Selecting Antimicrobial Susceptibility Testing Methods

By Dr. Zubair Khalid, DVM, MS, PhD · 2026-08-27

Quick Answer

• Choose disk diffusion for low-cost, high-throughput surveillance of many isolates against many drugs, and broth microdilution when you need quantitative minimum inhibitory concentration values.
• Match the method to your study objective, isolate count, and budget before you order media, panels, or automated instruments.
• No single phenotypic method fits every surveillance design, and results from different methods are not always interchangeable without standardized interpretation.

At a Glance

Antimicrobial Susceptibility Testing in Research Surveillance

Antimicrobial susceptibility testing (AST) is the laboratory measurement of a bacterial isolate's response to one or more antimicrobial agents. In research surveillance, AST produces the data that describe how resistance is distributed across populations, how it changes over time, and how it relates to clinical outcomes. The World Health Organization identifies antimicrobial resistance as a global public health concern that requires coordinated surveillance and stewardship efforts, and laboratory-based susceptibility data are the foundation of those efforts.

The choice of AST method is a study design decision, also a laboratory procedure. The method determines the resolution of your data, the number of isolates you can process, the cost per isolate, and the comparability of your results to other studies. Researchers who select a method without considering their study objectives often end up with data that cannot answer the research question or that cannot be compared to published literature.

This article provides a framework for selecting AST methods for research surveillance. It covers the main phenotypic methods, their strengths and limitations, the practical decisions involved in method selection, and the quality controls that make results trustworthy.

Core Principles of Susceptibility Testing

What Susceptibility Testing Measures

Susceptibility testing measures the effect of a drug on a bacterial isolate under standardized conditions. The most common output is the minimum inhibitory concentration, which is the lowest drug concentration that prevents visible growth of the organism. The minimum inhibitory concentration is a quantitative value, usually expressed in micrograms per milliliter.

Disk diffusion produces a zone of inhibition around a drug-impregnated disk. The zone diameter is measured in millimeters and interpreted as susceptible, intermediate, or resistant according to established interpretive criteria. The Clinical and Laboratory Standards Institute publishes standards for susceptibility testing methods and interpretive criteria, and these standards are widely used in clinical and research laboratories.

Why Standardization Matters

Susceptibility testing is highly sensitive to test conditions. The size of the inoculum, the composition of the medium, the incubation temperature, and the duration of incubation can all affect the result. A method that is not standardized can produce different results for the same isolate tested on different days or in different laboratories.

Standardization is the reason that organizations like the Clinical and Laboratory Standards Institute publish detailed procedures for each method. These procedures specify the medium, the inoculum density, the incubation conditions, and the quality control organisms that must be used. Following a standard method is the only way to ensure that your results are reproducible and comparable to other studies.

The Role of Interpretive Criteria

Interpretive criteria, often called breakpoints, are the thresholds that classify an isolate as susceptible, intermediate, or resistant. These criteria are established by organizations such as CLSI and are based on a combination of microbiological, pharmacokinetic, and clinical data.

Breakpoints are not universal. They can differ between organizations, between countries, and over time. A study that uses CLSI breakpoints may produce different resistance rates than a study that uses European Committee on Antimicrobial Susceptibility Testing breakpoints. Researchers must report the interpretive criteria they used and must be aware that changing breakpoints can change the interpretation of historical data.

Selecting a Method for Your Study

Define the Study Objective First

The first decision in selecting an AST method is to define the study objective. Are you measuring the prevalence of resistance in a population? Are you comparing resistance rates between two groups? Are you investigating the mechanism of resistance in a specific isolate? Are you evaluating a new drug or a new combination?

Each objective places different demands on the method. A prevalence study with a large number of isolates may prioritize throughput and cost. A mechanism study with a small number of isolates may prioritize accuracy and the ability to detect low-level resistance. A study of a new drug may require a method that can produce quantitative minimum inhibitory concentration values.

Estimate the Sample Size

The number of isolates you plan to test is a major determinant of method selection. Disk diffusion is the most cost-effective method for large collections because the materials are inexpensive and the process is simple. Broth microdilution is more expensive per isolate but produces quantitative data. Automated systems are the most expensive per isolate but require the least hands-on time.

