# Broth Microdilution vs. Agar Dilution ## Quick Answer - Choose broth microdilution when you need quantitative MIC values with higher throughput and standardized 96-well plate formats for most clinically relevant bacteria. - Choose agar dilution when you need to test many isolates against a single agent simultaneously or when testing fastidious organisms that grow poorly in broth. - Both methods require strict adherence to CLSI reference procedures, authenticated control strains, and documented quality control to produce reliable susceptibility data. ## At a Glance | Feature | Broth Microdilution | Agar Dilution | |---------|---------------------|---------------| | Format | 96-well microtiter plates with serial twofold dilutions in liquid media | Agar plates with serial twofold dilutions incorporated into solid media | | Throughput | High for testing one isolate against many antimicrobials | High for testing many isolates against one antimicrobial | | Quantitative result | Yes, provides exact MIC value | Yes, provides exact MIC value | | Automation potential | High, compatible with automated readers and liquid handlers | Low, requires manual plate preparation and spot inoculation | | Cost per test | Moderate, consumables include plates and liquid media | Higher for small panels, lower for large isolate batches | | Fastidious organism support | Good with supplemented media such as CAMHB with lysed horse blood | Good with supplemented agar such as Mueller-Hinton agar with blood | | Reproducibility | High when standardized | High when standardized | | CLSI reference status | Reference method for many organism groups | Reference method for many organism groups | | Common use cases | Clinical laboratories, research MIC panels, automated systems | Reference laboratories, surveillance studies, testing many isolates | ## Antimicrobial Susceptibility Testing Context Antimicrobial susceptibility testing provides the laboratory evidence that guides treatment decisions, resistance surveillance, and stewardship programs. The global threat of antimicrobial resistance makes accurate and reproducible susceptibility data a public health priority. The World Health Organization describes antimicrobial resistance as a process where microorganisms change over time and stop responding to medicines, which complicates treatment and increases the risk of disease spread, severe illness, and death. Laboratories that produce reliable MIC values contribute directly to this surveillance effort because their data feeds local, national, and global resistance tracking systems. The Clinical and Laboratory Standards Institute publishes the reference standards that define how susceptibility testing should be performed and interpreted. CLSI documents specify inoculum preparation, media formulation, incubation conditions, and quality control requirements for both broth and agar dilution methods. These standards exist so that a MIC measured in one laboratory can be compared with a MIC measured in another laboratory with confidence. The CLSI standards are the practical backbone of clinical susceptibility testing, and laboratories that implement either dilution method must follow the current edition of the relevant standard. The choice between broth microdilution and agar dilution is not a matter of one method being universally superior. Each method has strengths that fit particular laboratory workflows, sample volumes, and organism types. A clinical laboratory processing hundreds of patient isolates per day needs a method that supports automation and rapid turnaround. A reference laboratory testing large collections of isolates against a panel of new drug candidates needs a method that can process many isolates at once. Understanding the operational differences between the two methods is the first step in selecting the right approach. ## Core Principles of Dilution Susceptibility Testing Both broth microdilution and agar dilution measure the minimum inhibitory concentration, which is the lowest concentration of an antimicrobial agent that prevents visible growth of a microorganism under defined test conditions. The MIC is a quantitative endpoint that tells the clinician or researcher how much drug is needed to inhibit the organism. This value is then interpreted against clinical breakpoints to classify the isolate as susceptible, intermediate, or resistant. The principle behind both methods is identical. A standardized inoculum of the test organism is exposed to a series of twofold dilutions of the antimicrobial agent. After incubation under defined conditions, the lowest concentration that shows no visible growth is recorded as the MIC. The difference between the two methods lies in the physical format of the test. Broth microdilution uses liquid media in wells, while agar dilution uses antimicrobial incorporated into solid agar plates. The inoculum preparation is a critical step in both methods. The organism must be grown to a defined turbidity that corresponds to a known cell density, typically matched to a 0.5 McFarland standard. This standardization ensures that the number of bacteria exposed to each drug concentration is consistent between tests and between laboratories. The CLSI standards specify the exact inoculum preparation procedure for each organism group. Media formulation is equally important. The choice of media affects the