# Susceptibility Testing of Slow-Growing Bacteria

## Quick Answer

- Slow-growing bacteria such as *Brucella* and *Francisella* require extended incubation periods, enriched media, and modified susceptibility testing protocols because standard overnight methods produce unreliable results.
- Select a validated susceptibility testing method designed for fastidious organisms, extend incubation to 24 to 72 hours or longer, and verify growth adequacy before reading endpoints.
- Biosafety containment must match the organism risk group, and testing should occur only in facilities with appropriate controls because these pathogens pose serious laboratory hazards.

## Why Standard Susceptibility Testing Fails for Slow-Growing Bacteria

Clinical microbiology laboratories routinely perform antimicrobial susceptibility testing using standardized methods developed for rapidly growing pathogens. These methods assume that an organism will produce visible growth within 16 to 24 hours on standard Mueller-Hinton agar. Slow-growing bacteria violate this assumption. Organisms such as *Brucella* species, *Francisella tularensis*, *Nocardia* species, and certain *Mycobacterium* species require substantially longer incubation periods, often 48 to 72 hours or more, before colonies become visible and test results can be interpreted.

The problem is not simply that the bacteria grow slowly. The physiological state of slow-growing organisms differs from that of fast-growing pathogens. Their metabolic rates, cell wall synthesis kinetics, and response to antimicrobial agents are altered under standard test conditions. A minimum inhibitory concentration (MIC) measured after 16 to 20 hours of incubation may reflect the activity of the drug against a population that has not yet entered logarithmic growth, producing results that do not predict clinical response.

Standard susceptibility testing media also lack the supplements that many slow-growing organisms require. *Brucella* species grow poorly on unsupplemented Mueller-Hinton agar. *Francisella tularensis* requires cysteine and other growth factors that are absent from routine media. When the organism cannot grow adequately, the test produces either no result or a result that is not reproducible.

The consequences of unreliable susceptibility data are serious. Clinicians may select an antimicrobial agent that appears active in the test but fails in the patient, or they may avoid a drug that is actually effective. For organisms that cause severe or potentially fatal infections, such as *Brucella* and *Francisella*, the stakes are high. The World Health Organization identifies antimicrobial resistance as a global public health threat, and accurate susceptibility testing is a cornerstone of resistance surveillance and stewardship programs. When testing methods fail for slow-growing organisms, resistance trends may be missed, and treatment decisions may be made without adequate evidence.

## Organism Biology and Testing Challenges

### Growth Characteristics of Slow-Growing Pathogens

Slow-growing bacteria share several biological features that complicate susceptibility testing. Their generation times are measured in hours instead of minutes. A typical fast-growing organism such as *Escherichia coli* divides every 20 to 30 minutes under optimal conditions, while *Brucella* species may require several hours per generation. *Francisella tularensis* also grows slowly, and *Mycobacterium tuberculosis* has a generation time of approximately 15 to 20 hours. These extended generation times mean that visible growth appears only after prolonged incubation.

The growth requirements of these organisms also differ from those of standard test organisms. Many slow-growing pathogens are fastidious, meaning they require specific nutrients, growth factors, or atmospheric conditions. *Brucella* species grow best in media supplemented with serum or other enrichment factors. *Francisella tularensis* requires cysteine and other supplements for growth. Some slow-growing organisms also prefer reduced oxygen conditions or increased carbon dioxide concentrations. The American Type Culture Collection provides authenticated biological materials and strain information that laboratories use to establish and maintain quality control cultures for these organisms [ATCC culture collections](https://www.atcc.org/).

### Why Standard Media and Incubation Are Inadequate

Standard antimicrobial susceptibility testing uses Mueller-Hinton agar or broth, which supports the growth of most nonfastidious bacteria. This medium does not contain the supplements that slow-growing fastidious organisms require. When a laboratory attempts to test *Brucella* or *Francisella* on unsupplemented Mueller-Hinton agar, the organism may fail to grow or may grow so poorly that the test cannot be interpreted.

The incubation time is another critical limitation. Standard protocols call for reading results after 16 to 24 hours. Slow-growing organisms may not produce visible growth within this period. If the test is read too early, the result may show no growth, which could be misinterpreted as susceptibility to the antimicrobial agent. If the test is read too late, the organism may have overgrown the test system, producing results that are not reliable.

The Clinical and Laboratory Standards Institute publishes standards for antimicrobial susceptibility testing that address the special requirements of fastidious organisms [CLSI antimicrobial susceptibility testing standards](https://clsi.org/standards/products/microbiology). These standards provide guidance on media selection, incubation conditions, and interpretation criteria for organisms that do not grow under standard conditions. Laboratories that test slow-growing bacteria must follow these standards to produce reliable results.