The sample size also affects the logistics of the study. A study with 10,000 isolates will require a different workflow than a study with 200 isolates. The method must be compatible with the number of isolates you can process in the time available.

Consider the Organisms and Drugs

The method must be appropriate for the organisms you are testing. Some methods are not suitable for certain organisms. For example, disk diffusion is not recommended for some fastidious organisms, and broth microdilution may require special media for certain species.

The drugs you are testing also matter. Some drugs are not stable in the conditions required for certain methods. The method must be able to test the drugs you need to include in your panel.

Assess Available Resources

The resources available in your laboratory will constrain your method choice. These include the equipment, the personnel, the budget, and the time. A laboratory with an automated system may be able to process a large number of isolates quickly, but the cost per isolate may be higher than disk diffusion. A laboratory without specialized equipment may need to use disk diffusion or broth microdilution.

Disk Diffusion Testing

How Disk Diffusion Works

Disk diffusion testing, also known as the Kirby-Bauer method, is a qualitative method that measures the zone of inhibition around a drug-impregnated disk. A standardized inoculum of the test organism is spread on the surface of an agar plate, and disks containing known concentrations of drugs are placed on the surface. After incubation, the diameter of the zone of inhibition is measured and interpreted.

The zone diameter is inversely related to the susceptibility of the organism. A larger zone indicates greater susceptibility, and a smaller zone indicates greater resistance. The zone diameter is compared to interpretive criteria to classify the isolate as susceptible, intermediate, or resistant.

Strengths of Disk Diffusion

Disk diffusion is the most cost-effective method for testing a large number of isolates against a panel of drugs. The materials are inexpensive, and the method does not require specialized equipment. It is also flexible, because the panel of drugs can be changed easily by selecting different disks.

Disk diffusion is well suited for surveillance studies that aim to describe the resistance profile of a population. The method is simple enough to be performed in laboratories with limited resources, and the results are reproducible when the method is performed according to the standard.

Limitations of Disk Diffusion

The main limitation of disk diffusion is that it produces a qualitative result. The zone diameter is a continuous measurement, but the interpretation is categorical. The method does not provide a quantitative minimum inhibitory concentration value, which can be a limitation for studies that need to track changes in susceptibility over time.

Disk diffusion is also not suitable for all organisms. Some fastidious organisms do not grow well on the medium used for disk diffusion, and the method is not recommended for organisms that require special growth conditions.

When to Use Disk Diffusion

Disk diffusion is the method of choice for surveillance studies that need to test a large number of isolates against a panel of drugs. It is also a good choice for studies that are conducted in laboratories with limited resources. The method is less suitable for studies that need quantitative minimum inhibitory concentration values or that involve organisms that do not grow well on the standard medium.

Broth Microdilution Methods

How Broth Microdilution Works

Broth microdilution is a quantitative method that measures the minimum inhibitory concentration of a drug. The method uses a microtiter plate with wells containing serial dilutions of the drug. A standardized inoculum is added to each well, and the plate is incubated. The minimum inhibitory concentration is the lowest drug concentration that prevents visible growth.

The method can be performed manually or with automated systems. The manual method requires the preparation of the drug dilutions and the inoculation of the plate. The automated method uses a commercial panel that contains the drug dilutions and a reader that determines the result.

Strengths of Broth Microdilution

Broth microdilution is the reference method for susceptibility testing. It produces a quantitative minimum inhibitory concentration that can be used to compare the activity of different drugs, to detect low-level resistance, and to monitor changes in susceptibility over time.

The method is flexible because the drug panel can be customized to the needs of the study. The method is also suitable for a wide range of organisms, including fastidious species that do not grow well on the medium used for disk diffusion.

Limitations of Broth Microdilution

Broth microdilution is more expensive and more labor-intensive than disk diffusion. The preparation of the drug dilutions is time-consuming, and the method requires more technical skill. The method is also less efficient for testing a large number of isolates against a panel of drugs.

The method can be affected by the same factors that affect other susceptibility testing methods, including the inoculum, the medium, and the incubation conditions. The results must be interpreted using the appropriate interpretive criteria.