activity of the antimicrobial agent and the growth of the test organism. Mueller-Hinton broth and Mueller-Hinton agar are the standard media for most non-fastidious organisms. For fastidious organisms that require additional nutrients, the media are supplemented with blood or other growth factors. The CLSI standards specify the exact media formulations for each organism group. Incubation conditions are standardized to ensure reproducibility. Most tests are incubated at 35 degrees Celsius for 16 to 20 hours in ambient air. Some organisms require extended incubation or special atmospheric conditions such as increased carbon dioxide. The CLSI standards specify the incubation conditions for each organism group. ## Broth Microdilution Method Broth microdilution is the most widely used dilution method in clinical laboratories because it is adaptable to automation and high-throughput workflows. The test is performed in 96-well microtiter plates, where each well contains a different concentration of the antimicrobial agent in liquid broth. The test organism is added to each well, and the plate is incubated under standardized conditions. The preparation of a broth microdilution panel begins with the preparation of the antimicrobial stock solutions. The antimicrobial agent is weighed and dissolved in the appropriate solvent and then diluted to the desired concentration range. The stock solution is then diluted in broth to create the working concentrations that will be dispensed into the wells. The CLSI standards provide the recommended solvent and dilution procedures for each antimicrobial agent. The 96-well plate format allows a single isolate to be tested against many antimicrobial agents at once. Each row of the plate can contain a different antimicrobial agent, and each column contains a different concentration. This format is highly efficient for clinical laboratories that need to test each patient isolate against a panel of 15 to 20 drugs. The plate can be prepared in advance and stored frozen, which allows the laboratory to have panels ready for immediate use. The inoculum is prepared by suspending colonies from an overnight culture in broth or saline to match a 0.5 McFarland standard. The suspension is then diluted to the final test concentration, typically 5 times 10 to the fifth colony-forming units per milliliter. The diluted inoculum is added to each well of the plate. The plate is then incubated under the conditions specified by the CLSI standard. After incubation, the MIC is read as the lowest concentration of the antimicrobial agent that shows no visible growth. The reading can be performed visually or with an automated plate reader. Automated readers provide an objective measurement of growth and can reduce the time required for reading. The MIC value is then interpreted against the CLSI breakpoints for the organism and the antimicrobial agent. The broth microdilution method is the basis for many commercial susceptibility testing systems. These systems use the same principle but package the test in a format that is easier to use in a clinical laboratory. The commercial systems are validated against the reference broth microdilution method, and they provide results that are comparable to the reference method. ## Agar Dilution Method Agar dilution is the method of choice for testing large numbers of isolates against a single antimicrobial agent. The method involves incorporating the antimicrobial agent into molten agar at a series of twofold concentrations, pouring the agar into plates, and then spot inoculating the test organisms onto the surface of the agar. After incubation, the MIC is read as the lowest concentration of the antimicrobial agent that completely inhibits visible growth. The preparation of agar dilution plates begins with the preparation of the antimicrobial stock solution. The stock is then added to molten agar that has been cooled to a temperature that will not degrade the antimicrobial agent. The agar is mixed thoroughly to ensure even distribution of the drug, and then poured into petri dishes. The plates are allowed to solidify and can be stored for a short period before use. The inoculum for agar dilution is prepared in the same way as for broth microdilution. The organism is suspended to match a 0.5 McFarland standard, and the suspension is then diluted to the required test concentration. The inoculum is applied to the agar surface using a replicator device that transfers a fixed volume of the suspension to each plate. The replicator allows multiple isolates to be tested on a single plate, which is the key advantage of the agar dilution method. The plates are incubated under the conditions specified by the CLSI standard. After incubation, the MIC is read as the lowest concentration of the antimicrobial agent that completely inhibits the growth of the test organism. A single colony or a faint haze of growth is considered to be no growth for the purpose of the MIC reading. The agar dilution method is particularly useful for testing large collections of isolates, such as those collected during surveillance studies or during the evaluation of a new antimicrobial agent. A single agar plate can be used to test up to 32 isolates at one concentration of the drug. This makes the method highly efficient when the goal is to determine the MIC distribution of a large number of isolates