### Biosafety Considerations

Slow-growing bacteria that require special susceptibility testing are often also dangerous pathogens. *Brucella* species are classified as biosafety level 3 (BSL-3) agents, and *Francisella tularensis* is also a BSL-3 organism. The Centers for Disease Control and Prevention and the National Institutes of Health publish the Biosafety in Microbiological and Biomedical Laboratories manual, which provides risk assessment, containment, decontamination, and laboratory biosafety principles for work with these organisms [CDC and NIH biosafety manual](https://www.cdc.gov/labs/bmbl/index.html).

Laboratories that perform susceptibility testing on these organisms must have appropriate containment facilities, including biological safety cabinets, negative air pressure, and personal protective equipment. The risk assessment must consider the potential for aerosol transmission, the severity of the disease, and the availability of effective treatment. The biosafety manual provides the framework for this risk assessment and for the selection of appropriate containment levels.

## Core Principles of Susceptibility Testing for Slow-Growing Bacteria

### The Minimum Inhibitory Concentration Concept

The minimum inhibitory concentration (MIC) is the lowest concentration of an antimicrobial agent that prevents visible growth of a microorganism under defined test conditions. The MIC is the foundation of antimicrobial susceptibility testing. For slow-growing bacteria, the MIC must be determined under conditions that allow the organism to grow adequately while still providing a meaningful measure of antimicrobial activity.

The MIC is not a fixed property of the organism. It depends on the test method, the medium, the inoculum size, the incubation time, and the interpretation criteria. For slow-growing bacteria, each of these variables must be adjusted to produce reliable results. The MIC is also not a direct measure of clinical efficacy. It is a laboratory measurement that must be interpreted in the context of the drug's pharmacokinetics, the site of infection, and the patient's condition.

### Broth Dilution and Agar Dilution Methods

Broth dilution and agar dilution are the two primary methods for determining MIC values. In broth dilution, the antimicrobial agent is diluted in a series of tubes or wells containing broth medium, and the organism is added to each tube. After incubation, the lowest concentration that prevents visible growth is the MIC. In agar dilution, the antimicrobial agent is incorporated into agar plates at various concentrations, and the organism is spotted onto the plates. The lowest concentration that prevents growth is the MIC.

For slow-growing bacteria, both methods require modifications. The broth must be supplemented with the nutrients required by the organism. The incubation time must be extended to allow adequate growth. The inoculum must be prepared from a culture that is in the appropriate growth phase. The American Type Culture Association provides authenticated strains and quality control information that laboratories use to validate their testing methods [ATCC culture collections](https://www.atcc.org/).

### Disk Diffusion Testing

Disk diffusion testing, also known as the Kirby-Bauer method, is a simpler approach that is widely used for fast-growing organisms. In this method, paper disks containing antimicrobial agents are placed on an agar plate that has been inoculated with the organism. The antimicrobial agent diffuses from the disk into the agar, creating a concentration gradient. After incubation, the zone of inhibition around each disk is measured and interpreted.

Disk diffusion is less commonly used for slow-growing bacteria because the extended incubation time can alter the diffusion of the antimicrobial agent and the growth of the organism. However, some slow-growing organisms can be tested by disk diffusion if the method is validated and the interpretation criteria are appropriate. The Clinical and Laboratory Standards Institute provides guidance on the use of disk diffusion for fastidious organisms [CLSI antimicrobial susceptibility testing standards](https://clsi.org/standards/products/microbiology).

### Gradient Diffusion Methods

Gradient diffusion methods, such as the Etest, use a plastic strip that contains a gradient of antimicrobial concentrations. The strip is placed on an agar plate that has been inoculated with the organism. After incubation, the point where the growth intersects the strip indicates the MIC. Gradient diffusion methods are convenient because they provide a quantitative MIC result with a single test.

For slow-growing bacteria, gradient diffusion methods require the same adjustments as other methods. The agar must be supplemented, the incubation time must be extended, and the inoculum must be standardized. The gradient diffusion method is often used for organisms that are difficult to test by broth dilution, but the results must be validated against a reference method.

## Media and Supplementation Requirements

### Mueller-Hinton Agar and Its Limitations

Mueller-Hinton agar is the standard medium for antimicrobial susceptibility testing. It is a simple medium that supports the growth of most fastidious organisms. However, it does not contain the supplements required by many slow-growing bacteria. *Brucella* species grow poorly on unsupplemented Mueller-Hinton agar, and *Francisella tularensis* requires cysteine and other growth factors.

The Clinical and Laboratory Standards Institute provides guidance on the use of supplemented media for fastidious organisms [CLSI antimicrobial susceptibility testing standards](https://clsi.org/standards/products/microbiology). The specific supplements depend on the organism being tested. For *Brucella* species, the medium may be supplemented with serum or other enrichment factors. For *Francisella*, the medium must contain cysteine and other supplements.

### Supplemented Media for *Brucella* and *Francisella*

*Brucella* species require a medium that supports their growth while allowing the antimicrobial agent to act. The recommended medium for *Brucella* susceptibility testing is Mueller-Hinton agar supplemented with serum and other factors. The serum provides the nutrients that *Brucella* requires for growth, while the antimicrobial agent diffuses through the agar.