Clinical Considerations for Broth Microdilution

Broth microdilution is the method of choice when the study needs quantitative minimum inhibitory concentration values. It is also the method of choice when the study involves fastidious organisms or when the study needs to detect low-level resistance. The method is less suitable for large surveillance studies that need to test many isolates against a panel of drugs.

Gradient Diffusion Methods

How Gradient Diffusion Works

Gradient diffusion is a method that combines the simplicity of disk diffusion with the quantitative output of broth microdilution. A strip with a gradient of drug concentrations is placed on an agarose plate that has been inoculated with the test organism. The strip releases the drug into the agarose, creating a gradient of concentrations. The minimum inhibitory concentration is read at the point where the zone of inhibition intersects the strip.

Strengths of Gradient Diffusion

Gradient diffusion is a simple method that produces a quantitative minimum inhibitory concentration. The method is easier to perform than broth microdilution, and it does not require the preparation of drug dilutions. The method is also flexible, because the drug panel can be customized by selecting the appropriate strips.

Limitations of Gradient Diffusion

Gradient diffusion is more expensive per isolate than disk diffusion, and the strips are not available for all drugs. The method is also less efficient than disk diffusion for testing a large number of isolates against a panel of drugs.

The method is not suitable for all organisms. The gradient diffusion strips are designed for specific organisms, and the method may not be appropriate for fastidious organisms or for organisms that require special media.

Clinical Considerations for Gradient Diffusion

Gradient diffusion is a good choice for studies that need a quantitative minimum inhibitory concentration for a small number of isolates. The method is also useful for confirming the result of a disk diffusion test or for testing a drug that is not available in a broth microdilution panel. The method is less suitable for large surveillance studies.

Automated Susceptibility Testing Systems

How Automated Systems Work

Automated susceptibility testing systems are instruments that perform susceptibility testing using preconfigured panels. The panels contain a set of drugs in a range of concentrations, and the instrument reads the results automatically. The instrument can process a large number of isolates in a short time, and the results are reported as minimum inhibitory concentrations.

Strengths of Automated Systems

Automated systems are the most efficient method for testing a large number of isolates. The systems require less hands-on time than manual methods, and the results are read and recorded automatically. The systems are also less prone to errors in reading and recording.

Limitations of Automated Systems

Automated systems are the most expensive method per isolate. The panels are costly, and the instrument requires a significant capital investment. The method is also less flexible than manual methods, because the drug panel is fixed by the manufacturer.

The results from automated systems must be validated against a reference method. The system may not be suitable for all organisms, and the results may need to be confirmed with a manual method for some isolates.

Clinical Considerations for Automated Systems

Automated systems are a good choice for clinical laboratories that process a high volume of samples. The method is less suitable for surveillance studies that need to test a large number of isolates against a customized panel of drugs. The cost per isolate may be prohibitive for research studies with limited budgets.

Selecting a Method for Your Study

A Decision Framework

The selection of an AST method can be guided by a series of questions. The first question is whether the study needs a quantitative minimum inhibitory concentration or a qualitative category. The second question is the number of isolates to be tested. The third question is the organisms to be tested. The fourth question is the resources available in the laboratory.

The following flowchart summarizes the decision process.

flowchart TD
    A[Define study objective] --> B{Quantitative MIC needed?}
    B -- Yes --> C{High throughput needed?}
    C -- Yes --> D[Automated system or broth microdilution]
    C -- No --> E[Broth microdilution or gradient diffusion]
    B -- No --> F{High throughput needed?}
    F -- Yes --> G[Disk diffusion]
    F -- No --> H[Disk diffusion or gradient diffusion]

Matching the Method to the Study

A study that aims to describe the prevalence of resistance in a large population is best served by disk diffusion. The method is cost-effective, and the qualitative result is sufficient to classify isolates as susceptible or resistant.

A study that aims to compare the minimum inhibitory concentration of a new drug to an existing drug is best served by broth microdilution. The method produces quantitative values that can be compared statistically.

A study that aims to detect low-level resistance or to investigate the mechanism of resistance is best served by broth microdilution. The method can detect small differences in susceptibility that may not be apparent with disk diffusion.

