain Enterococci, Lactobacillus, Leuconostoc, and Pediococcus spp.) are susceptible to vancomycin, an antimicrobial agent that acts on the bacterial cell wall. In contrast, most clinically important gram-negative bacteria are resistant to vancomycin. Therefore, when organisms with uncertain Gram stain results are encountered, susceptibility to vancomycin can be used to help establish the organism’s Gram “status.” Any zone of inhibition around a vancomycin-impregnated disk after overnight incubation is usually indicative of a gram-positive bacterium (Figure 7-15). With few exceptions (e.g., certain Chryseobacterium, Moraxella, or Acinetobacter spp. isolates may be vancomycin susceptible), truly gram-negative bacteria are resistant to vancomycin. Conversely, most gram-negative bacteria are susceptible to the antibiotics colistin or polymyxin, whereas gram-positive bacteria are frequently resistant to these agents.

Nutritional Requirements and Metabolic Capabilities

Determining the nutritional and metabolic capabilities of a bacterial isolate is the most common approach used for determining the genus and species of an organism. The methods available for making these determinations share many commonalties but also have some important differences. In general, all methods use a combination of tests to establish the enzymatic capabilities of a given bacterial isolate as well as the isolate’s ability to grow or survive the presence of certain inhibitors (e.g., salts, surfactants, toxins, and antibiotics).

Oxidation and Fermentation Tests.

As discussed in Chapter 2, bacteria use various metabolic pathways to produce biochemical building blocks and energy. For most clinically relevant bacteria, this involves utilization of carbohydrates (e.g., sugar or sugar derivatives) and protein substrates. Determining whether substrate utilization is an oxidative or fermentative process is important for the identification of several different bacteria.

Oxidative processes require oxygen; fermentative ones do not. The clinical laboratory determines how an organism utilizes a substrate by observing whether acid byproducts are produced in the presence or absence of oxygen. In most instances, the presence of acid byproducts is detected by a change in the pH indicator incorporated into the medium. The color changes that occur in the presence of acid depend on the type of pH indicator used.

Oxidation-fermentation determinations are usually accomplished using a special semi-solid medium (oxidative-fermentative [O-F] medium) that contains low concentrations of peptone and a single carbohydrate substrate such as glucose. The organism to be identified is inoculated into two glucose O-F tubes, one of which is then overlaid with mineral oil as a barrier to oxygen. Common pH indicators used for O-F tests, and the color changes they undergo with acidic conditions, include bromcresol purple, which changes from purple to yellow; Andrade’s acid fuchsin indicator, which changes from pale yellow to pink; phenol red, which changes from red to yellow; and bromthymol blue, which changes from green to yellow.

As shown in Figure 7-16, when acid production is detected in both tubes, the organism is identified as a glucose fermenter because fermentation can occur with or without oxygen. If acid is only detected in the open, aerobic tube, the organism is characterized as a glucose-oxidizer. As a third possibility, some bacteria do not use glucose as a substrate and no acid is detected in either tube (a nonutilizer). The glucose fermentative or oxidative capacity is generally used to separate organisms into major groups (e.g., Enterobacteriaceae are fermentative; Pseudomonas spp. are oxidative). However, the utilization pattern for several other carbohydrates (e.g., lactose, sucrose, xylose, maltose) is often needed to help identify an organism’s genus and species.

Type of Bacteria to Be Identified

Certain organisms have such unique features that relatively few tests are required to establish identity. For example, Staphylococcus aureus is essentially the only gram-positive coccus that appears microscopically in clusters, is catalase-positive, and produces coagulase. Therefore, identification of this common pathogen usually requires the use of only two tests coupled with colony and microscopic morphology. In contrast, identification of most clinically relevant gram-negative bacilli, such as those of the Enterobacteriaceae family, requires establishing metabolic profiles often involving 20 or more tests.

Clinical Significance of the Bacterial Isolate

Although a relatively large number of tests may be required to identify a particular bacterial species, the number of tests actually inoculated may depend on the clinical significance of an isolate. For instance, if a gram-negative bacillus is mixed with five other bacterial species in a urine culture, it is likely to be a contaminant. In this setting, multiple tests to establish species identity are not warranted and should not routinely be performed. However, if this same organism is isolated in pure culture from cerebrospinal fluid, the full battery of tests required for definitive identification should be performed.