*Francisella tularensis* requires a medium that contains cysteine, which is essential for its growth. The recommended medium for *Francisella* susceptibility testing is Mueller-Hinton agar supplemented with cysteine and other growth factors. The medium must be prepared carefully to ensure that the supplements are present in the correct concentrations.

The American Type Culture Association provides authenticated strains and quality control information for these organisms [ATCC culture collections](https://www.atcc.org/). Laboratories use these strains to verify that their media and methods produce reliable results.

### Incubation Time and Atmosphere

The incubation time for slow-growing bacteria must be extended beyond the standard 16 to 20 hours. For *Brucella* species, the incubation time is typically 48 hours, and for *Francisella tularensis*, the incubation time is typically 48 to 72 hours. The incubation time must be long enough to allow the organism to produce visible growth but not so long that the antimicrobial agent degrades or the organism overgrows.

The incubation atmosphere is also important. Many slow-growing bacteria require increased carbon dioxide concentrations for optimal growth. The incubation chamber must be set to provide the appropriate atmosphere. The temperature is typically 35 to 37 degrees Celsius, but the optimal temperature may vary by organism.

## Practical Workflow for Susceptibility Testing

### Step 1: Confirm the Organism Identification

Before performing susceptibility testing, the laboratory must confirm the identity of the organism. This is important because the testing method, media, and incubation conditions depend on the species. A misidentified organism can produce misleading susceptibility results.

The identification should be based on a combination of phenotypic and genotypic methods. The American Type Culture Association provides authenticated strains that can be used as controls for identification [ATCC culture collections](https://www.atcc.org/). The laboratory should also follow the biosafety requirements for the organism, as described in the CDC and NIH biosafety manual [CDC and NIH biosafety manual](https://www.cdc.gov/labs/bmbl/index.html).

### Step 2: Select the Testing Method

The choice of testing method depends on the organism, the laboratory resources, and the clinical need. Broth dilution and agar dilution are the reference methods for MIC determination. Gradient diffusion methods are convenient for single tests. Disk diffusion is less commonly used for slow-growing organisms.

The Clinical and Laboratory Standards Institute provides standards for the selection and use of susceptibility testing methods [CLSI antimicrobial susceptibility testing standards](https://clsi.org/standards/products/microbiology). The laboratory should select a method that is validated for the organism being tested and that produces reproducible results.

### Step 3: Prepare the Inoculum

The inoculum is the suspension of bacteria that is added to the test system. The inoculum size must be standardized to produce reliable results. For slow-growing bacteria, the inoculum is typically prepared from a culture that is in the logarithmic growth phase. The culture is suspended in a suitable broth, and the turbidity is adjusted to a standard value.

The inoculum size is critical. Too many bacteria may produce a false-resistant result, while too few bacteria may produce a false-susceptible result. The laboratory must follow the standard procedures for inoculum preparation, and the inoculum must be used within a short time after preparation.

### Step 4: Set Up the Test

The test is set up according to the method selected. For broth dilution, the antimicrobial agent is diluted in a series of wells, and the inoculum is added to each well. For agar dilution, the antimicrobial agent is incorporated into the agar, and the inoculum is spotted onto the plates. For gradient diffusion, the strip is placed on the agar plate.

The test must be set up in a biosafety cabinet to protect the laboratory personnel from the organism. The biosafety requirements are described in the CDC and NIH biosafety manual [CDC and NIH biosafety manual](https://www.cdc.gov/labs/bmbl/index.html). The test plates and tubes must be labeled clearly, and the test must be incubated under the appropriate conditions.

### Step 5: Incubate and Read the Test

The test is incubated for the appropriate time and under the appropriate atmosphere. The incubation time is typically 24 to 72 hours, depending on the organism. The test is read by examining the wells or plates for visible growth. The MIC is the lowest concentration of the antimicrobial agent that prevents visible growth.

The reading must be performed by a trained laboratory professional. The results must be recorded in the laboratory information system, and the interpretation must be based on the appropriate breakpoints. The breakpoints are provided by the Clinical and Laboratory Standards Institute [CLSI antimicrobial susceptibility testing standards](https://clsi.org/standards/products/microbiology).

### Step 6: Interpret and Report the Result

The MIC is interpreted using the breakpoints for the organism and the antimicrobial agent. The result is reported as susceptible, intermediate, or resistant. The interpretation must be based on the clinical context, and the report must include the MIC value and the interpretation.

The report must also include the method used, the incubation time, and any limitations of the test. The clinician uses the report to select the appropriate antimicrobial therapy. The report must be clear and complete to support the clinical decision.

