Rationale for Approaching Organism Identification

It is challenging to determine how most effectively to present and teach diagnostic microbiology in a way that is sufficiently comprehensive and yet not excessively cluttered with rare and seldom-needed facts about bacterial species uncommonly encountered. Approximately 530 different bacterial species or taxa are reported by clinical microbiology laboratories across the United States (Figure 13-1). Yet 95% of the bacterial identifications reported are distributed across only 27 of these taxa. This is an indication of how infrequently the other 500 or more taxa are identified and reported. Therefore, although the chapters in Part III, Bacteriology, are intended to be comprehensive in terms of the variety of bacterial species presented, it is helpful to keep in perspective which taxa are most likely to be encountered in the clinical environment. The relative frequencies with which the common bacterial species and organism groups are reported in clinical laboratories are presented in Figure 13-2.

Historically, most chapters in microbiology texts have been organized by genus name; however, they failed to provide information and processes needed to understand what is involved in analyzing information from the clinical specimen to the identification of the correct genus. Many texts (including this one) provide flow charts containing algorithms or identification schemes for organism workup. Although these are helpful, one must be aware of the limitations of flow charts. In some cases they may be too general to be helpful; that is, they may lack sufficient detail to be useful for discriminating among key microbial groups and species. In other cases, they may be too esoteric to be of practical use in routine clinical practice (e.g., identification schemes based on cellular analysis of fatty acid analysis). In addition, many other criteria that must be incorporated into the identification process are too complex to be included in most flow charts. Thus, flow charts are only one of many tools used in the field of diagnostic microbiology.

Also, as discussed later in this chapter, organism taxonomy and profiles continuously change. Detailed flow charts are at risk of quickly becoming outdated. Furthermore, as is evident throughout the chapters in Part III, diagnostic microbiology is full of exceptions to rules, and flow charts are not constructed in a manner that readily captures many of the important exceptions.

To meet the challenges of bacterial identification processes beyond what can be portrayed in flow charts, the chapters in Part III have been arranged to guide the student through the entire workup of a microorganism, beginning with initial culture of the specimen. In most instances, the first information a microbiologist uses in the identification process is the macroscopic description of the colony, or colony morphology. This includes the type of hemolysis (if any), pigment (if present), size, texture (opaque, translucent, or transparent), adherence to agar, pitting of agar, and many other characteristics (see Chapter 7). After careful observation of the colony, the Gram stain is used to separate the organism into a variety of broad categories based on Gram stain reaction and the cellular morphology of gram-positive or gram-negative bacteria (e.g., gram-positive cocci, gram-negative rods; see Chapter 6). For gram-positive organisms, the catalase test should follow the Gram stain, and testing on gram-negative organisms should begin with the oxidase test. These simple tests, plus growth on MacConkey agar, if the isolate is a gram-negative rod or coccobacillus, help the microbiologist assign the organism to one of the primary categories (organized here as subsections). Application of the various identification methods and systems outlined in this chapter generate the data and criteria discussed in each chapter for the definitive identification of clinically relevant bacteria. Most of the procedures described in the following chapters can be found at the end of this chapter. In this chapter, each procedure includes a photograph of positive and negative reactions. Chapter 6 includes photographs of some commonly used bacteriologic stains. In addition, Table 13-1 lists several commonly used commercial identification systems for a variety of the microorganisms discussed in the following pages.

Because diagnostic microbiology is centered around the identification of organisms based on common phenotypic traits shared with known members of the same genus or family, microbiologists “play the odds” every day by finding the best biochemical “fit” and assigning the most probable identification. For example, the gram-negative rod known as CDC group EF-4a may be considered with either MacConkey-positive or MacConkey-negative organisms, because it grows on MacConkey agar 50% of the time. Therefore, although CDC group EF-4a has been arbitrarily assigned to the section on oxidase-positive, MacConkey-positive, gram-negative bacilli and coccobacilli in this text, it is also included in the discussion of oxidase-positive, MacConkey-negative, gram-negative bacilli and coccobacilli. This example clearly demonstrates the limitations of solely depending on flow charts for the identification process.