Matching the Method to the Organism

The method must be appropriate for the organism being tested. Disk diffusion is not recommended for organisms that do not grow well on the standard medium. Broth microdilution is more flexible and can be adapted to the growth requirements of the organism.

The Clinical and Laboratory Standards Institute provides standards for susceptibility testing of different organisms. These standards specify the medium, the inoculum, and the incubation conditions for each organism. The researcher should consult these standards when selecting a method.

Quality Controls and Assurance

The Role of Quality Control

Quality control is the process of ensuring that the susceptibility testing method is performing correctly. Quality control involves testing a reference strain with a known susceptibility profile alongside the test isolates. The result of the reference strain is compared to the expected result, and the test is considered valid if the reference strain produces the expected result.

The American Type Culture Collection (ATCC) provides authenticated reference strains that are used for quality control. These strains have known susceptibility profiles and are used to validate the performance of the method.

Quality Control Procedures

The quality control procedure depends on the method. For disk diffusion, the reference strain is tested with the same disks and the same medium as the test isolates. The zone diameter for the reference strain must be within the expected range for the test to be valid.

For broth microdilution, the reference strain is tested with the same panel and the same conditions as the test isolates. The minimum inhibitory concentration for the reference strain must be within the expected range for the test to be valid.

Recording Quality Control Results

The results of the quality control tests must be recorded. The record should include the date of the test, the reference strain, the drug, and the result. The record is used to monitor the performance of the method over time and to identify any problems.

The quality control results should be reviewed regularly. If the results are outside the expected range, the method must be investigated and corrected before the test results are reported.

Records and Measurements

Data to Record

The data recorded for each isolate should include the identification of the isolate, the source of the isolate, the date of the test, the method used, and the result. The result should be recorded as the zone diameter for disk diffusion or the minimum inhibitory concentration for broth microdilution.

The interpretive category should also be recorded. The category is based on the interpretive criteria used, and the criteria should be recorded with the result.

Data Management

The data should be managed in a way that allows for analysis. The data should be entered into a database or a spreadsheet, and the data should be checked for errors. The data should be backed up regularly.

Data Analysis

The data can be analyzed to describe the resistance profile of the population. The resistance rate can be calculated as the proportion of isolates that are resistant to a drug. The data can also be analyzed to compare the resistance rates between two groups.

The data can be analyzed to detect trends over time. The resistance rate can be plotted over time to see if it is increasing or decreasing.

Common Failure Patterns

Failure to Standardize the Method

A common failure is to use a method that is not standardized. The method must be performed according to the standard procedure, and the conditions must be controlled. A method that is not standardized can produce results that are not reproducible.

Failure to Use Quality Control

A common failure is to skip the quality control. The quality control is essential to ensure that the method is performing correctly. A method that is not quality controlled can produce results that are not reliable.

Failure to Record the Breakpoints

A common failure is to not record the breakpoints used to interpret the results. The breakpoints can change over time, and the results can be interpreted differently with different breakpoints. The breakpoints must be recorded with the results.

Failure to Validate the Method

A common failure is to use a method that has not been validated for the organism or the drug. The method must be validated for the specific organism and drug before it is used for the study.

Biosafety and Containment

Risk Assessment

The susceptibility testing of bacterial isolates involves the handling of potentially hazardous microorganisms. The laboratory must conduct a risk assessment to identify the hazards and to determine the appropriate containment measures. The Biosafety in Microbiological and Biomedical Laboratories provides guidance on the risk assessment and the containment of microorganisms.

Containment

The containment measures depend on the risk group of the organism. The laboratory must use the appropriate biosafety level for the organism being tested. The biosafety level is determined by the risk group of the organism and the procedures being performed.

Decontamination

The laboratory must have procedures for the decontamination of the work area and the waste. The decontamination procedures must be effective against the organisms being handled. The waste must be disposed of according to the regulations.

Professional Escalation Criteria

When to Escalate

The researcher should escalate a problem to a supervisor or a specialist when the problem cannot be resolved with the available resources. The escalation criteria include the following:

• The quality control results are consistently outside the expected range.
• The method is not producing reproducible results.
• The results are not consistent with the expected susceptibility profile of the organism.
• The laboratory does not have the resources to perform the method correctly.