against a single drug. The agar dilution method is also useful for testing fastidious organisms that grow poorly in broth. The solid agar surface can be supplemented with blood or other growth factors to support the growth of these organisms. The CLSI standards provide the specific agar formulations for each organism group. ## Comparison of Workflow and Throughput The workflow differences between broth microdilution and agar dilution are substantial and directly affect the choice of method for a given laboratory. The broth microdilution method is designed for testing one isolate against many antimicrobial agents. The agar dilution method is designed for testing many isolates against one antimicrobial agent. The broth microdilution workflow begins with the preparation of the antimicrobial panels. The panels can be prepared in advance and stored frozen, which allows the laboratory to have a ready supply of panels for routine testing. The daily workflow involves preparing the inoculum, adding the inoculum to the panel, incubating the panel, and reading the results. The entire process can be completed in a single day, and the results are available within 24 hours. The agar dilution workflow begins with the preparation of the agar plates. The plates must be prepared on the day of the test or the day before, because the antimicrobial agent can degrade during storage. The daily workflow involves preparing the inoculum, inoculating the plates with the replicator, incubating the plates, and reading the results. The agar dilution method is more labor-intensive for a small number of isolates, but it becomes more efficient as the number of isolates increases. The throughput of the two methods is a key consideration. A broth microdilution plate can test one isolate against 12 different antimicrobial agents in a single plate. A single agar dilution plate can test 32 isolates against one antimicrobial agent. The choice of method depends on the balance between the number of isolates and the number of antimicrobial agents that need to be tested. The cost of the two methods also differs. The broth microdilution method requires the purchase of microtiter plates, broth media, and antimicrobial agents. The cost per test is moderate and is influenced by the number of antimicrobial agents in the panel. The agar dilution method requires the purchase of agar, petri dishes, and antimicrobial agents. The cost per test is lower when a large number of isolates are tested against a single drug, but the cost per test is higher when only a few isolates are tested. ## Automation and Commercial Systems The broth microdilution method is the foundation for most automated susceptibility testing systems used in clinical laboratories. These systems combine the broth microdilution format with automated inoculation, incubation, and reading. The automated systems provide results in a shorter time than the manual method and reduce the hands-on time required for the laboratory staff. The automated systems use a plastic panel that contains the antimicrobial agents in a dried or frozen format. The laboratory adds the test inoculum to the panel, and the system automatically incubates and reads the panel. The system uses a photometric or fluorometric reader to detect growth in each well. The results are then interpreted against the CLSI breakpoints and reported to the clinician. The automated systems are validated against the reference broth microdilution method. The validation ensures that the results from the automated system are comparable to the results from the reference method. The automated systems are widely used in clinical laboratories because they provide rapid and reliable results with minimal hands-on time. The agar dilution method is not easily automated. The preparation of the agar plates is labor-intensive, and the reading of the plates requires visual inspection. The agar dilution method is therefore used primarily in reference laboratories and research settings where the number of isolates is large and the need for automation is less critical. The choice between an automated broth microdilution system and the manual agar dilution method depends on the laboratory workflow. A clinical laboratory that processes a high volume of patient samples will benefit from the automation of the broth microdilution system. A reference laboratory that tests large collections of isolates for research or surveillance will benefit from the efficiency of the agar dilution method. ## Organism-Specific Considerations The choice between broth microdilution and agar dilution is also influenced by the type of organism being tested. The CLSI standards provide specific methods for different organism groups, and the choice of method depends on the growth requirements of the organism. For the common aerobic bacteria, such as Enterobacterales, Pseudomonas aeruginosa, and Staphylococcus aureus, both broth microdilution and agar dilution are acceptable methods. The broth microdilution method is the most commonly used in clinical laboratories because it is compatible with the automated systems. The agar dilution method is used in reference laboratories and for research purposes. For fastidious organisms, such as Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria gonorrhoeae, the media must be supplemented with blood or other growth factors. The broth