## At a Glance: Key Parameters for Slow-Growing Bacteria

| Parameter | Standard Fast-Growing Bacteria | Slow-Growing Bacteria | Practical Implication |
| --- | --- | --- | --- |
| Incubation time | 16 to 24 hours | 24 to 72 hours or longer | Test results are not valid if read too early |
| Media | Mueller-Hinton agar | Supplemented Mueller-Hinton agar or specialized media | Standard media may not support growth |
| Atmosphere | Ambient air | Increased CO2 or other conditions | Incubation chamber must be adjusted |
| Inoculum preparation | Standard turbidity | Adjusted for slow growth | Inoculum must be prepared from a log-phase culture |
| Interpretation | Standard breakpoints | Organism-specific breakpoints | Breakpoints may differ from standard |

## Quality Control and Assurance

### The Role of Reference Strains

Reference strains are essential for quality control in susceptibility testing. These strains have known susceptibility patterns and are used to verify that the testing method, media, and reagents are working correctly. The American Type Culture Association provides authenticated reference strains for many organisms, including slow-growing bacteria [ATCC culture collections](https://www.atcc.org/).

The reference strains must be tested in parallel with the clinical isolates. The results of the reference strains must be within the expected range. If the reference strain results are outside the expected range, the test is invalid, and the laboratory must investigate the cause.

### Quality Control Procedures

Quality control procedures must be performed on a regular basis. The laboratory must test the reference strains with each batch of media and reagents. The results must be recorded and reviewed. The laboratory must also participate in external quality assessment programs to verify the accuracy of its testing.

The Clinical and Laboratory Standards Institute provides guidance on quality control for susceptibility testing [CLSI antimicrobial susceptibility testing standards](https://clsi.org/standards/products/microbiology). The laboratory must follow these guidelines to ensure the reliability of its results.

### Biosafety and Containment

The biosafety requirements for slow-growing bacteria depend on the organism. *Brucella* and *Francisella* are BSL-3 agents, and the laboratory must have appropriate containment. The CDC and NIH biosafety manual provides the requirements for BSL-3 containment, including the use of biological safety cabinets, personal protective equipment, and decontamination procedures [CDC and NIH biosafety manual](https://www.cdc.gov/labs/bmbl/index.html).

The laboratory must also have a risk assessment that considers the potential for aerosol transmission, the severity of the disease, and the availability of treatment. The risk assessment must be reviewed regularly and updated as needed.

## Common Failure Patterns and How to Avoid Them

### Failure to Confirm the Organism

A common failure is to perform susceptibility testing without confirming the identity of the organism. This can lead to the use of the wrong media, the wrong incubation time, and the wrong interpretation criteria. The laboratory must confirm the identity of the organism before testing.

### Inadequate Growth

The organism may fail to grow in the test system, producing no result. This is often due to the use of the wrong media or the wrong incubation conditions. The laboratory must use the appropriate media and incubation conditions for the organism.

### Overgrowth of the Organism

The organism may overgrow the test system, producing a result that is not reliable. This can occur if the incubation time is too long or the inoculum is too large. The laboratory must use the appropriate incubation time and inoculum size.

### Misinterpretation of the Result

The result may be misinterpreted if the breakpoints are not appropriate for the organism. The laboratory must use the breakpoints that are defined for the organism and the antimicrobial agent.

## Limitations and Evidence Gaps

### Limited Breakpoint Data

The breakpoints for slow-growing bacteria are less well established than for fast-growing organisms. The Clinical and Laboratory Standards Institute provides breakpoints for some organisms, but the data may be limited. The laboratory must be aware of the limitations of the breakpoints and interpret the results with caution.

### Lack of Standardized Methods

The methods for susceptibility testing of slow-growing bacteria are less standardized than for fast-growing organisms. The laboratory must validate its methods and ensure that the results are reproducible. The lack of standardization can make it difficult to compare results between laboratories.

### Clinical Correlation

The MIC is a laboratory measurement, and the clinical correlation may be uncertain. The MIC does not always predict the clinical outcome, and the clinician must consider the patient's condition and the pharmacokinetics of the drug.

## Professional Escalation Criteria

The laboratory professional should escalate the case to a clinical microbiologist or an infectious disease specialist when the susceptibility testing results are unexpected or when the organism is a high-risk pathogen. The escalation should include the laboratory results, the clinical context, and the potential implications for patient care.

The laboratory should also escalate the case when the quality control results are outside the expected range, when the organism cannot be identified, or when the susceptibility testing method fails to produce a result.

## Building a Laboratory Decision Framework for Slow-Growing Organism Testing

### Defining the Scope of the Decision Framework

A practical decision framework helps the laboratory determine which susceptibility testing pathway to use for a slow-growing organism before committing reagents, time, and containment resources. The framework answers three questions in sequence. First, does the organism require susceptibility testing at all? Second, which testing method and medium combination is appropriate for the organism and the clinical question? Third, what is the minimum set of controls and records needed to produce a defensible result? The framework is not a substitute for published standards. It is a structured way to apply those standards to a specific isolate in a specific laboratory.

The Clinical and Laboratory Standards Institute publishes the standards that define acceptable methods for antimicrobial susceptibility testing [CLSI antimicrobial susceptibility testing standards](https://clsi.org/standards/products/microbiology). The framework described here helps the laboratory map a clinical isolate to the correct standard and identify when a result cannot be produced with available resources. The framework also helps the laboratory distinguish between a test that is not validated and a test that is not appropriate for the organism. These are different problems with different solutions.

