Laboratory Methods and Strategies for Antimicrobial Susceptibility Testing

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Laboratory Methods and Strategies for Antimicrobial Susceptibility Testing

Objectives

1. List the relevant factors considered for control and standardization of antimicrobial susceptibility testing.

2. Describe testing conditions (medium, inoculum size, incubation conditions, incubation duration, controls, and purpose) for the broth dilution, agar dilution, and disk diffusion methods.

3. Define a McFarland standard and explain how it is used to standardize susceptibility testing.

4. Explain how end points are determined for the broth dilution, agar dilution, and disk diffusion methods.

5. Define the minimal inhibitory concentration (MIC) break point and identify the types of testing used to determine an MIC.

6. Define peak and trough levels and describe the clinical application for the data associated with each level.

7. Define the susceptible, intermediate, and resistant interpretive categories of antimicrobial susceptibility testing.

8. Outline the basic principles for agar screens, disk screens, and the “D” test for antimicrobial resistance detection, including method, application, and clinical utility.

9. Explain the principle and purpose of the chromogenic cephalosporinase test.

10. Compare and contrast molecular methods to detect resistance mechanisms versus traditional susceptibility testing, including clinical utility, effectiveness, and specificity.

11. Restate the principle of the minimal bactericidal concentration, time-kill assay, serum bactericidal test, and synergy test.

12. Define synergy and indifferent and antagonistic interactions in drug combinations.

13. Define and describe the purpose of drug susceptibility testing as it relates to the use of predictor drugs and organismal identification.

14. List the criteria for determining when to perform susceptibility testing.

15. Describe the purpose of reviewing susceptibility profiles and provide examples of profiles requiring further evaluation.

As discussed in Chapter 11, most clinically relevant bacteria are capable of acquiring and expressing resistance to antimicrobial agents commonly used to treat infections. Therefore, once an organism is isolated in the laboratory, characterization frequently includes tests to detect antimicrobial resistance. In addition to identifying the organism, the antimicrobial susceptibility profile often is a key component of the clinical laboratory report produced for the physician. The procedures used to produce antimicrobial susceptibility profiles and detect resistance to therapeutic agents are referred to as antimicrobial susceptibility testing (AST) methods. The methods applied for profiling aerobic and facultative anaerobic bacteria are the focus of this chapter; strategies for when and how these methods should be applied are also considered. Procedures for antimicrobial susceptibility testing of clinical isolates of anaerobic bacteria and mycobacteria are discussed in Chapters 41 and 43, respectively.

Goal and Limitations

The primary goal of antimicrobial susceptibility testing is to determine whether the bacterial isolate is capable of expressing resistance to the therapeutic antimicrobial agents selected for treatment. Because intrinsic resistance is usually known for most organisms, testing for instrinsic resistance usually is not necessary and organism identification is sufficient. In essence, antimicrobial susceptibility tests are assays designed to determine the extent of acquired resistance in any clinically important organism for which the antimicrobial susceptibility profile is unpredictable.

Standardization

For laboratory tests to accurately determine organism-based resistances, the potential influence of environmental factors on antibiotic activity should be minimized (see Chapter 11). This is not to suggest that environmental resistance does not play a clinically relevant role; however, the major focus of the in vitro tests is to measure an organism’s expression of resistance. To control the impact of environmental factors, the conditions for susceptibility testing are extensively standardized. Standardization serves three important purposes:

Standard conditions for antimicrobial susceptibility testing methods have been established based on numerous laboratory investigations. The procedures, guidelines, and recommendations are published in documents from the Subcommittee on Antimicrobial Susceptibility Testing of the Clinical and Laboratory Standards Institute (CLSI). The CLSI documents that describe various methods of antimicrobial susceptibility testing are continuously updated and may be obtained by contacting CLSI, 940 W. Valley Road, Suite 1400, Wayne, Pennsylvania, 19087. www.clsi.org

The standardized components of antimicrobial susceptibility testing include:

Limitations of Standardization

Although standardization of in vitro conditions is essential, the use of standard conditions imparts some limitations. Most notably, the laboratory test conditions cannot reproduce the in vivo environment at the infection site where the antimicrobial agent and bacteria will actually interact. Factors such as the bacterial inoculum size, pH, cation concentration, and oxygen tension can differ substantially, depending on the site of infection. Additionally, several other important factors play key roles in the patient outcome and are not taken into account by susceptibility testing. Some of these factors include:

Despite these limitations, antimicrobial resistance can substantially alter the rates of morbidity and mortality in infected patients. Early and accurate recognition of resistant bacteria significantly aids the selection of antimicrobial therapy and optimal patient management. Thus, in vitro susceptibility testing provides valuable data that are used in conjunction with other diagnostic information to guide patient therapeutic options. Additionally, as discussed later in this chapter, in vitro susceptibility testing provides the data to track resistance trends among clinically relevant bacteria.

Testing Methods

Principles

Three general methods are available to detect and evaluate antimicrobial susceptibility:

The method used depends on factors such as clinical need, accuracy, and convenience. Given the complexities of antimicrobial resistance patterns, a laboratory may commonly use methods from more than one category.

Methods That Directly Measure Antimicrobial Activity

Methods that directly measure antimicrobial activity involve bringing the antimicrobial agents of interest and the infecting bacterium together in the same in vitro environment to determine the impact of the drug’s presence on bacterial growth or viability. The level of impact on bacterial growth is measured, and the organism’s resistance or susceptibility to each agent is reported to the clinician. Direct measures of antimicrobial activity are accomplished using:

Conventional Testing Methods: General Considerations

Some general considerations apply to all three methods, including inoculum preparation and selection of antimicrobial agents.

Inoculum Preparation.

Properly prepared inocula are the key to any antimicrobial susceptibility testing method. Inconsistencies in inoculum preparation may lead to inconsistencies and inaccuracies in susceptibility test results. The two important requirements for correct inoculum preparation are use of a pure culture and use of a standard-sized inoculum.

Interpretation of results obtained with a mixed culture is not reliable and can substantially delay reporting of results. Pure inocula are obtained by selecting four or five colonies of the same morphology, inoculating them into a broth medium, and allowing the culture to achieve active growth (i.e., midlogarithmic phase), as indicated by observable turbidity in the broth. For most organisms this requires 3 to 5 hours of incubation. Alternatively, four to five colonies 16 to 24 hours of age may be selected from an agar plate and suspended in broth or 0.9% saline solution to achieve a turbid suspension.

Use of a standard inoculum size is as important as culture purity and is accomplished by comparing the turbidity of the organism suspension with a turbidity standard. McFarland turbidity standards, prepared by mixing 1% sulfuric acid and 1.175% barium chloride to obtain a solution with a specific optical density, are commonly used. The 0.5 McFarland standard, which is commercially available, provides an optical density comparable to the density of a bacterial suspension of 1.5 × 108 colony forming units (CFU) per milliliter. Pure cultures are grown or are prepared directly from agar plates to match the turbidity of the 0.5 McFarland standard (Figure 12-1). The newly inoculated bacterial suspension and the McFarland standard are compared by examining turbidity against a dark background. Alternatively, any one of various commercially available instruments capable of measuring turbidity may be used to standardize the inoculum. If the bacterial suspension does not match the standard’s turbidity, the suspension may be further diluted or supplemented with more organisms as needed.

Selection of Antimicrobial Agents for Testing.

The antimicrobial agents chosen for testing against a particular bacterial isolate are referred to as the antimicrobial battery or panel. A laboratory may use different testing batteries, but the content and application of each battery are based on specific criteria. Although the criteria listed in Box 12-1 influence the selection of the panel’s content, the final decision should not be made by the laboratory independently; input from the medical staff (particularly infectious diseases specialists) and the pharmacy is imperative.