Availability of Reliable Testing Methods

Because of an increasing population of immunocompromised patients and the increasing multitude of complicated medical procedures, isolation of uncommon or unusual bacteria is occurring more frequently. Because of the unusual nature exhibited by some of these bacteria, reliable testing methods and identification criteria may not be established in most clinical laboratories. In these instances, only the genus of the organism may be identified (e.g., Bacillus spp.), or identification may not go beyond a description of the organism’s microscopic morphology (e.g., gram-positive, pleomorphic bacilli, or gram-variable, branching organism). When such bacteria are encountered and are thought to be clinically significant, they should be sent to a reference laboratory whose personnel are experienced in identifying unusual organisms.

Although the number of tests included in an identification battery may vary and different identification systems may require various inoculation techniques, the one common feature of all systems is the requirement for inoculation with a pure culture. Inoculation with a mixture of bacteria produces mixed and often uninterpretable results. To expedite identification, cultivation strategies (described earlier in this chapter) should focus on obtaining pure cultures as soon as possible. Furthermore, positive and negative controls should be ran in parallel with most identification systems as a check for purity of the culture used to inoculate the system.

Conventional Identification

Because the generation time (i.e., the time required for a bacterial population to double) for most clinically relevant bacteria is 20 to 30 minutes, growth-based tests usually require hours of incubation before the presence of an end product can be measured. Many conventional identification schemes require 18 to 24 hours of incubation, or longer, before the tests can be accurately interpreted. Although the conventional approach has been the standard for most bacterial identification schemes, the desire to produce results and identifications in a more timely fashion has resulted in the development of rapid identification strategies.

Rapid Identification

In the context of diagnostic bacteriology, the term rapid is relative. In some instances a rapid method is one that provides a result the same day that the test was inoculated. Alternatively, the definition may be more precise, whereby rapid is only used to describe tests that provide results within 4 hours of inoculation. It is important to note that rapid identification still requires overnight incubation of culture media from the primary specimen. Pure culture isolates grown on culture media are required for use in rapid identification systems.

Two general approaches have been developed to obtain more rapid identification results. One has been to vary the conventional testing approach by decreasing the test substrate medium volume and increasing the concentration of bacteria in the inoculum. Several conventional methods, such as carbohydrate fermentation profiles, use this strategy for more rapid results.

The second approach uses unique or unconventional substrates. Particular substrates are chosen, based on their ability to detect enzymatic activity at all times. That is, detection of the enzyme does not depend on multiplication of the organism (i.e., not a growth-based test) so that delays caused by depending on bacterial growth are minimized. The catalase, oxidase, and PYR tests discussed previously are examples of such tests, but many others are available as part of commercial testing batteries.

Still other rapid identification schemes are based on antigen-antibody reactions, such as latex agglutination tests, that are commonly used to quickly and easily identify certain beta-hemolytic streptococci and S. aureus (for more information regarding these test formats, see Chapter 10).

Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF)

MALDI-TOF is an advanced chemical technique that uses laser excitation to ionize chemical functional groups that are included in the proteins of an organism. MALDI-TOF has the potential to significantly reduce turnaround time and identification rates, while at the same time reducing the cost of consumables in the microbiology laboratory. The organism is either applied directly onto a plate from a pure culture or prepared as a protein extract prior to application. The sample is then mixed with a chemical matrix. The laser is applied to the sample and the matrix absorbs the energy transferring heat to the sample proteins and creating ions, this is essentially the desorption and ionization process. These ions are then separated in a tube referred to as a flight tube. The lighter the ions, the faster they will travel in the tube. The ions are then measured using a detector, and a protein spectrum for the specific organism is then created as a mass spectrum using a mass-to-charge ratio and signal intensity. Typically the proteins that are detected efficiently would include small relatively abundant proteins such as ribosomal proteins. This new organism protein profile can then be compared to other organisms included in a computerized database. As of this writing, there are a few commercially available MALDI-TOF systems including MALDI Biotyper (Bruker Daltonics Inc, Fremont, CA) and Vitek MS (BioMerieux, Etoile France). However, clinical identification of microorganisms including bacteria, fungi, and viruses is limited to the size of the current data base. The technique is also limited to the identification of organisms following pure colony isolation and is not useful on specimens containing contaminating microbiota or multiple species. More clinical data are needed before this technique becomes widely accepted within the microbiology laboratory.

Colorimetry

Several identification systems measure color change to detect the presence of metabolic end products. Most frequently the color change is produced using pH indicators included in the media. Depending on the byproducts to be measured and the testing method, additional reagents may need to be added to the reaction before the results are interpreted. An alternative to the use of pH indicators is the oxidation-reduction potential indicator tetrazolium violet. Organisms are inoculated into wells that contain a single, utilizable carbon source. Metabolism of that substrate generates electrons that reduce the tetrazolium violet, producing a purple color (positive reaction) that can be spectrophotometrically detected. In a third approach, the substrates themselves may be chromogenic so that when they are “broken down” by the organism, the altered substrate produces a color.