### Decision Point 1: Determine Whether Susceptibility Testing Is Clinically Necessary

The first decision is not about method selection. It is about whether testing should be performed at all. For some slow-growing organisms, the clinical treatment protocol is fixed, and susceptibility testing does not change the management plan. For other organisms, testing is essential because resistance is documented and treatment options are limited.

The laboratory should ask the clinical team to confirm that the susceptibility result will change patient management. If the answer is no, the laboratory should not consume resources on a test that will not be used. If the answer is yes, the laboratory proceeds to the next decision point.

The World Health Organization describes antimicrobial resistance as a global health threat and emphasizes that accurate susceptibility data are needed for both individual patient care and population-level surveillance [WHO antimicrobial resistance](https://www.who.int/health-topics/antimicrobial-resistance). A susceptibility result that will not be used for treatment may still be valuable for surveillance. The laboratory should distinguish between a clinical testing request and a surveillance testing request because the acceptable turnaround time and the reporting requirements differ.

### Decision Framework 2: Confirm the Organism Identity and Risk Group

The second decision point is organism identity. The laboratory must confirm the species before selecting a testing pathway because the medium, incubation time, and biosafety containment all depend on the species. A presumptive identification from a primary culture is not sufficient for susceptibility testing. The laboratory should confirm the identity using a validated method that includes the appropriate biochemical and genetic markers.

The American Type Culture Collection provides authenticated strains and strain information that laboratories use to confirm the identity of their isolates and to validate their identification methods [ATCC culture collections](https://www.atcc.org/). The laboratory should compare the clinical isolate to a reference strain to confirm that the identification method is working correctly.

The risk group determines the biosafety containment level. The CDC and NIH biosafety manual provides the risk assessment framework and the containment requirements for work with biological agents [CDC and NIH biosafety manual](https://www.cdc.gov/labs/bmbl/index.html). *Brucella* species and *Francisella tularensis* are BSL-3 agents. The laboratory must confirm that its containment facilities meet the requirements before handling the isolate for susceptibility testing. If the containment is not adequate, the laboratory must not proceed with the test and should refer the isolate to a reference laboratory.

### Decision Framework 3: Select the Testing Method Based on Organism and Laboratory Capability

The third decision is method selection. The laboratory must choose between broth dilution, agar dilution, gradient diffusion, and disk diffusion. The choice depends on the organism, the laboratory resources, and the clinical need.

Broth dilution and agar dilution are the reference methods for MIC determination. They provide quantitative results and are the most reproducible methods for slow-growing organisms. They require more labor and more reagents than other methods. Gradient diffusion methods provide a quantitative MIC with a single test and are convenient for small numbers of isolates. Disk diffusion is the least labor-intensive method but is less commonly used for slow-growing organisms because the extended incubation time can alter the diffusion of the antimicrobial agent.

The Clinical and Laboratory Standards Institute provides guidance on the selection and use of susceptibility testing methods [CLSI antimicrobial susceptibility testing standards](https://clsi.org/standards/products/microbiology). The laboratory should select a method that is validated for the organism being tested. If the laboratory does not have a validated method for the organism, the laboratory should not perform the test and should refer the isolate to a reference laboratory.

The decision framework should also consider the number of isolates to be tested. For a single isolate, a gradient diffusion method may be the most efficient choice. For a batch of isolates, broth dilution or agar dilution may be more efficient. The laboratory should document the method selection and the reason for the selection in the laboratory record.

### Decision Framework 4: Verify the Medium and Supplementation

The fourth decision is the medium. The laboratory must confirm that the medium supports the growth of the organism and that the supplements are present at the correct concentrations. The medium must also allow the antimicrobial agent to act. Some supplements can interfere with the activity of certain antimicrobial agents, and the laboratory must be aware of these interactions.

For *Brucella* species, the recommended medium is Mueller-Hinton agar or broth supplemented with serum or other enrichment factors. For *Francisella tularensis*, the recommended medium is Mueller-Hinton agar or broth supplemented with cysteine and other growth factors. The laboratory must prepare the medium according to the manufacturer instructions and verify that the supplements are present in the correct concentrations.

The American Type Culture Collection provides authenticated strains and quality control information that laboratories use to verify that their media and methods produce reliable results [ATCC culture collections](https://www.atcc.org/). The laboratory should test the medium with a reference strain before using it for clinical isolates.

### Decision Framework 5: Confirm the Incubation Time and Atmosphere

The fifth decision is the incubation time and atmosphere. The laboratory must confirm that the incubation time is long enough to allow the organism to produce visible growth but not so long that the antimicrobial agent degrades or the organism overgrows. The incubation atmosphere must support the growth of the organism.

For *Brucella* species, the incubation time is typically 48 hours. For *Francisella tularensis*, the incubation time is typically 48 to 72 hours. The incubation atmosphere is typically increased carbon dioxide for these organisms. The laboratory must verify that the incubation chamber is set to the correct temperature and atmosphere.