Box 12-1   Criteria for Antimicrobial Battery Content and Use

CLSI publishes up-to-date tables listing potential antimicrobial agents recommended for inclusion in batteries for testing against specific organisms or organism groups. Two tables are of particular interest: Table 1, “Suggested Groupings of U.S. FDA–Approved Antimicrobial Agents That Should Be Considered for Routine Testing and Reporting on Nonfastidious Organisms by Clinical Microbiology Laboratories,” and Table 1A, “Suggested Groupings of U.S. FDA–Approved Antimicrobial Agents That Should Be Considered for Routine Testing and Reporting on Fastidious Organisms by Clinical Microbiology Laboratories.” Because revisions are made annually, laboratory protocols should be reviewed and modified accordingly (see the Bibliography). Further considerations about antibiotics that may be used for a specific organism or group are presented later in this chapter and in various chapters in Part III of this text.

Testing profiles are considered for each of the common organism groupings:

Conventional Testing Methods: Broth Dilution

Broth dilution testing involves challenging the organism of interest with antimicrobial agents in a liquid environment. Each antimicrobial agent is tested using a range of concentrations, commonly expressed as micrograms (µg) of active drug per milliliter (mL) of broth (i.e., µg/mL). The concentration range examined for a particular drug depends on specific criteria, including the safest therapeutic concentration possible in a patient’s serum. Therefore, the concentration range examined often varies from one drug to the next, depending on the pharmacologic properties of the antimicrobial agent. Additionally, the concentration range may be based on the level of drug required to reliably detect a particular resistance mechanism. In this case, the test concentration for a drug may vary depending on the organism and its associated resistances. For example, to detect clinically significant resistance to cefotaxime in S. pneumoniae, the dilution scheme uses a maximum concentration of 2 µg/mL; however, to detect cefotaxime resistance in Escherichia coli, the required maximum concentration is 16 µg/mL or higher.

Typically, the range of concentrations examined for each antibiotic is a series of doubling dilutions (e.g., 16, 8, 4, 2, 1, 0.5, 0.25 µg/mL); the lowest antimicrobial concentration that completely inhibits visible bacterial growth, as detected visually or with an automated or semiautomated method, is recorded as the minimal inhibitory concentration (MIC).

Procedures.

The key features of broth dilution testing procedures are shown in Table 12-1. Because changes are made in these procedural recommendations, the CLSI M07 series, “Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically,” should be consulted annually.

TABLE 12-1

Summary of Broth Dilution Susceptibility Testing Conditions

Organism Groups Test Medium Inoculum Size (CFU/mL) Incubation Conditions Incubation Duration
Enterobacteriaceae Mueller-Hinton 5 × 105 35°C; air 16-20 hr
Staphylococci (to detect methicillin-resistant staphylococci) Mueller-Hinton plus 2% NaCl   30°-35°C; air 24 hr
Streptococcus pneumoniae and other streptococci Mueller-Hinton plus 2%-5% lysed horse blood 5 × 105 35°C; 5%-10% CO2 20-24 hr
Haemophilus influenzae Haemophilus test medium 5 × 105 35°C; 5%-10% CO2 20-24 hr
Neisseria meningitidis Mueller-Hinton plus 2%-5% lysed horse blood 5 × 105 35°C; 5%-7% carbon dioxide (CO2) 24 hr

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Medium and Antimicrobial Agents.

With in vitro susceptibility testing methods, certain conditions must be altered when examining fastidious organisms to optimize growth and facilitate expression of bacterial resistance. For example, the Mueller-Hinton preparation is the standard medium used for most broth dilution testing, and conditions in the medium (e.g., pH, cation concentration, thymidine content) are well controlled by commercial manufacturers. However, media supplements or different media are required to obtain good growth and reliable susceptibility profiles for bacteria such as S. pneumoniae and H. influenzae. Although staphylococci are not considered fastidious organisms, media supplemented with sodium chloride (NaCl) enhance the expression and detection of methicillin-resistant isolates (see Table 12-1).