microdilution method can be used with the supplemented broth, and the agar dilution method can be used with the supplemented agar. The CLSI standards provide the specific media formulations for each organism group. For anaerobes, the agar dilution method is the reference method. The agar dilution method is preferred for anaerobes because the solid agar provides a stable environment for the growth of these organisms. The broth microdilution method can also be used for anaerobes, but the agar dilution method is the reference method. For mycobacteria, the broth microdilution method is used for the testing of the rapidly growing mycobacteria. The agar dilution method is used for the testing of the slowly growing mycobacteria. The CLSI standards provide the specific methods for the mycobacteria. The choice of method for a given organism is determined by the CLSI standards. The laboratory must follow the recommended method for the organism being tested to ensure that the results are reliable and comparable to the results from other laboratories. ## Quality Control and Reference Strains Quality control is an essential component of any susceptibility testing method. The laboratory must include quality control strains in each batch of testing to ensure that the method is performing correctly. The quality control strains are well-characterized strains with known MIC values for the antimicrobial agents being tested. The CLSI standards provide the recommended quality control strains and the acceptable MIC ranges for each strain and antimicrobial agent. The laboratory must test the quality control strains in the same way as the patient isolates. The results from the quality control strains must fall within the acceptable ranges for the test to be valid. The American Type Culture Collection provides authenticated biological materials, including the quality control strains used in susceptibility testing. The use of authenticated strains is essential for the reliability of the testing. The ATCC strains are characterized and documented, and they provide a consistent reference for the laboratory. The quality control strains are tested on a regular basis. The frequency of the quality control testing depends on the laboratory protocol and the requirements of the accreditation body. The laboratory must document the quality control results and take corrective action if the results are out of range. The quality control is also important for the preparation of the antimicrobial agents. The laboratory must verify the purity and the concentration of the antimicrobial stock solutions. The stock solutions must be prepared according to the manufacturer instructions and the CLSI standards. ## Common Failure Patterns and Troubleshooting The most common failure in dilution susceptibility testing is the contamination of the test system. The contamination can be introduced during the preparation of the inoculum, the addition of the inoculum to the test, or the reading of the results. The laboratory must follow the aseptic technique to minimize the risk of contamination. The second common failure is the incorrect preparation of the antimicrobial stock. The antimicrobial agent must be weighed accurately and dissolved in the correct solvent. The stock solution must be diluted to the correct concentration. An error in the preparation of the stock will result in an incorrect MIC value. The third common failure is the incorrect preparation of the inoculum. The inoculum must be prepared to the correct density. An inoculum that is too dense will result in a MIC that is too high. An inoculum that is too dilute will result in a MIC that is too low. The fourth common failure is the incorrect reading of the results. The MIC is read as the lowest concentration that shows no visible growth. The reading must be performed carefully to avoid the misinterpretation of the growth. The use of an automated reader can reduce the risk of a reading error. The fifth common failure is the use of the wrong media or the wrong incubation conditions. The media and the incubation conditions must be matched to the organism being tested. The use of the wrong media or the wrong incubation conditions will result in an unreliable MIC. The laboratory must have a system for the detection and the correction of these failures. The quality control results are used to detect the failures. The laboratory must take the corrective action when the quality control results are out of range. ## Biosafety and Laboratory Practices The susceptibility testing involves the handling of the bacterial cultures, which requires the appropriate biosafety practices. The laboratory must follow the biosafety guidelines for the containment of the microorganisms. The CDC and NIH provide the biosafety guidelines for the laboratories that handle the microorganisms. The risk assessment is the first step in the biosafety. The laboratory must assess the risk of the organisms being handled. The risk assessment considers the pathogenicity of the organism, the route of the transmission, and the laboratory procedures being performed. The risk assessment determines the biosafety level required for the laboratory. The biosafety level for the susceptibility testing is typically the biosafety level 2. The biosafety level 2 requires the use of the biological safety cabinet for the procedures that can generate the