The laboratory should document the incubation time and atmosphere for each test. The documentation should include the start time, the read time, and the conditions. The documentation is important for quality control and for troubleshooting when the result is unexpected.

### Decision Framework 6: Confirm the Inoculum and the Growth Control

The sixth decision is the inoculum. The laboratory must confirm that the inoculum is prepared from a culture in the appropriate growth phase and that the turbidity is adjusted to the standard value. The inoculum size is critical. Too many bacteria can produce a false-resistant result, and too few bacteria can produce a false-susceptible result.

The laboratory must also confirm that the growth control is adequate. The growth control is a well or plate that contains the organism but no antimicrobial agent. The growth control must show visible growth for the test to be valid. If the growth control does not show growth, the test is invalid, and the laboratory must investigate the cause.

### Decision Framework 7: Interpret the Result and Report

The seventh decision is the interpretation and reporting. The laboratory must interpret the MIC using the breakpoints that are defined for the organism and the antimicrobial agent. The breakpoints are provided by the Clinical and Laboratory Standards Institute [CLSI antimicrobial susceptibility testing standards](https://clsi.org/standards/products/microbiology). The laboratory must not use breakpoints that are not defined for the organism.

The report must include the MIC value, the interpretation, the method used, the incubation time, and any limitations of the test. The report must be clear and complete to support the clinical decision. The laboratory should also report the biosafety level of the organism and any special handling requirements.

### A Practical Decision Matrix for the Laboratory

The following decision matrix summarizes the framework. The laboratory should use the matrix as a checklist before performing susceptibility testing on a slow-growing organism.

| Decision Point | Question to Answer | Action if Yes | Action if No |
| --- | --- | --- | --- |
| 1. Clinical need | Will the result change patient management? | Proceed to decision 2 | Do not test, document the decision |
| 2. Organism identity | Is the species confirmed? | Proceed to decision 3 | Confirm the identity before testing |
| 3. Risk group | Is the containment adequate for the risk group? | Proceed to decision 4 | Refer to a reference laboratory |
| 4. Method | Is a validated method available? | Proceed to decision 5 | Refer to a reference laboratory |
| 5. Medium | Does the medium support growth and allow antimicrobial activity? | Proceed to decision 6 | Prepare the correct medium |
| 6. Incubation | Is the incubation time and atmosphere correct? | Proceed to decision 7 | Adjust the incubation conditions |
| 7. Inoculum | Is the inoculum standardized and the growth control adequate? | Proceed to interpretation | Repeat the test with the correct inoculum |
| 8. Interpretation | Are breakpoints defined for the organism and the drug? | Report the result | Report the MIC without interpretation |

### Records and Measurements for the Decision Framework

The laboratory should maintain a record for each susceptibility test performed on a slow-growing organism. The record should include the following elements.

The organism identification and the method used to confirm the identity. The source of the isolate and the date of collection. The testing method and the medium used. The supplements added to the medium and the concentration of each supplement. The incubation time and the atmosphere. The inoculum preparation method and the turbidity reading. The growth control result. The MIC value for each antimicrobial agent tested. The interpretation for each antimicrobial agent. The breakpoints used for the interpretation. The name of the person who performed the test and the date of the test. The name of the person who reviewed the result and the date of the review.

The laboratory should also record the reference strain results for each batch of media and reagents. The reference strain results must be within the expected range. If the reference strain results are outside the expected range, the test is invalid, and the laboratory must investigate the cause.

The American Type Culture Collection provides authenticated reference strains and quality control information that the laboratory can use to verify the accuracy of its testing [ATCC culture collections](https://www.atcc.org/). The laboratory should maintain a log of the reference strain results and review the log regularly.

### Troubleshooting the Decision Framework

The decision framework can fail at any point. The laboratory should have a troubleshooting plan for each decision point.

If the organism does not grow in the test system, the laboratory should check the medium, the supplements, the incubation time, and the atmosphere. The laboratory should also check the inoculum size. If the organism still does not grow, the laboratory should refer the isolate to a reference laboratory.

If the organism overgrows the test system, the laboratory should check the incubation time and the inoculum size. The laboratory may need to reduce the incubation time or the inoculum size. The laboratory should also check the medium for contamination.

If the reference strain results are outside the expected range, the laboratory should check the medium, the reagents, and the method. The laboratory should also check the reference strain for contamination or mutation. The laboratory should repeat the test with a fresh reference strain.

If the result is unexpected, the laboratory should review the entire record. The laboratory should check the organism identification, the method, the medium, the incubation, the inoculum, and the interpretation. The laboratory should also consider the clinical context. The laboratory should escalate the case to a clinical microbiologist or an infectious disease specialist when the result is unexpected or when the organism is a high-risk pathogen.

### Common Failure Patterns in the Decision Framework

The most common failure pattern is to skip the first decision and proceed directly to testing. The laboratory may test an organism that does not require susceptibility testing, wasting time and resources. The laboratory should always confirm the clinical need before testing.

