Acineto Bacteria Morphology On Cled Agar

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Enterobacteriaceae Haemophilus spp. Pathogenic Other

Pseudomonas spp. Brucella spp. Neisseria spp. Neisseria spp. Legionella spp.

Catalase

Corynebacterium spp. Lactobacillus spp. Listeria spp. Actinomyces spp.

Others

Oxidase

Pseudomonas spp. Enterobacteriaceae

Selection and performance of appropriate definitive bacterial identification schemes or systems

(see Figure 11-12)

Figure 7-13 Flowchart example of a bacterial identification scheme.

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, identification 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 HI); this flowchart simply illustrates how information about microorganisms is integrated into subsequent identification schemes that are usually 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 of this 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, color (pigment), surface appearance,

Figure 7-14 Microscopic examination of a wet preparation demonstrates the size difference between most yeast cells, such as those of Candida albicans (arrow A), and bacteria, such as Staphylococcus aureus (arrow B).

Figure 7-15 A, Zone of growth inhibition around the 5-mg vancomycin disk is indicative of a gram-positive bacterium. B, The gram-negative organism is not inhibited by this antibiotic, and growth extends to the edge of the disk.

and any changes that colony growth produces in the surrounding agar medium (e.g., hemolysis of blood in blood agar plates).

Although these characteristics usually are not sliffident 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 rot that different from many other speaes, especially if the colonies are relativdy young (i.e., less than 14 hours old). Therefore, unless colony morphology is distinctive er unless growth occurs on a particular selective medium, other characteristics must be induded in the identification scheme.

Environmental Requirements for Growth

Bnvironmental conditions required for growth can be used to supplement other identification criteria. However, as with colony morphologies, this information alone is not suffident 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 spedfic 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)

Figure 7-15 A, Zone of growth inhibition around the 5-mg vancomycin disk is indicative of a gram-positive bacterium. B, The gram-negative organism is not inhibited by this antibiotic, and growth extends to the edge of the disk.

As discussed earlier in this chapter, most clinical specimens are inoculated to several media, induding some selective or differential agars. Therefore, the first due 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 exdusion 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 Neisseria spp., the antibiotic-supplemented Thayer-Martin formulation will almost exdusively support the growth of the pathogenic spedes 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 grampositive bacteria (with a few exceptions, such as certain enterococd, lactobadlli, 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 vancomydn-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).

Establishing Enzymatic Capabilities. As discussed in Chapter 2, enzymes are the driving force in bacterial metabolism. Because enzymes are genetically encoded, the enzymatic content of an organism is a direct reflection of the organism's genetic makeup, which, in turn, is specific for individual bacterial species.

Types of Enzyme-Based Tests. In diagnostic bacteriology, enzyme-based tests are designed to measure the presence of one specific enzyme or a complete metabolic pathway that may contain several different enzymes. Although the specific tests most useful for the identification of particular bacteria are discussed in Part m, some examples of tests commonly used to characterize a wide spectrum of bacteria are reviewed here.

Single Enzyme Tests. Several tests are commonly used to determine the presence of a single enzyme. These tests usually provide rapid results because they can be performed on organisms already grown in culture. Of importance, these tests are easy to perform and interpret and often play a key role in the identification scheme. Although most single enzyme tests do not yield sufficient information to provide spedes identification, they are used extensively to determine which subsequent identification steps should be followed. For example, the catalase test can provide pivotal information and is commonly used in schemes for gram-positive identifications. The oxidase test is of comparable importance in identification schemes for gram-negative bacteria (see Figure 7-13).

Catalase Test. The enzyme catalase catalyzes the release of water and oxygen from hydrogen peroxide (H202 + catalase = H20 + 02); its presence is determined by direct analysis of a bacterial culture (see Procedure 13-8), The rapid production of bubbles (effervescence) when bacterial growth is mixed with a hydrogen peroxide solution is interpreted as a positive test (i.e„ the presence of catalase). Failure to produce effervescence or weak effervescence is interpreted as negative. If the bacterial inoculum is inadvertently contaminated with red blood cells when the test inoculum is collected from a sheep blood agar plate, weak production of bubbles may occur, but this should not be interpreted as a positive test.

Because the catalase test is key to the identification scheme of many gram-positive oiganisms, interpretation must be done carefully. For example, staphylococci are catalase-positive, whereas streptococci and enterococd are negative; similarly, the catalase reaction differentiates Listeria monocytogenes and coryne-bacteria (catalase-positive) from other gram-positive, non-spore-forming bacilli (see Figure 7-13).