The identification process often can be arduous and a drain on resources. Laboratorians must make every effort to identify only those organisms most likely to be involved in the infection process. To that end, the chapters in Part III have also been designed to provide guidance for determining whether a clinical isolate is relevant and requires full identification. Furthermore, the clinical diagnosis and the source of the specimen can help determine which group of organisms to consider. For example, if a patient has endocarditis or the specimen source is blood and a small, gram-negative rod is observed on Gram stain, the microbiologist should consider a group of gram-negative bacilli known as the HACEK (Aggrega-tibacter [formerly the aphrophilus group of Haemophilus and Actinobacillus], Cardiobacterium hominis, Eikenella corrodens, and Kingella spp.), which are not commonly encountered in clinical specimens. Similarly, if a patient has suffered an animal bite, the microbiologist should think of Pasteurella multocida if the isolate is gram negative and Staphylococcus hyicus and Staphylococcus intermedius if the organism is gram positive. Finally, in consideration of an isolate’s clinical relevance, each chapter also provides information on whether antimicrobial susceptibility testing is indicated and, if needed, the way it should be performed.

Future Trends of Organism Identification

Several dynamics are involved in clinical microbiology and infectious diseases that continue to challenge bacterial identification practices. For instance, new species associated with human infections will continue to be discovered, and well-known species may change their characteristics, affecting the criteria used to identify them. For these reasons, identification schemes and strategies for both conventional methods and commercial systems must be continually reviewed and updated. Also, although most identification schemes are based on the phenotypic characteristics of bacteria, the use of molecular and advanced chemical methods (e.g., matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) to detect, identify, and characterize bacteria continues to expand and play a greater role in diagnostic microbiology. In addition, phenotypic, molecular, and advanced chemical methods increasingly will become incorporated into simpler automated systems.

Procedure 13-1   Acetamide Utilization

Purpose

Differentiate microorganisms based on the ability to use acetamide as the sole source of carbon.

Principle

Bacteria capable of growth on this medium produce the enzyme acylamidase, which deaminates acetamide to release ammonia. The production of ammonia results in an alkaline pH, causing the medium to change color from green to royal blue.

Media: NaCl (5 g), NH4H2PO4 (1 g), K2HPO4 (1 g), agar (15 g), bromthymol blue indicator (0.8 g), per 1000 mL, acetamide (10 g), pH 6.8.

Method

1. Inoculate acetamide slant with a needle using growth from an 18- to 24-hour culture. Do not inoculate from a broth culture, because the growth will be too heavy.

2. Incubate aerobically at 35°-37°C for up to 4 days. If equivocal, the slant may be reincubated for 2 additional days.

Expected Results

Positive: Deamination of the acetamide, resulting in a blue color (Figure 13-3, A).

Negative: No color change (Figure 13-3, B).

Limitations

Growth without a color change may indicate a positive test result. If further incubation results in no color change, repeat test with less inoculum.

Quality Control

Positive: Pseudomonas aeruginosa (ATCC 27853)—growth; blue color

Negative: Escherichia coli (ATCC 25922)—no growth; green color

Procedure 13-2   Acetate Utilization

Purpose

Differentiate organisms based on ability to use acetate as the sole source of carbon. Generally used to differentiate Shigella sp. from Escherichia coli.

Principle

This test is used to differentiate an organism capable of using acetate as the sole source of carbon. Organisms capable of using sodium acetate grow on the medium, resulting in an alkaline pH, turning the indicator from green to blue.

Media: NaC2H3O2 (2 g); MgSO4 (0.1 g); NaCl (5 g); NH4H2PO4 (1 g); agar (20 g); bromthymol blue indicator (0.8 g), per 1000 mL, pH 6.7.

Method

1. With a straight inoculating needle, inoculate acetate slant lightly from an 18- to 24-hour culture. Do not inoculate from a broth culture, because the growth will be too heavy.

2. Incubate at 35°-37°C for up to 7 days.

Expected Results

Positive: Medium becomes alkalinized (blue) as a result of the growth and use of acetate (Figure 13-4, A).

Negative: No growth or growth with no indicator change to blue (Figure 13-4, B).

Limitations

Some strains of E. coli may use acetate at a very slow rate or not at all, resulting in a false negative reaction in the identification process.