How to Escalate

The escalation should be documented. The documentation should include the problem, the steps taken to resolve the problem, and the outcome. The escalation should be made to the appropriate person or organization.

Building a Laboratory Verification Plan for Research Surveillance

Why Verification Matters Before Data Collection

A verification plan is the set of documented checks that confirm your chosen AST method performs as expected in your specific laboratory before you test study isolates. This step is distinct from routine quality control, which runs during data collection. Verification answers a different question: can this method, as executed by your staff with your equipment and your reagents, produce results that match the expected performance of the method?

Many surveillance studies fail not because the method was wrong but because the method was never verified in the laboratory where the work took place. A broth microdilution panel that performs well in a reference laboratory may produce different results when prepared with a different water source, a different lot of media, or a different incubator. A disk diffusion method that works for one technician may not work for another who prepares the inoculum differently. Verification is the process that catches these problems before they contaminate your dataset.

The Clinical and Laboratory Standards Institute publishes standards for susceptibility testing methods, and these standards include expectations for method performance. The American Society for Microbiology provides professional education and laboratory practice context that supports the development of verification protocols. The verification process you design should be documented in your study protocol and completed before any study isolate is tested.

Verification Steps for a New Method

The first step in verification is to confirm that you have the correct materials. This includes the media, the disks or panels, the reference strains, and the equipment. The American Type Culture Collection provides authenticated reference strains with known susceptibility profiles, and these strains are the foundation of a verification plan. You should confirm that the reference strains you plan to use are appropriate for the method and the organisms you will test.

The second step is to test the reference strains using the exact procedure you will use for study isolates. This means using the same media lot, the same inoculum preparation, the same incubation conditions, and the same reading method. You should test each reference strain multiple times, ideally on different days, to assess the reproducibility of the method.

The third step is to compare your results to the expected results for the reference strains. The expected results are published by the organization that provides the interpretive criteria, such as the Clinical and Laboratory Standards Institute. Your results must fall within the acceptable range for the method to be considered verified.

The fourth step is to document the verification results. The documentation should include the date of the verification, the reference strains used, the drugs tested, the results obtained, and the expected results. This documentation is part of your study records and should be retained with the study data.

Verification Steps for an Existing Method

If you are using a method that is already established in your laboratory, you still need to verify that the method is performing correctly for your study. The verification process is less extensive than for a new method, but it is still necessary.

The first step is to review the existing quality control records. The records should show that the method has been performing within the expected range for the reference strains. If the records show any out-of-range results, you need to investigate and correct the problem before you start the study.

The second step is to confirm that the method has not changed. If you have changed the media, the disks, the panels, or any other component of the method, you need to verify the method again. A change in any component can affect the performance of the method.

The third step is to confirm that the personnel who will perform the method are trained and competent. The American Society for Microbiology provides professional education resources that can support the training of laboratory personnel. The training should include the specific procedures you will use for the study.

Verification for Automated Systems

Automated susceptibility testing systems require a different verification process than manual methods. The system must be verified to produce results that are comparable to a reference method. The verification process for an automated system typically involves testing a set of isolates with both the automated system and a reference method, such as broth microdilution.

The comparison should include isolates with a range of susceptibility profiles, including susceptible, intermediate, and resistant isolates. The results should be compared to determine the level of agreement between the automated system and the reference method. The agreement should be high for the method to be considered verified.

The verification process for an automated system should also include a check of the system's software and data management. The system should be configured to report the results in the format you need for your study. The system should also be configured to record the interpretive criteria you will use.

Verification for Research-Only Methods

Some surveillance studies use methods that are not standard clinical methods. These methods may be developed for a specific research question or for a specific organism. The verification process for these methods is more complex because there may not be published expected values for the reference strains.

For a research-only method, the verification process should include a comparison to a reference method. The reference method is typically broth microdilution, which is the reference method for susceptibility testing. The comparison should include a set of isolates that represent the range of susceptibility you expect to see in your study.