The second most common failure pattern is to use the wrong medium. The laboratory may use unsupplemented Mueller-Hinton agar for a *Brucella* isolate, producing no growth or a result that is not reliable. The laboratory should always verify the medium before testing.

The third most common failure pattern is to read the test too early. The laboratory may read the test after 24 hours when the organism requires 48 to 72 hours of incubation. The result may show no growth, which could be misinterpreted as susceptibility. The laboratory should always confirm the incubation time before reading the test.

The fourth most common failure pattern is to use breakpoints that are not defined for the organism. The laboratory may use breakpoints for a fast-growing organism to interpret a slow-growing organism. The result may be misinterpreted. The laboratory should always use the breakpoints that are defined for the organism.

### Professional Escalation Criteria for the Decision Framework

The laboratory should escalate the case to a clinical microbiologist or an infectious disease specialist when the decision framework cannot be completed. The escalation should include the laboratory results, the clinical context, and the potential implications for patient care.

The laboratory should also escalate the case when the quality control results are outside the expected range, when the organism cannot be identified, or when the susceptibility testing method fails to produce a result. The escalation should be documented in the laboratory record.

The laboratory should also escalate the case when the organism is a high-risk pathogen and the containment is not adequate. The laboratory should refer the isolate to a reference laboratory that has the appropriate containment facilities.

### The Role of the Decision Framework in Antimicrobial Stewardship

The decision framework supports antimicrobial stewardship by ensuring that susceptibility testing is performed only when it is clinically necessary and that the results are reliable. The World Health Organization describes antimicrobial resistance as a global public health threat and emphasizes the importance of accurate susceptibility data for stewardship programs [WHO antimicrobial resistance](https://www.who.int/health-topics/antimicrobial-resistance). The decision framework helps the laboratory produce accurate data that can be used for both patient care and surveillance.

The framework also supports the laboratory workforce by providing a structured approach to a complex task. The American Society for Microbiology provides professional education and laboratory practice resources that support the development of the laboratory workforce [ASM microbiology resources](https://asm.org/). The framework can be used as a training tool for new laboratory professionals.

### The Decision Framework in the Context of the Laboratory Quality System

The decision framework is part of the laboratory quality system. The quality system includes the policies, procedures, and records that ensure the reliability of the laboratory results. The framework should be documented in the laboratory quality manual and reviewed regularly.

The Clinical and Laboratory Standards Institute provides guidance on the quality system for the microbiology laboratory [CLSI antimicrobial susceptibility testing standards](https://clsi.org/standards/products/microbiology). The laboratory should follow the CLSI guidance and the framework should be consistent with the CLSI standards.

The laboratory should also participate in external quality assessment programs to verify the accuracy of its testing. The external quality assessment programs provide samples with known results that the laboratory tests and reports. The laboratory results are compared to the expected results, and the laboratory receives feedback on its performance.

### The Decision Framework for Surveillance and Research

The decision framework can also be used for surveillance and research. The framework ensures that the testing is performed consistently and that the results are comparable across laboratories. The World Health Organization describes the importance of surveillance for antimicrobial resistance [WHO antimicrobial resistance](https://www.who.int/health-topics/antimicrobial-resistance). The framework supports the surveillance by providing a consistent approach to testing.

The framework should be adapted for the specific needs of the surveillance or research project. The laboratory should document the adaptations and the rationale for the adaptations. The adaptations should not compromise the reliability of the results.

### The Decision Framework for the Food Laboratory

The food laboratory may also perform susceptibility testing on slow-growing organisms. The U.S. Food and Drug Administration publishes the Bacteriological Analytical Manual, which provides official methods for food microbiology [FDA Bacteriological Analytical Manual](https://www.fda.gov/food/laboratory-methods-food/bacteriological-analytical-manual-bam). The food laboratory should follow the BAM methods and the decision framework should be consistent with the BAM methods.

The food laboratory should also follow the biosafety requirements for the organisms it tests. The CDC and NIH biosafety manual provides the requirements for the containment of biological agents [CDC and NIH biosafety manual](https://www.cdc.gov/labs/bmbl/index.html). The food laboratory should confirm that the containment is adequate before testing.

### The Decision Framework as a Continuous Improvement Tool

The decision framework should be reviewed regularly and updated as needed. The laboratory should review the framework after each testing event and identify any failures or near misses. The laboratory should also review the framework when new standards are published or when new methods become available.

The laboratory should document the review and the updates. The documentation should include the date of the review, the name of the reviewer, and the changes that were made. The documentation should be part of the laboratory quality record.

The decision framework is a practical tool that helps the laboratory produce reliable susceptibility results for slow-growing organisms. The framework is not a substitute for published standards. It is a structured way to apply the standards to a specific isolate in a specific laboratory. The laboratory should use the framework as a checklist and a record and should escalate the case when the framework cannot be satisfied.