Quality Control

Positive: Escherichia coli (ATCC 25922)—growth; blue

Negative: Shigella sonnei (ATCC 25931)—small amount of growth; green

Procedure 13-3   Bacitracin Susceptibility

Purpose

This test is used for presumptive identification and differentiation of beta-hemolytic group A streptococci (Streptococcus pyogenes–susceptible) from other beta-hemolytic streptococci. It is also used to distinguish staphylococci species (resistant) from micrococci (susceptible).

Principle

The antibiotic bacitracin inhibits the synthesis of bacterial cell walls. A disk (TaxoA) impregnated with a small amount of bacitracin (0.04 units) is placed on an agar plate, allowing the antibiotic to diffuse into the medium and inhibit the growth of susceptible organisms. After incubation, the inoculated plates are examined for zones of inhibition surrounding the disks.

Method

1. Using an inoculating loop, streak two or three suspect colonies of a pure culture onto a blood agar plate.

2. Using heated forceps, place a bacitracin disk in the first quadrant (area of heaviest growth). Gently tap the disk to ensure adequate contact with the agar surface.

3. Incubate the plate for 18 to 24 hours at 35°-37°C in ambient air for staphylococci and in 5% to 10% carbon dioxide (CO₂) for streptococci differentiation.

4. Look for a zone of inhibition around the disk.

Expected Results

Positive: Any zone of inhibition greater than 10 mm; susceptible (Figure 13-5, A).

Negative: No zone of inhibition; resistant (Figure 13-5, B).

Limitations

Performance depends on the integrity of the disk. Proper storage and expiration dates should be maintained.

Quality Control

Positive: Streptococcus pyogenes (ATCC19615)—susceptible

Micrococcus luteus (ATCC10240)—susceptible

Negative: Streptococcus agalactiae (ATCC27956)—resistant

Staphylococcus aureus (ATCC25923)—resistant

Procedure 13-4   Bile Esculin Test

Purpose

This test is used for the presumptive identification of enterococci and organisms in the Streptococcus bovis group. The test differentiates enterococci and group D streptococci from non–group D viridans streptococci.

Principle

Gram-positive bacteria other than some streptococci and enterococci are inhibited by the bile salts in this medium. Organisms capable of growth in the presence of 4% bile and able to hydrolyze esculin to esculetin. Esculetin reacts with Fe3+ and forms a dark brown to black precipitate.

Media: Beef extract (11 g), enzymatic digest of gelatin (34.5 g), esculin (1 g), ox bile (2 g), ferric ammonium citrate (0.5 g), agar (15 g), per 1000 mL, pH 6.6.

Method

1. Inoculate one to two colonies from an 18- to 24-hour culture onto the surface of the slant.

2. Incubate at 35°-37°C in ambient air for 48 hours.

Expected Results

Positive: Growth and blackening of the agar slant (Figure 13-6, A)

Negative: Growth and no blackening of medium (Figure 13-6, B)

No growth (not shown)

Limitations

As a result of nutritional requirements, some organisms may grow poorly or not at all on this medium.

Quality Control

Positive: Enterococcus faecalis (ATCC19433)—growth; black precipitate

Negative: Escherichia coli (ATCC25922)—growth; no color change

Streptococcus pyogenes (ATCC19615)—no growth; no color change

Procedure 13-5   Bile Solubility Test

Purpose

This test differentiates Streptococcus pneumoniae (positive–soluble) from alpha-hemolytic streptococci (negative–insoluble).

Principle

Bile or a solution of a bile salt (e.g., sodium desoxycholate) rapidly lyses pneumococcal colonies. Lysis depends on the presence of an intracellular autolytic enzyme, amidase. Bile salts lower the surface tension between the bacterial cell membrane and the medium, thus accelerating the organism’s natural autolytic process.

Method

1. After 12 to 24 hours of incubation on 5% sheep blood agar, place 1 to 2 drops of 10% sodium desoxycholate on a well-isolated colony.

    Note: A tube test is performed with 2% sodium desoxycholate.

2. Gently wash liquid over the colony without dislodging the colony from the agar.

3. Incubate the plate at 35°-37°C in ambient air for 30 minutes.

4. Examine for lysis of colony.

Expected Results

Positive: Colony disintegrates; an imprint of the lysed colony may remain in the zone (Figure 13-7, A).

Negative: Intact colonies (Figure 13-7, B).