The comparison should be analyzed to determine the level of agreement between the research method and the reference method. The analysis should include the number of isolates that are classified in the same category by both methods and the number of isolates that are classified differently. The analysis should also include the number of isolates that have a minimum inhibitory concentration that is within one dilution of the reference method.

The verification process for a research-only method should be documented in the study protocol. The documentation should include the comparison method, the isolates used, the results, and the analysis. The documentation should also include the criteria for acceptable agreement.

Building a Verification Record System

What to Record

The verification record should include the date of the verification, the method being verified, the reference strains used, the drugs tested, the results, and the expected values. The record should also include the name of the person who performed the verification and the name of the person who reviewed the results.

The verification record should be part of the study records. The record should be retained with the study data and should be available for review by the study team and by any external reviewers.

How to Organize the Record

The verification record should be organized in a way that is easy to review. The record should be organized by method and by date. The record should include a summary of the verification results and the conclusion of the verification.

The record should also include the details of the verification. The details should include the media lot numbers, the disk or panel lot numbers, the incubator temperatures, and the incubation times. These details are important because they allow the verification to be repeated if necessary.

How to Use the Record

The verification record is used to confirm that the method is performing correctly before the study data is collected. The record is also used to monitor the performance of the method over time. If the method begins to produce results that are outside the expected range, the verification record can be used to identify the cause of the problem.

The verification record should be reviewed before the study data is analyzed. The review should confirm that the method was verified and that the verification was successful. The review should also confirm that the method has not changed since the verification was performed.

Troubleshooting Verification Failures

Out-of-Range Reference Strain Results

The most common verification failure is an out-of-range result for a reference strain. This failure indicates that the method is not performing correctly. The first step in troubleshooting is to identify the cause of the failure.

The cause of the failure can be in the inoculum, the media, the disks or panels, the incubation conditions, or the reading of the results. The troubleshooting process should be systematic. The process should start with the simplest possible cause and work toward the more complex causes.

The inoculum is the most common cause of out-of-range results. The inoculum must be prepared to the correct density. The density is typically measured with a turbidity standard. The inoculum must be used within the time specified by the method.

The media is the second most common cause of out-of-range results. The media must be prepared correctly and must be within the expiration date. The media must be stored correctly. The media must be the correct type for the method and the organism.

The disks are the third most common cause of out-of-range results. The disks must be stored correctly. The disks must be within the expiration date. The disks must be applied to the plate correctly.

The incubation is the fourth most common cause of out-of-range results. The incubation temperature must be correct. The incubation time must be correct. The incubator must be calibrated.

The reading of the result is the fifth most common cause of out-of-range results. The zone diameter must be measured correctly. The minimum inhibitory concentration must be read correctly. The reading must be done by a person who is trained and competent.

Inconsistent Results Between Runs

If the verification results are inconsistent between runs, the method is not reproducible. The cause of the inconsistency is likely in the inoculum or the media. The inoculum preparation should be reviewed. The media preparation should be reviewed.

The inconsistency can also be caused by the equipment. The incubator temperature may vary. The pipettes may not be calibrated. The balance may not be calibrated.

The inconsistency can also be caused by the personnel. The personnel may not be performing the method consistently. The personnel may need additional training.

Results That Do Not Match the Reference Method

If the verification results do not match the reference method, the method is not performing correctly. The cause of the mismatch is likely in the method itself. The method may not be appropriate for the organism or the drug.

The method may need to be modified. The modification should be based on the standard method. The modification should be documented.

Verification and the Study Protocol

The verification process should be described in the study protocol. The protocol should describe the verification steps, the reference strains, the expected values, and the criteria for acceptable performance. The protocol should also describe the troubleshooting process and the escalation criteria.

The verification process should be completed before the study data is collected. The verification should be documented in the study record. The verification should be reviewed by the study team.

The verification process is a critical step in the design of a surveillance study. The process ensures that the method is performing correctly and that the data is reliable. The process also ensures that the data is comparable to other studies that use the same method.

The verification process is also a key part of the quality system of the laboratory. The process is consistent with the principles of the Clinical and Laboratory Standards Institute and the American Society for Microbiology. The process is also consistent with the principles of the World Health Organization for the surveillance of antimicrobial resistance.