Broth dilution testing is divided into two general categories: microdilution and macrodilution. The principle of each test is the same; the only difference is the volume of broth in which the test is performed. For microdilution testing, the total broth volume is 0.05 to 0.1 mL; for macrodilution testing, the broth volumes are usually 1 mL or greater. Because most susceptibility test batteries require testing of several antibiotics at several different concentrations, the smaller volume used in microdilution allows this to be conveniently accomplished in a single microtiter tray (Figure 12-2).

The need for multiple large test tubes in the macrodilution method makes that technique substantially cumbersome and labor intensive when several bacterial isolates are tested simultaneously. For this reason, macrodilution is rarely used in most clinical laboratories, and subsequent comments about broth dilution focuses on the microdilution approach.

A key component of broth testing is proper preparation and dilution of the antimicrobial agents incorporated into the broth medium. Most laboratories that perform broth microdilution use commercially supplied microdilution panels in which the broth is already supplemented with appropriate antimicrobial concentrations. Therefore, antimicrobial preparation and dilution are not commonly carried out in most clinical laboratories (the details of this procedure are outlined in the CLSI M07-A6 document). In most instances, each antimicrobial agent is included in the microtiter trays as a series of doubling twofold dilutions. To ensure against loss of antibiotic potency, the antibiotic microdilution panels are stored at −20°C or lower, if possible, and are thawed immediately before use. Once thawed the panels should never be refrozen, which may result in substantial loss of antimicrobial action and potency. Alternatively, the antimicrobial agents may be lyophilized or freeze dried with the medium or drug in each well; upon inoculation with the bacterial suspension, the medium and drug are simultaneously reconstituted to the appropriate concentration.

Inoculation and Incubation.

Standardized bacterial suspensions that match the turbidity of the 0.5 McFarland standard (i.e., 1.5 × 108 CFU/mL) usually serve as the starting point for dilutions ultimately achieving the required final standard bacterial concentration of 5 × 105 CFU/mL in each microtiter well. It is essential to prepare the standard inoculum from a fresh, overnight, pure culture of the test organism. Inoculation of the microdilution panel is accomplished using manual or automated multiprong inoculators calibrated to deliver the precise volume of inoculum to each well in the panel simultaneously (see Figure 12-2).

Inoculated trays are incubated under optimal environmental conditions to optimize bacterial growth without interfering with the antimicrobial activity (i.e., avoiding environmentally mediated results). For the most commonly tested bacteria (e.g., Enterobacteriaceae, P. aeruginosa, staphylococci, and enterococci), the environmental condition consists of room air at 35°C (see Table 12-1). Fastidious bacteria, such as H. influenzae, require incubation in 5% to 10% carbon dioxide (CO2). Similarly, incubation durations for some organisms may need to be extended beyond the usual 16 to 20 hours (see Table 12-1). However, prolonged incubation times beyond recommended limits should be avoided, because antimicrobial deterioration may result in false or elevated resistance patterns. This is a primary factor that limits the ability to perform accurate testing with some slow-growing bacteria.

Reading and Interpretation of Results.

After incubation, the microdilution trays are examined for bacterial growth. Each tray should include a growth control that does not contain antimicrobial agent and a sterility control that was not inoculated. Once growth in the growth control and no growth in the sterility control wells have been confirmed, the growth profiles for each antimicrobial dilution can be established and the MIC determined. The detection of growth in microdilution wells is often augmented through the use of light boxes and reflecting mirrors. When a panel is placed in these devices, bacterial growth, manifested as light to heavy turbidity or a button of growth on the well bottom, is more reliably visualized (Figure 12-3).