Limitations

Enzyme activity may be reduced in old cultures. Therefore, negative results with colonies resembling S. pneumoniae should be further tested for identification with alternate methods.

Quality Control

Positive: Streptococcus pneumoniae (ATCC49619)—bile soluble

Negative: Enterococcus faecalis (ATCC29212)—bile insoluble

Procedure 13-6   Butyrate Disk

Purpose

This is a rapid test to detect the enzyme butyrate esterase, to aid identification of Moraxella (Branhamella) catarrhalis.

Principle

Organisms capable of producing butyrate esterase hydrolyze bromochlorindolyl butyrate. Hydrolysis of the substrate in the presence of butyrate esterase releases indoxyl, which in the presence of oxygen spontaneously forms indigo, a blue to blue-violet color.

Method

1. Remove a disk from the vial and place on a glass microscope slide.

2. Add 1 drop of reagent-grade water. This should leave a slight excess of water on the disk.

3. Using a wooden applicator stick, rub a small amount of several colonies from an 18- to 24-hour pure culture onto the disk.

4. Incubate at room temperature for up to 5 minutes.

Expected Results

Positive: Development of a blue color during the 5-minute incubation period (Figure 13-8, A).

Negative: No color change (Figure 13-8, B).

Limitations

Incubation longer than 5 minutes may result in a false-positive reaction.

False-negative reactions may occur if the inoculum is too small. If the organism is negative, repeat with a larger inoculum and follow-up with additional methods.

Quality Control

Positive: Moraxella catarrhalis (ATCC25240)—formation of blue color

Negative: Neisseria gonorrhoeae (ATCC43069)—no color change

Procedure 13-7   CAMP Test

Purpose

The Christie, Atkins, and Munch-Peterson (CAMP) test is used to differentiate group B streptococci (Streptococcus agalactiae–positive) from other streptococcal species. Listeria monocytogenes also produces a positive CAMP reaction.

Principle

Certain organisms (including group B streptococci) produce a diffusible extracellular hemolytic protein (CAMP factor) that acts synergistically with the beta-lysin of Staphylococcus aureus to cause enhanced lysis of red blood cells. The group B streptococci are streaked perpendicular to a streak of S. aureus on sheep blood agar. A positive reaction appears as an arrowhead zone of hemolysis adjacent to the place where the two streak lines come into proximity.

Method

1. Streak a beta-lysin–producing strain of S. aureus down the center of a sheep blood agar plate.

2. Streak test organisms across the plate perpendicular to the S. aureus streak within 2 mm. (Multiple organisms can be tested on a single plate).

3. Incubate overnight at 35°-37°C in ambient air.

Expected Results

Positive: Enhanced hemolysis is indicated by an arrowhead-shaped zone of beta-hemolysis at the juncture of the two organisms (Figure 13-9, A).

Negative: No enhancement of hemolysis (Figure 13-9, B).

Limitations

A small percentage of group A streptococci may have a positive CAMP reaction. The test should be limited to colonies with the characteristic group B streptococci morphology and narrow zone beta-hemolysis on sheep blood agar.

Quality Control

Positive: Streptococcus agalactiae (ATCC13813)—enhanced arrowhead hemolysis

Negative: Streptococcus pyogenes (ATCC19615)—beta-hemolysis without enhanced arrowhead formation

Procedure 13-8   Catalase Test

Purpose

This test differentiates catalase-positive micrococcal and staphylococcal species from catalase-negative streptococcal species.

Principle

Aerobic and facultative anaerobic organisms produce two toxins during normal metabolism, hydrogen peroxide (H₂O₂) and superoxide radical (O2−). These bacteria have two enzymes that detoxify the products of normal metabolism. One of these enzymes, catalase, is capable of converting hydrogen peroxide to water and oxygen. The presence of the enzyme in a bacterial isolate is evidenced when a small inoculum introduced into hydrogen peroxide (30% for the slide test) causes rapid elaboration of oxygen bubbles. The lack of catalase is evident by a lack of or weak bubble production.

Method

1. Use a loop or sterile wooden stick to transfer a small amount of colony growth to the surface of a clean, dry glass slide.

2. Place a drop of 30% hydrogen peroxide (H₂O₂) onto the medium.

3. Observe for the evolution of oxygen bubbles (Figure 13-10).

Expected Results

Positive: Copious bubbles are produced (Figure 13-10, A).