aerosols. The laboratory must also use the personal protective equipment, such as the gloves and the lab coat. The decontamination of the waste is an important part of the biosafety. The cultures and the contaminated materials must be decontaminated before the disposal. The decontamination is typically performed by the autoclaving. The laboratory must have a written protocol for the decontamination of the waste. The laboratory must also have a written biosafety manual that describes the procedures for the safe handling of the microorganisms. The manual must be reviewed and updated on a regular basis. The laboratory staff must be trained in the biosafety procedures. ## Professional Escalation Criteria The laboratory must have a system for the escalation of the results that are unusual or that require the additional investigation. The escalation criteria are used to ensure that the results are reliable and that the appropriate action is taken. The first escalation criterion is the result that is outside the expected range for the organism. The result may indicate a problem with the test or a new resistance mechanism. The laboratory must investigate the result and repeat the test if necessary. The second escalation criterion is the result that is inconsistent with the previous results for the same isolate. The laboratory must investigate the discrepancy and determine the cause. The third escalation criterion is the result that is inconsistent with the clinical picture. The laboratory must communicate the result to the clinician and discuss the implications. The fourth escalation criterion is the result that is from a quality control strain that is out of range. The laboratory must investigate the cause of the out-of-range result and take the corrective action. The escalation of the results is an important part of the laboratory quality. The laboratory must have a written protocol for the escalation of the results. The protocol should define the criteria for the escalation and the steps to be taken. ## Records and Documentation The laboratory must maintain the records of the susceptibility testing. The records include the patient information, the organism identification, the antimicrobial agents tested, the MIC values, and the interpretation of the results. The records must be accurate and complete. The records are used for the clinical decision-making and for the surveillance of the antimicrobial resistance. The records are also used for the quality assurance and for the accreditation of the laboratory. The laboratory must have a system for the storage and the retrieval of the records. The records must be stored in a secure location and must be available for the review by the authorized personnel. The laboratory must also maintain the records of the quality control. The quality control records include the results of the quality control strains and the corrective actions taken. The quality control records are used to demonstrate the reliability of the testing. The laboratory must also maintain the records of the equipment maintenance and the calibration. The equipment used for the susceptibility testing must be maintained and calibrated on a regular basis. The records of the maintenance and the calibration are used to demonstrate the reliability of the equipment. ## Limitations and Evidence Gaps The susceptibility testing methods have limitations that must be considered when interpreting the results. The MIC is a measure of the in vitro activity of the antimicrobial agent. The MIC does not always predict the clinical outcome. The clinical outcome depends on the pharmacokinetics and the pharmacodynamics of the drug, the site of the infection, and the immune status of the patient. The MIC is also a measure of the activity of the antimicrobial agent under the standardized conditions. The MIC may be different under the different conditions, such as the different pH or the different oxygen tension. The MIC is a laboratory measure and must be interpreted in the context of the clinical situation. The susceptibility testing methods are also limited by the availability of the breakpoints. The breakpoints are the values that are used to interpret the MIC as susceptible, intermediate, or resistant. The breakpoints are defined by the CLSI and are based on the clinical and the microbiological data. The breakpoints are updated on a regular basis, and the laboratory must use the current breakpoints. The evidence for the susceptibility testing methods is also limited by the lack of the standardization for some organism groups. The CLSI standards provide the methods for the most common organisms, but the methods for the less common organisms may not be well-defined. The laboratory must use the best available evidence when testing the less common organisms. ## Decision Framework for Method Selection Based on Laboratory Capacity Selecting between broth microdilution and agar dilution requires a structured evaluation of laboratory capacity, specimen volume, and reporting obligations. A practical decision framework helps laboratory directors move beyond general preferences and match the method to the actual operational environment. The framework below uses four assessment domains that can be scored and weighted according to the laboratory mission. ### Domain 1: Specimen Volume and Isolate Diversity The first assessment domain