Some commercial systems use a miniaturized modification of conventional biochemical batteries, with the color change being detectable with the unaided eye. Alternatively, in certain automated systems, a photoelectric cell measures the change in the wavelength of light transmitted through miniaturized growth cuvettes or wells, thus eliminating the need for direct visual interpretation by laboratory personnel. Additionally, a complex combination of dyes and filters may be used to enhance and broaden the scope of substrates and color changes that can be used in such systems. These combinations hasten identification and increase the variety of organisms that can be reliably identified.

Fluorescence

There are two basic strategies for using fluorescence to measure metabolic activity. In one approach, substrate-fluorophore complexes are used. If a bacterial isolate processes the substrate, the fluorophore is released and assumes a fluorescent configuration. Alternatively, pH changes resulting from metabolic activity can be measured by changes in fluorescence of certain fluorophore markers. In these pH-driven, fluorometric reactions, pH changes result in either the fluorophore becoming fluorescent or, in other instances, fluorescence being quenched or lost. To detect fluorescence, ultraviolet light of appropriate wavelength is focused on the reaction mixture and a special kind of photometer, a fluorometer, measures fluorescence.

Turbidity

Turbidity measurements are not commonly used for bacterial identifications but do have widespread application for determining growth in the presence of specific growth inhibitors, including antimicrobial agents, and for detecting bacteria present in certain clinical specimens.

Turbidity is the ability of particles in suspension to refract and deflect light rays passing through the suspension such that the light is reflected back into the eyes of the observer. The optical density (OD), a measurement of turbidity, is determined in a spectrophotometer. This instrument compares the amount of light that passes through the suspension (the percent transmittance) with the amount of light that passes through a control suspension without particles. A photoelectric sensor, or photometer, converts the light that impinges on its surface to an electrical impulse, which can be quantified. A second type of turbidity measurement is obtained by nephelometry or light scatter. In this case, the photometers are placed at angles to the suspension, and the scattered light, generated by a laser or incandescent bulb, is measured. The amount of light scattered depends on the number and size of the particles in suspension.

Identification Databases

Reference databases are available for clinical use. These databases are maintained by manufacturers of identification systems and are based on the continuously updated taxonomic status of clinically relevant bacteria. Although microbiologists typically do not establish and maintain their own databases, an overview of the general approach provides background information.

The first step in developing a database is to accumulate many bacterial strains of the same species. Each strain is inoculated to an identical battery of metabolic tests to generate a positive-negative test profile. The cumulative results of each test are expressed as a percentage of each genus or species that possesses that characteristic. For example, suppose that 100 different known E. coli strains and 100 known Shigella spp. strains are tested in four biochemicals, yielding the results illustrated in Table 7-3. In reality, many more strains and tests would be performed. However, the principle—to generate a database for each species that contains the percentage probability for a positive result with each test in the battery—is the same.

Manufacturers develop databases for each of the identification systems they produce for diagnostic use (e.g., Enterobacteriaceae, gram-positive cocci, nonfermentative gram-negative bacilli). Because the data are based on organism “behavior” in a particular commercial system, the databases cannot and should not be applied to interpret profiles obtained by other testing methods.

Furthermore, most databases are established with the assumption that the isolate to be identified has been appropriately characterized using adjunctive tests. For example, if a S. aureus isolate is mistakenly tested using a system for identification of Enterobacteriaceae, the database will not identify the gram-positive cocci because the results obtained will only be compared with data available for enteric bacilli. This underscores the importance of accurately performing preliminary tests and observations, such as colony and Gram stain morphologies, before selecting a particular identification battery.

Use of the Database to Identify Unknown Isolates

Once a metabolic profile has been obtained with a bacterial isolate of unknown identity, the profile must be converted to a numeric code that will facilitate comparison of the unknown’s phenotypic fingerprint with the appropriate database.

To exemplify this step in the identification process, a binary code conversion system that uses the numerals 0 and 1 to represent negative and positive metabolic reactions, respectively, is used as an example (although other strategies are now used). As shown in Figure 7-18, using binary code conversion, a 21-digit binomial number (e.g., 101100001001101111010, as read from top to bottom in the figure) is produced from the test result. This number is then used in an octal code conversion scheme to produce a mathematic number (octal profile [see Figure 7-18]). The octal profile number is used to generate a numerical profile distinctly related to a specific bacterial species. As shown in Figure 7-18, the octal profile for the unknown organism is 5144572. This profile would then be compared with database profiles to determine the most likely identity of the organism. In this example, the octal profile indicates the unknown organism is E. coli.