Negative: No or few bubbles are produced (Figure 13-10, B).

Limitations

Some organisms (enterococci) produce a peroxidase that slowly catalyzes the breakdown of H₂O₂, and the test may appear weakly positive. This reaction is not a truly positive test.

False positives may occur if the sample is contaminated with blood agar.

Quality Control

Positive: Staphylococcus aureus (ATCC25923)

Negative: Streptococcus pyogenes (ATCC19615)

Procedure 13-9   Cetrimide Agar

Purpose

This test is primarily used to isolate and purify Pseudomonas aeruginosa from contaminated specimens.

Principle

The test is used to determine the ability of an organism to grow in the presence of cetrimide, a toxic substance that inhibits the growth of many bacteria by causing the release of nitrogen and phosphorous, which slows or kills the organism. P. aeruginosa is resistant to cetrimide.

Media: Enzymatic digest of gelatin (20 g), MgCl2 (1.4 g), K2SO4 (10 g), cetrimide (cetyltrimethylammonium bromide) (0.3 g), agar (13.6), pH 7.2.

Method

1. Inoculate a cetrimide agar slant with 1 drop of an 18- to 24-hour brain-heart infusion broth culture.

2. Incubate at 35°-37°C for up to 7 days.

3. Examine the slant for bacterial growth.

Expected Results

Positive: Growth, variation in color of colonies (Figure 13-11, A).

Negative: No growth (Figure 13-11, B).

Limitations

Some enteric organisms will grow and exhibit a weak yellow color in the media. This color change is distinguishable from the production of fluorescein.

Additional testing is necessary to confirm a diagnosis of P. aeruginosa.

Quality Control

Positive: Pseudomonas aeruginosa (ATCC27853)—growth and color change; yellow-green to blue-green colonies

Negative: Escherichia coli (ATCC25922)—no growth and no color change

Procedure 13-10   Citrate Utilization

Purpose

The purpose of this test is to identify organisms capable of using sodium citrate as the sole carbon source and inorganic ammonium salts as the sole nitrogen source. The test is part of a series referred to as IMViC (indole, methyl red, Voges-Proskauer, and citrate), which is used to differentiate Enterobacteriaceae from other gram-negative rods.

Principle

Bacteria that can grow on this medium produce an enzyme, citrate-permease, capable of converting citrate to pyruvate. Pyruvate can then enter the organism’s metabolic cycle for the production of energy. Bacteria capable of growth in this medium use the citrate and convert ammonium phosphate to ammonia and ammonium hydroxide, creating an alkaline pH. The pH change turns the bromthymol blue indicator from green to blue.

Media: NH₄H₂PO₄ (1 g), K₂HPO₄ (1 g), NaCl (5 g), sodium citrate (2 g), MgSO₄ (0.2 g), agar (15 g), bromthymol blue (0.08 g), per 1000 mL, pH 6.9.

Method

1. Inoculate Simmons citrate agar lightly on the slant by touching the tip of a needle to a colony that is 18 to 24 hours old. Do not inoculate from a broth culture, because the inoculum will be too heavy.

2. Incubate at 35°-37°C for up to 7 days.

3. Observe for growth and the development of blue color, denoting alkalinization.

Expected Results

Positive: Growth on the medium, with or without a change in the color of the indicator. Growth typically results in the bromthymol blue indicator turning from green to blue (Figure 13-12, A).

Negative: Absence of growth (Figure 13-12, B).

Limitations

Some organisms are capable of growth on citrate and do not produce a color change. Growth is considered a positive citrate utilization test, even in the absence of a color change.

Quality Control

Positive: Enterobacter aerogenes (ATCC13048)—growth, blue color

Negative: Escherichia coli (ATCC25922)—little to no growth, no color change

Procedure 13-11   Coagulase Test

Purpose

The test is used to differentiate Staphylococcus aureus (positive) from coagulase-negative staphylococci (negative).