examines the daily and monthly isolate counts that the laboratory expects to process. A clinical laboratory receiving 100 or more patient isolates per day for susceptibility testing will need a method that supports rapid turnaround and batch processing. The broth microdilution format allows one isolate to be tested against a panel of 15 to 20 antimicrobial agents in a single 96-well plate. This means a laboratory processing 100 isolates per day would need approximately 100 plates if each isolate is tested against a full panel. The agar dilution method would require a separate plate for each antimicrobial agent concentration, which becomes impractical at this volume. A reference laboratory or surveillance program that receives large collections of isolates for testing against a single agent will find the agar dilution method more efficient. A single agar plate can accommodate up to 32 isolates at one drug concentration. Testing 1,000 isolates against a single agent requires approximately 32 plates for each concentration in the dilution series. The agar dilution method becomes increasingly efficient as the isolate count grows and the number of agents remains small. The decision framework should include a simple calculation. Estimate the average number of isolates processed per week and the average number of antimicrobial agents tested per isolate. Multiply these values to obtain the total isolate-agent combinations per week. If the product exceeds 500 combinations per week, the broth microdilution method with automation will likely reduce hands-on time. If the product is below 200 combinations per week and the number of agents per isolate is low, the agar dilution method may be more cost effective. ### Domain 2: Turnaround Time Requirements The second assessment domain addresses the time between specimen receipt and result reporting. Clinical laboratories serving hospitals and outpatient clinics must produce susceptibility results within 24 to 48 hours to guide treatment decisions. The broth microdilution method supports this timeline because the panels can be prepared in advance and stored frozen. The daily workflow requires only the inoculum preparation, plate inoculation, incubation, and reading. Automated systems can complete the entire process within 16 to 20 hours of incubation. The agar dilution method requires the preparation of agar plates on the day of the test or the day before. The antimicrobial agent must be incorporated into molten agar, and the plates must be poured and allowed to solidify before use. This preparation step adds several hours to the workflow. The agar dilution method is therefore less suitable for laboratories that must produce results within a strict 24-hour window. The decision framework should include a turnaround time requirement. If the laboratory must report results within 24 hours of specimen receipt, the broth microdilution method is the practical choice. If the laboratory can accept a 48 to 72 hour turnaround time, the agar dilution method becomes feasible. ### Domain 3: Staff Expertise and Training The third assessment domain evaluates the technical skill level of the laboratory staff. The broth microdilution method requires proficiency in preparing antimicrobial stock solutions, dispensing reagents into 96-well plates, and operating automated readers. The staff must also be trained in the interpretation of the visual or automated growth readings. The American Society for Microbiology provides professional education and laboratory practice resources that can support the training of the laboratory staff in these techniques. The agar dilution method requires additional skills in preparing agar plates, incorporating the antimicrobial agent into the molten agar, and using a replicator device for the spot inoculation. The replicator device must be calibrated to deliver a consistent inoculum volume to each plate. The staff must also be trained in the visual reading of the agar plates, which requires the ability to distinguish a single colony or a faint haze of growth from the confluent growth. The decision framework should include an assessment of the current staff skill level and the training resources available. A laboratory with limited staff and no dedicated training time may prefer the broth microdilution method because the automated systems reduce the hands-on time and the training burden. A laboratory with experienced staff and a training program may be able to implement the agar dilution method effectively. ### Domain 4: Budget and Consumable Costs The fourth assessment domain examines the total cost of the method, including the consumables, the equipment, and the labor. The broth microdilution method requires the purchase of 96-well microtiter plates, broth media, and antimicrobial agents. The cost per test is moderate and is influenced by the number of antimicrobial agents in the panel. The automated systems require a significant capital investment, but they reduce the hands-on time and the labor cost. The agar dilution method requires the purchase of agar, petri dishes, and antimicrobial agents. The cost per test is lower when a large number of isolates are tested against a single drug. The cost per test is higher when only a few isolates are tested because the agar plates must be prepared for each concentration level. The agar dilution method also requires