## Frequently Asked Questions

### Why do slow-growing bacteria require longer incubation times for susceptibility testing?

Slow-growing bacteria have generation times that are much longer than fast-growing organisms. They require more time to produce visible growth in the test system. The incubation time must be extended to allow the organism to grow adequately and to produce a reliable MIC.

### What media are used for susceptibility testing of *Brucella* species?

*Brucella* species require a medium that is supplemented with serum or other enrichment factors. The recommended medium is Mueller-Hinton agar or broth supplemented with the appropriate factors. The medium must support the growth of the organism while allowing the antimicrobial agent to act.

### What media are used for susceptibility testing of *Francisella tularensis*?

*Francisella tularensis* requires a medium that contains cysteine and other growth factors. The recommended medium is Mueller-Hinton agar or broth supplemented with cysteine and other factors. The medium must be prepared carefully to ensure that the supplements are present in the correct concentrations.

### What are the biosafety requirements for susceptibility testing of *Brucella* and *Francisella*?

*Brucella* and *Francisella* are BSL-3 organisms. The laboratory must have appropriate containment, including biological safety cabinets, personal protective equipment, and decontamination procedures. The CDC and NIH biosafety manual provides the requirements for BSL-3 containment.

### How is the MIC interpreted for slow-growing bacteria?

The MIC is interpreted using the breakpoints that are defined for the organism and the antimicrobial agent. The result is classified as susceptible, intermediate, or resistant. The breakpoints may differ from those used for fast-growing organisms.

### What are the limitations of susceptibility testing for slow-growing bacteria?

The limitations include the lack of standardized methods, the limited breakpoint data, and the potential for the MIC to not predict the clinical outcome. The laboratory must be aware of these limitations and use the results with caution.

### What is the role of reference strains in susceptibility testing?

Reference strains are used for quality control. They have known antimicrobial patterns and are used to verify that the testing method, media, and reagents are working correctly. The American Type Culture Collection provides authenticated reference strains for many organisms.

### When should I escalate a susceptibility testing case?

The case should be escalated when the result is unexpected, when the organism is a high-risk pathogen, or when the quality control is outside the expected range. The escalation should be to a laboratory microbiologist or an infectious disease specialist.

## Related Bacteriology Guides

- [Mycoplasma gallisepticum Vaccine in Poultry: Protocols and Efficacy](/knowledge/bacteria/avian-bacteria/mycoplasma-gallisepticum-vaccine-poultry-protocols-efficacy)
- [Which Bacteria Are Common to Raw Poultry? A Safety and Pathogen Guide](/knowledge/bacteria/avian-bacteria/bacteria-common-to-raw-poultry-safety-guide)
- Bacteria on Chicken: Common Pathogens and Mitigation in Poultry Production
- [Bumblefoot in Chickens: Bacterial Pododermatitis and Management](/knowledge/bacteria/avian-bacteria/bumblefoot-chicken-bacteria)
- [Chicken Ka Bacteria: Understanding Bacterial Pathogens in Poultry](/knowledge/bacteria/avian-bacteria/chicken-ka-bacteria-poultry-pathogens)

## References and Further Reading

- [Biosafety in Microbiological and Biomedical Laboratories](https://www.cdc.gov/labs/bmbl/index.html). CDC and NIH.
- [Microbiology Resources](https://asm.org/). American Society for Microbiology.
- [Antimicrobial Resistance](https://www.who.int/health-topics/antimicrobial-resistance). World Health Organization.
- [Bacteriological Analytical Manual](https://www.fda.gov/food/laboratory-methods-food/bacteriological-analytical-manual-bam). U.S. Food and Drug Administration.
- [Antimicrobial Susceptibility Testing](https://clsi.org/standards/products/microbiology). Clinical and Laboratory Standards Institute.
- [Culture Collections](https://www.atcc.org/). American Type Culture Collection.
- [NCBI Pathogen Detection](https://www.ncbi.nlm.nih.gov/pathogens). National Center for Biotechnology Information.
- [Disk susceptibility testing of slow-growing anaerobic bacteria.](https://pubmed.ncbi.nlm.nih.gov/1137353). Antimicrobial agents and chemotherapy, 1975.
- [Automated antimicrobial susceptibility testing of slow-growing Pseudomonas aeruginosa strains in the presence of tetrazolium salt WST-1.](https://pubmed.ncbi.nlm.nih.gov/34051278). Journal of microbiological methods, 2021.
- [Nanomotion Detection Method for Testing Antibiotic Resistance and Susceptibility of Slow-Growing Bacteria.](https://pubmed.ncbi.nlm.nih.gov/29205867). Small (Weinheim an der Bergstrasse, Germany), 2018.
- [In vitro susceptibility testing of tetracycline-class antibiotics against slowly growing non-tuberculous mycobacteria.](https://pubmed.ncbi.nlm.nih.gov/37086075). Clinical and experimental pharmacology & physiology, 2023.

> This article is educational and does not replace validated laboratory procedures, clinical diagnosis, institutional biosafety review, or public-health guidance.