Verification and the Study Timeline

The verification process should be planned into the study timeline. The verification should be completed before the study data is collected. The verification should be completed in time to allow for any troubleshooting that is needed.

The verification process should be planned for the number of isolates that will be tested. The verification should be planned for the number of drugs that will be tested. The verification should be planned for the number of personnel who will perform the method.

The verification process should be planned for the resources that are available. The verification should be planned for the budget that is available. The verification should be planned for the time that is available.

The verification process is a key part of the study design. The process is a key part of the study protocol. The process is a key part of the study record. The process is a key part of the study analysis.

Frequently Asked Questions

What is the difference between a qualitative and a quantitative susceptibility test?

A qualitative test produces a categorical result, such as susceptible, intermediate, or resistant. A quantitative test produces a numerical value, such as the minimum inhibitory concentration. Disk diffusion is a qualitative method, while broth microdilution is a quantitative method.

Which method is the reference method for susceptibility testing?

Broth microdilution is the reference method for susceptibility testing. It produces a quantitative minimum inhibitory concentration and is used to validate other methods.

Can I use disk diffusion for all organisms?

Disk diffusion is not suitable for all organisms. The method is not recommended for organisms that do not grow well on the standard medium. The Clinical and Laboratory Standards Institute provides standards for the susceptibility testing of different organisms.

How do I choose the breakpoints for my study?

The breakpoints are chosen based on the standards of the organization that you are using. The Clinical and Laboratory Standards Institute provides breakpoints for susceptibility testing. The breakpoints must be recorded with the results.

How do I perform quality control for my susceptibility testing?

Quality control is performed by testing a reference strain with a known susceptibility profile. The reference strain is tested alongside the test isolates, and the result is compared to the expected result. The test is valid if the reference strain produces the expected result.

How do I record the results of my susceptibility testing?

The results are recorded as the zone diameter for disk diffusion or the minimum inhibitory concentration for broth microdilution. The interpretive category is also recorded. The breakpoints used to interpret the results must be recorded.

What is the role of the Clinical and Laboratory Standards Institute in susceptibility testing?

The Clinical and Laboratory Standards Institute provides standards for susceptibility testing methods and interpretive criteria. The standards are used to ensure that the results are reproducible and comparable.

How do I choose the method for my surveillance study?

The method is chosen based on the study objective, the sample size, the organisms, and the resources. A study that needs a quantitative value is best served by broth microdilution. A study that needs to test a large number of isolates is best served by disk diffusion.

Using the Evidence

Related Bacteriology Guides

• Salmonellosis in Poultry: Serovar Surveillance, Antimicrobial Resistance, and Egg Safety
• Biological Foundation Models for Antimicrobial Peptide Discovery in Veterinary Pathogens
• Aeromonas hydrophila in Aquaculture: Virulence Factors and Antimicrobial Resistance
• Swine Bacterial Diseases: Colibacillosis, UTI, and Antimicrobial Resistance
• Avian Colibacillosis: Pathogenesis, Diagnosis, and Antimicrobial Resistance Patterns in Poultry

References and Further Reading

• Biosafety in Microbiological and Biomedical Laboratories. CDC and NIH.
• Microbiology Resources. American Society for Microbiology.
• Antimicrobial Resistance. World Health Organization.
• Bacteriological Analytical Manual. U.S. Food and Drug Administration.
• Antimicrobial Susceptibility Testing. Clinical and Laboratory Standards Institute.
• Culture Collections. American Type Culture Collection.
• NCBI Pathogen Detection. National Center for Biotechnology Information.
• Invasive Group A Streptococcal Infections in 10 US States.. JAMA, 2025.
• Treatment of enteric fever (typhoid and paratyphoid fever) with cephalosporins.. The Cochrane database of systematic reviews, 2022.
• A fatal outbreak of ST11 carbapenem-resistant hypervirulent Klebsiella pneumoniae in a Chinese hospital: a molecular epidemiological study.. The Lancet. Infectious diseases, 2018.
• Rapid versus standard antimicrobial susceptibility testing to guide treatment of bloodstream infection.. The Cochrane database of systematic reviews, 2021.

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