When the dilution series for each antibiotic is inspected, the microdilution well containing the lowest drug concentration that completely inhibits visible bacterial growth is recorded as the MIC. Once the MICs for the antimicrobials in the test battery for an organism have been recorded, they are usually translated into one of the interpretive categories, specifically susceptible, intermediate, or resistant (Box 12-2). The interpretive criteria for these categories are based on extensive studies that correlate the MIC with serum-achievable levels for each antimicrobial agent, particular resistance mechanisms, and successful therapeutic outcomes. The interpretive criteria for an array of antimicrobial agents are published in the CLSI M07 series document, “Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically (M100 supplements).” For example, using these standards, an isolate of P. aeruginosa with an imipenem MIC of less than or equal to 4 µg/mL would be classified as susceptible; one with an MIC of 8 µg/mL would be classified as intermediate; and one with an MIC of 16 µg/mL or greater would be classified as resistant to imipenem.

After the MICs are determined and their respective and appropriate interpretive categories assigned, the laboratory may report the MIC, the category, or both. Because the MIC alone will not provide most physicians with a meaningful interpretation of data, either the category result with or without the MIC is usually reported.

In some settings, the full range of antimicrobial dilutions is not used; only the concentrations that separate the categories of susceptible, intermediate, and resistant are used. The specific concentrations that separate or define the different categories are known as breakpoints, and panels that only contain these antimicrobial concentrations are referred to as breakpoint panels. In this case, only category results are produced; precise MICs are not available, because the full range of dilutions is not tested.

Advantages and Disadvantages.

Broth dilution methods provide data for both quantitative results (i.e., MIC) and qualitative results (i.e., category interpretation). Whether this is an advantage is the subject of debate. On one hand, the MIC can be helpful in establishing the level of resistance of a particular bacterial strain and can substantially affect the decision to treat a patient with a specific antimicrobial agent. For example, the penicillin MIC for S. pneumoniae may determine whether penicillin or alternative agents will be used to treat a patient with meningitis. On the other hand, for most antimicrobial susceptibility testing methods, a category report is sufficient and the actual MIC data are superfluous. This is one reason other methods (e.g., disk diffusion) that focus primarily on producing interpretive categories have been maintained among clinical microbiologists.

Conventional Testing Methods: Agar Dilution

With agar dilution the antimicrobial concentrations and organisms to be tested are brought together on an agar-based medium rather than in liquid broth. Each doubling dilution of an antimicrobial agent is incorporated into a single agar plate; therefore, testing of a series of six dilutions of one drug requires the use of six plates, plus one positive growth control plate without antibiotic. The standard conditions and media for agar dilution testing are shown in Table 12-2. The surface of each plate is inoculated with 1 × 104 CFU (Figure 12-4). This method allows examination of one or more bacterial isolates per plate. After incubation the plates are examined for growth; the MIC is the lowest concentration of an antimicrobial agent in agar that completely inhibits visible growth. The same MIC breakpoints and interpretive categories used for broth dilution are applied for interpretation of agar dilution methods. Similarly, test results may be reported as the MICs only, the category only, or both.

TABLE 12-2

Summary of Agar Dilution Susceptibility Testing Conditions

Organism Groups Test Medium Inoculum Size (CFU/spot) Incubation Conditions Incubation Duration
Enterobacteriaceae Mueller-Hinton 1 × 104 35°C; air 16-20 hr
Enterococci        
Staphylococci (to detect methicillin-resistant staphylococci) Mueller-Hinton plus 2% NaCl   30°-35°C; air 24 hr
Neisseria meningitidis Mueller-Hinton plus 5% sheep blood 1 × 104 35°C; 5%-7% carbon dioxide (CO2) 24 hr
Streptococcus pneumoniae Agar dilution not recommended method for testing this organism        
Other streptococci Mueller-Hinton plus 5% sheep blood 1 × 104 35°C; air, CO2 may be needed for some isolates 20-24 hr
Neisseria gonorrhoeae GC agar plus supplements 1 × 104 35°C; 5%-X% CO2 24 hr

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The preparation of agar dilution plates (see CLSI M07-A6 series document, “Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically”) is sufficiently labor intensive to preclude the use of this method in most clinical laboratories in which multiple antimicrobial agents must be tested, even though several isolates may be tested per plate. As with broth dilution, the standard medium is the Mueller-Hinton preparation, but supplements and substitutions are made as needed to facilitate growth of more fastidious organisms. In fact, one advantage of this method is that it provides a means for determining MICs for N. gonorrhoeae, which does not grow sufficiently in broth to be tested by broth dilution methods.