Principle

S. aureus produces two forms of coagulase, bound and free. Bound coagulase, or “clumping factor,” is bound to the bacterial cell wall and reacts directly with fibrinogen. This results in precipitation of fibrinogen on the staphylococcal cell, causing the cells to clump when a bacterial suspension is mixed with plasma. The presence of bound coagulase correlates with free coagulase, an extracellular protein enzyme that causes the formation of a clot when S. aureus colonies are incubated with plasma. The clotting mechanism involves activation of a plasma coagulase-reacting factor (CRF), which is a modified or derived thrombin molecule, to form a coagulase-CRF complex. This complex in turn reacts with fibrinogen to produce the fibrin clot.

Method

A Slide Test (Detection of ?)

1. Place a drop of coagulase plasma (preferably rabbit plasma with ethylenediaminetetraacetic acid [EDTA]) on a clean, dry, glass slide.

2. Place a drop of distilled water or saline next to the drop of plasma as a control.

3. With a loop, straight wire, or wooden stick, emulsify a portion of the isolated colony being tested in each drop, inoculating the water or saline first. Try to create a smooth suspension.

4. Mix well with a wooden applicator stick.

5. Rock the slide gently for 5 to 10 seconds.

Expected Results

Positive: Macroscopic clumping in 10 seconds or less in coagulated plasma drop and no clumping in saline or water drop (Figure 13-13, A, left side).

Negative: No clumping in either drop.

Note: All negative slide tests must be confirmed using the tube test (Figure 13-13, B, right side).

B Tube Test

1. Emulsify several colonies in 0.5 mL of rabbit plasma (with EDTA) to give a milky suspension.

2. Incubate tube at 35°-37°C in ambient air for 4 hours.

3. Check for clot formation.

Expected Results

Positive: Clot of any size (Figure 13-13, A, left side).

Negative: No clot (Figure 13-13, B, right side).

Limitations

Slide Test

Equivocal: Clumping in both drops indicates that the organism autoagglutinates and is unsuitable for the slide coagulase test.

Tube Test

1. Test results can be positive at 4 hours and then revert to negative after 24 hours.

2. If negative at 4 hours, incubate at room temperature overnight and check again for clot formation.

Quality Control

Positive: Staphylococcus aureus (ATCC25923)

Negative: Staphylococcus epidermidis (ATC12228)

Procedure 13-12   Decarboxylase Tests (Moeller’s Method)

Purpose

This test is used to differentiate decarboxylase-producing Enterobacteriaceae from other gram-negative rods.

Principle

This test measures the enzymatic ability (decarboxylase) of an organism to decarboxylate (or hydrolyze) an amino acid to form an amine. Decarboxylation, or hydrolysis, of the amino acid results in an alkaline pH and a color change from orange to purple.

Media: Peptic digest of animal tissue (5 g), beef extract (5 g), bromcresol purple (0.1 g), cresol red (0.005 g), dextrose (0.5 g), pyridoxal (0.005 g), amino acid (10 g), pH 6.0.

Method

A Glucose-Nonfermenting Organisms

1. Prepare a suspension (≥McFarland No. 5 turbidity standard) in brain-heart infusion broth from an overnight culture (18 to 24 hours old) growing on 5% sheep blood agar.

2. Inoculate each of the three decarboxylase broths (arginine, lysine, and ornithine) and the control broth (no amino acid) with 4 drops of broth.

3. Add a 4-mm layer of sterile mineral oil to each tube.

4. Incubate the cultures at 35°-37°C in ambient air. Examine the tubes at 24, 48, 72, and 96 hours.

B Glucose-Fermenting Organisms

1. Inoculate tubes with 1 drop of an 18- to 24-hour brain-heart infusion broth culture.

2. Add a 4-mm layer of sterile mineral oil to each tube.

3. Incubate the cultures for 4 days at 35°-37°C in ambient air. Examine the tubes at 24, 48, 72, and 96 hours.

Expected Results

Positive: Alkaline (purple) color change compared with the control tube (Figure 13-14, A).

Negative: No color change or acid (yellow) color in test and control tube. Growth in the control tube.

Limitations

The fermentation of dextrose in the medium causes the acid color change. However, it would not mask the alkaline color change brought about by a positive decarboxylation reaction (Figure 13-14, B). An uninoculated tube is shown in Figure 13-14, C.

Quality Control

Positive:

Lysine—Klebsiella pneumoniae (ATCC33495)

Ornithine—Enterobacter aerogenes (ATCC13048)

Arginine—Enterobacter cloacae (ATCC13047)

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