Oxidase Test. Cytochrome oxidase partiapates in electron transport and in the nitrate metabolic pathways of certain bacteria. The test for the presence of oxidase can be performed by flooding bacterial colonies on the agar surface with the reagent 1% tetramethyl-p-phenylenediamine ¿¡hydrochloride. Alternatively, a sample of the bacterial colony can be rubbed onto filter paper impregnated with the reagent (see Procedure 13-33). If an iron-containing wire is used to transfer growth, a false-positive reaction may result; therefore, platinum wire or wooden sticks are recommended. Certain organisms may show slight positive reactions after the initial 10 seconds have passed; such results are not considered definitive.

The test is initially used for differentiating between groups of gram-negative bacteria. Among the commonly encountered gram-negative bacilli Entero-bacteriaceae, Stenotrophomonas maltophilia, and Acineto-bacter spp. are oxidase-negative, whereas many other badlli, such as Pseudomortas spp. and Aeromonas spp., are positive (see Figure 7-13). The oxidase test is also a key reaction for the identification of Neisseria spp. (oxidase-positive).

Indole Test Bacteria that produce the enzyme tryptophanase are able to degrade the amino add tryptophan into pyruvic add, ammonia, and indole. Indole is detected by combining with an indicator, aldehyde (1% paradimethylaminocinnamaldehyde), that results in a blue color formation (see Procedure 13-20). This test is used in numerous identification schemes, espedally to presumptively identify Escherichia coli, the gram-negative bacillus most commonly encountered in diagnostic bacteriology.

Urease Test. Urease hydrolyzes the substrate urea into ammonia, water, and carbon dioxide. The presence of the enzyme is determined by inoculating an organism to broth or agar that contains urea as the primary carbon source and detecting the production of ammonia (see Procedure 13-41). Ammonia increases the pH of the medium so its presence is readily detected using a pH indicator. Change in medium pH is a common indicator of metabolic process and, because pH indicators change color with increases (alkalinity) or decreases (acidity) in the medium's pH, they are commonly used in many identification test schemes. The urease test helps identify certain species of Bnterobacteriaceae, such as Proteus spp., and other important bacteria such as Corynebacterium urealyticum and Helicobacter pylori.

PYR Test The enzyme L-pyrroglutamyl-aminopeptidase hydrolyzes the substrate i-pyrrolidonyl-p-naphthylamide (PYR) to produce a p-naphthylamine. When the 0-naphthylamine combines with a cinna-maldehyde reagent, a bright red color is produced (see Procedure 13-36). The PYR test is particularly helpful in identifying gram-positive cocci such as Streptococcus pyogenes and Enterococcus spp., which are positive, whereas other streptococci are negative.

Hippurate Hydrolysis. Hippuricase is a constitutive enzyme that hydrolyzes the substrate hippurate to produce the amino add glycine. Glycine is detected by oxidation with ninhydrin reagent that results in the production of a deep purple color (see Procedure 1319). The hippurate test is most frequently used in the identification of Streptococcus agalactiae, Campylobacter jejuni, and Listeria monocytogenes.

Tests for Presence of Metabolic Pathways. Several identification schemes are based on determining what metabolic pathways an organism uses and the substrates processed by these pathways. In contrast to single enzyme tests, these pathways may involve several interactive enzymes. The presence of an end product resulting from these interactions is measured in the testing system. Assays for metabolic pathways can be classified into three general categories: carbohydrate oxidation and fermentation, amino acid degradation, and single substrate utilizations.

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 dinical laboratory determines how an organism utilizes a substrate by observing whether add byproducts are produced in the presence or absence of oxygen. In most instances, the presence of add byproducts is detected by a change in the pH indicator incorporated into the medium. The color changes that occur in the presence of add depend on the type of pH indicator used.

Oxidation-fermentation determinations are usually accomplished using a spedal medium (oxidative-fermentative [O-FJ 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 add 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 add 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 add is detected in either tube (a nonutilizer). The glucose fermentative or oxidative capadty is generally used to separate organisms into major groups (e.g., Bnterobacteriaceae 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 speaes.

Amino Acid Degradation. Determining the ability of bacteria to produce enzymes that either deaminate, dihydrolyze, or decarboxylate certain amino adds is often used in identification schemes. The amino add substrates most often tested indude lysine, ornithine, arginine, and phenylalanine. (The indole test for tryptophan cleavage is presented earlier in this chapter.)

Decarboxylases cleave the carboxyl group from amino adds so that amino adds are converted into amines; lysine is converted to cadaverine, and ornithine is converted to putresdne. Because amines increase medium pH, they are readily detected by color changes in a pH indictor indicative of alkalinity. Decarboxylation is an anaerobic process that requires an add environment for activation. The most common medium used for this test is Moeller decarboxylase base, whose components indude glucose, the amino add substrate

Mineral oil overlay

Mineral oil overlay

Figure 7-16 Principle of glucose oxidative-fermentation (O-F) lest. Fermentation patterns shown in O-F tubes are examples of oxidative, fermentative, and nonutilizing bacteria.

Both tubes of O-F glucose inoculated with test organism

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