Conventional Testing Methods: Disk Diffusion

As more antimicrobial agents were created to treat bacterial infections, the limitations of the macrobroth dilution method became apparent. Before microdilution technology became widely available, it became clear that a more practical and convenient method of testing multiple antimicrobial agents against bacterial strains was needed. Out of this need the disk diffusion test was developed, emerging from the landmark study by Bauer et al.1 in 1966. These investigators standardized and correlated the use of antibiotic-impregnated filter paper disks (i.e., antibiotic disks) with MICs using many bacterial strains. With the disk diffusion susceptibility test, antimicrobial resistance is detected by challenging bacterial isolates with antibiotic disks placed on the surface of an agar plate that has been seeded with a lawn of bacteria (Figure 12-5).

When disks containing a known concentration of antimicrobial agent are placed on the surface of a freshly inoculated plate, the agent immediately begins to diffuse into the agar and establish a concentration gradient around the paper disk. The highest concentration is closest to the disk. Upon incubation, the bacteria grow on the surface of the plate except where the antibiotic concentration in the gradient around each disk is sufficiently high to inhibit growth. After incubation, the diameter of the zone of inhibition around each disk is measured in millimeters (see Figure 12-5).

To establish reference inhibitory zone–size breakpoints to define the susceptible, intermediate, and resistant categories for each antimicrobial agent/bacterial species combination, hundreds of strains are tested. The inhibition zone sizes obtained are then correlated with MICs obtained by broth or agar dilution, and a regression analysis is completed comparing the zone size in millimeters against the MIC (Figure 12-6). As the MICs of the bacterial strains tested increase (i.e., the more resistant bacterial strains), the corresponding inhibition zone sizes (i.e., diameters) decrease. Using Figure 12-6 to illustrate, horizontal lines are drawn from the MIC resistant breakpoint and the susceptible MIC breakpoint, 8 µg/mL and 2 µg/mL, respectively. Where the horizontal lines intersect the regression line, vertical lines are drawn to delineate the corresponding inhibitory zone size breakpoints (in millimeters). Using this approach, zone size interpretive criteria have been established for most of the commonly tested antimicrobial agents and are published in the CLSI M02 series, “Performance Standards for Antimicrobial Disk Susceptibility Tests.”

Procedures.

The key features of disk diffusion testing procedures are summarized in Table 12-3, with more details and updates available through CLSI.

TABLE 12-3

Summary of Disk Diffusion Susceptibility Testing Conditions

Organism Groups Test Medium Inoculum Size (CFU/mL) Incubation Conditions Incubation Duration
Enterobacteriaceae Mueller-Hinton agar Swab from 1.5 × 108 35°C; air 16-18 hr
Pseudomonas aeruginosa Mueller-Hinton agar Swab from 1.5 × 108 suspension 35°C; air 16-18 hr
Enterococci Mueller-Hinton agar Swab from 1.5 × 108 suspension 35°C; air 16-18 hr (24 hr for vancomycin)
Staphylococci (to detect methicillin-resistant staphylococci) Mueller-Hinton agar Swab from 1.5 × 108 suspension 30°-35°C; air 24 hr
Streptococcus pneumoniae and other streptococci Mueller-Hinton agar plus 5% sheep blood Swab from 1.5 × 108 suspension 35°C; 5%-7% carbon dioxide (CO2) 20-24 hr
Haemophilus influenzae Haemophilus test medium Swab from 1.5 × 108 suspension 35°C; 5%-7% CO2 16-18 hr
Neisseria gonorrhoeae GC agar plus supplements Swab from 1.5 × 108 suspension 35°C; 5%-7% CO2 20-24 hr

image

Medium and Antimicrobial Agents.