the purchase of a replicator device, which is a one-time capital investment. The decision framework should include a cost comparison for the expected volume of testing. The laboratory should calculate the cost per isolate-agent combination for each method. The calculation should include the consumables, the labor, and the equipment amortization. The method with the lower cost per isolate-agent combination is the more economical choice for the laboratory. ### Implementation Steps for the Decision Framework The implementation of the decision framework follows a structured sequence. The first step is to collect the operational data for the laboratory. The data includes the number of isolates processed per week, the number of antimicrobial agents tested per isolate, the turnaround time requirements, the staff training level, and the budget. The second step is to score each of the four domains on a scale of 1 to 5. A score of 5 indicates that the domain strongly favors the broth microdilution method. A score of 1 indicates that the domain strongly favors the agar dilution method. The scores are then weighted according to the laboratory mission. A clinical laboratory will weight the turnaround time and the staff training more heavily. A reference laboratory will weight the isolate throughput and the cost more heavily. The third step is to calculate the weighted total for each method. The method with the higher weighted total is the recommended choice. The fourth step is to validate the recommendation with a pilot study. The laboratory should run a small batch of isolates using the recommended method and compare the results with the results from the current method. The pilot study should include the quality control strains and the clinical isolates. The fifth step is to document the decision and the rationale. The documentation should include the operational data, the scoring, the weighted total, and the pilot study results. The documentation is used for the accreditation and for the future review of the method selection. ## Records and Measurements for the Decision Framework The laboratory should maintain the records of the decision framework and the operational data. The records should include the weekly isolate count, the number of antimicrobial agents tested per isolate, the turnaround time, the staff training records, and the cost data. The records should be reviewed on a quarterly basis to ensure that the method selection remains appropriate. The laboratory should also track the quality control results for the selected method. The quality control results should be within the acceptable ranges for the quality control strains. The laboratory should document the quality control results and the corrective actions taken when the results are out of range. The laboratory should also track the turnaround time for the selected method. The turnaround time should be measured from the receipt of the specimen to the final report. The laboratory should document the turnaround time for each batch of testing and compare the results with the requirement. The laboratory should also track the cost per isolate-agent test for the selected method. The cost should be calculated on a monthly basis and compared with the budget. The laboratory should document the cost data and the reasons for any variance. ## Common Failure Patterns in Method Selection The most common failure in method selection is the choice of the method based on the preference of the laboratory director instead of the operational data. The laboratory director may prefer the broth microdilution method because of the familiarity with the method, even when the agar dilution method would be more efficient for the isolate volume. The decision framework should be used to avoid this bias. The second common failure is the choice of the method based on the cost of the consumables alone. The laboratory may choose the agar dilution method because the consumables are cheaper, but the labor cost may be higher. The decision framework should include the labor cost in the cost comparison. The third common failure is the choice of the method based on the availability of the automated system. The laboratory may choose the broth microdilution method because the automated system is available, but the laboratory may not have the volume to justify the cost of the system. The decision framework should include the volume assessment in the selection. The fourth common failure is the lack of a pilot study. The laboratory may implement the method without validating the method with a pilot study. The pilot study is essential to confirm that the method works in the laboratory environment. ## Biosafety and Containment in Method Selection The method selection should also consider the biosafety requirements of the laboratory. The broth microdilution method involves the handling of the liquid cultures in the 96-well plates. The agar dilution method involves the handling of the solid agar plates. Both methods require the use of the biological safety cabinet for the procedures that can generate the aerosols. The CDC and NIH provide the biosafety guidelines for the laboratories that handle the microorganisms. The risk assessment should be performed to determine the biosafety level required for the laboratory. The biosafety level 2 is typically required for the susceptibility testing. The