The Mueller-Hinton preparation is the standard agar-base medium used for testing of most bacterial organisms, although certain supplements and substitutions are required for testing of fastidious organisms. In addition to factors such as the pH and cation content, the depth of the agar medium can affect test accuracy and must be carefully controlled. Because antimicrobial agents diffuse in all directions from the surface of the agar plate, the thickness of the agar affects the antimicrobial drug concentration gradient. If the agar is too thick, the antimicrobial agent diffuses down through the agar as well as outward, resulting in smaller zone sizes; if the agar is too thin, the inhibition zones are larger. For many laboratories that perform disk diffusion testing, commercial manufacturers are reliable sources for properly prepared and controlled Mueller-Hinton plates.

The appropriate concentration of drug for each disk is predetermined and set by the U.S. Food and Drug Administration (FDA). The disks are available from various commercial sources and should be stored at the recommended temperature in a desiccator until used. Inappropriate storage can lead to deterioration of the antimicrobial agents and result in misleading zone size results.

To ensure equal diffusion of the drug into the agar, the disks must be placed flat on the surface and be firmly applied to ensure adhesion. This is most easily accomplished by using any one of several disk dispensers that are available through commercial disk manufacturers. With these dispensers, all disks in the test battery are simultaneously delivered to the inoculated agar surface and are adequately spaced to minimize the chances for inhibition zone overlap and significant interactions between antimicrobials. In most instances, a maximum of 12 antibiotic disks may be applied to the surface of a single 150-mm Mueller-Hinton agar plate (see Figure 12-5).

Inoculation and Incubation.

Before disk placement, the plate surface is inoculated using a swab that has been submerged in a bacterial suspension standardized to match the turbidity of the 0.5 McFarland turbidity standard, equivalent to 1.5 × 108 CFU/mL. The surface of the plate is swabbed in three directions to ensure even and complete distribution of the inoculum over the entire plate. Within 15 minutes of inoculation, the antimicrobial disks are applied and the plates are inverted for incubation to prevent the accumulation of moisture on the agar surface, which would interfere with the interpretation of test results.

Most organisms are incubated at 35°C in room air, but increased CO2 is used for testing of specific fastidious bacteria (see Table 12-3). Similarly, the incubation time may be increased beyond 16 hours to enhance detection of certain resistance patterns (e.g., methicillin resistance in staphylococci and vancomycin resistance in enterococci) and to ensure accurate results in general for fastidious organisms such as N. gonorrhoeae.

The dynamics and timing of antimicrobial agent diffusion required for establishing a concentration gradient, in addition to growth of the organisms over 18 to 24 hours, are critical for reliable results. Therefore, incubation of disk diffusion plates beyond the allotted time should be avoided, and disk diffusion generally is not an acceptable method for testing slow-growing organisms that require extended incubation such as mycobacteria and anaerobes.

Reading and Interpretation of Results.

Before results with individual antimicrobial agent disks are read, the plate is examined to confirm that a confluent lawn of growth has been obtained (see Figure 12-5). If growth between inhibitory zones around each disk is poor and nonconfluent, the test should not be interpreted and should be repeated. The lack of confluent growth may be due to insufficient inoculum. Alternatively, a particular isolate may have undergone mutation, and growth factors supplied by the standard medium are no longer sufficient to support robust growth. In the latter case, medium supplemented with blood and/or incubation in CO2 may enhance growth. However, caution in interpreting results is required when extraordinary measures are used to obtain good growth and the standard medium recommended for testing a particular type of organism is not used. Plates should also be examined for purity. Mixed cultures are evident through the appearance of different colony morphologies scattered throughout the lawn of bacteria (Figure 12-7). Mixed cultures require purification and repeat testing.

A dark background and reflected light are used to examine a disk diffusion plate (Figure 12-8

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