laboratory must follow the containment and the decontamination procedures described in the biosafety guidelines. The method selection should also consider the decontamination of the waste. The broth microdilution plates and the agar plates must be decontaminated before the disposal. The decontamination is typically performed by the autoclaving. The laboratory must have a written protocol for the decontamination of the waste. ## Professional Escalation Criteria for Method Selection The laboratory should have a system for the escalation of the method selection issues. The escalation criteria are used to ensure that the method selection is reviewed and the appropriate action is taken. The first escalation criterion is the failure of the quality control results for the selected method. The laboratory must investigate the cause of the failure and take the corrective action. If the corrective action does not resolve the issue, the laboratory should escalate the issue to the laboratory director. The second escalation criterion is the change in the operational data. If the isolate volume or the number of antimicrobial agents tested per isolate changes significantly, the laboratory should review the method selection. The laboratory should escalate the issue to the laboratory director for the review. The third escalation criterion is the change in the budget. If the cost of the consumables or the equipment changes significantly, the laboratory should review the method selection. The laboratory should escalate the issue to the laboratory director for the review. The fourth escalation criterion is the change in the regulatory requirements. If the CLSI standards or the accreditation requirements change, the laboratory should review the method selection. The laboratory should escalate the issue to the laboratory director for the review. The escalation of the method selection issues is an important part of the laboratory quality. The laboratory must have a written protocol for the escalation of the issues. The protocol should define the criteria for the escalation and the steps to be taken. ## Frequently Asked Questions ### What is the difference between broth microdilution and agar dilution? Broth microdilution uses liquid broth in a 96-well plate to test one isolate against many antimicrobial agents. Agar dilution uses solid agar plates with the antimicrobial incorporated into the agar to test many isolates against one agent. Both methods produce a quantitative MIC value. ### Which method is more suitable for a clinical laboratory? Broth microdilution is more suitable for a clinical laboratory because it is compatible with automated systems and can test a single isolate against many antimicrobial agents in one plate. The automated systems reduce the hands-on time and provide results within a short time. ### Which method is more suitable for a reference laboratory? Agar dilution is more suitable for a reference laboratory that tests large collections of isolates against a single antimicrobial agent. The agar dilution method allows many isolates to be tested on a single plate, which is more efficient for large-scale testing. ### Can both methods be used for fastidious organisms? Yes, both methods can be used for fastidious organisms. The broth microdilution method requires the use of a supplemented broth, and the agar dilution method requires the use of a supplemented agar. The CLSI standards provide the recommended media formulations for each organism group. ### What is the role of quality control strains in susceptibility testing? Quality control strains are used to ensure that the susceptibility testing is performing correctly. The quality control strains have known MIC values for the antimicrobial agents being tested. The results from the quality control strains must be within the acceptable range for the test to be valid. ### How do I choose between broth microdilution and agar dilution? The choice depends on the laboratory workflow. If you need to test a single isolate against many antimicrobial agents, use broth microdilution. If you need to test many isolates against a single antimicrobial agent, use agar dilution. The choice also depends on the organism being tested and the availability of the automated systems. ### What are the common causes of errors in susceptibility testing? The common causes of error include contamination of the test, incorrect preparation of the antimicrobial stock, incorrect preparation of the inoculum, incorrect reading of the results, and the use of the wrong media or incubation conditions. The quality control results are used to detect the errors. ### How do I interpret the MIC result? The MIC is the lowest concentration of the antimicrobial agent that inhibits the visible growth of the organism. The MIC is interpreted against the CLSI breakpoints to determine the susceptibility category. The breakpoints are updated on a regular basis, and the laboratory must use the current breakpoints. ## Using the Evidence | Source | Best use in this topic | Important limitation | |---|---|---| | [Biosafety in Microbiological and Biomedical Laboratories](https://www.cdc.gov/labs/bmbl/index.html) | official guidance | Check the linked page for current local requirements | | [Microbiology Resources](https://asm.org/) | official guidance | Check the linked page for current local requirements | | [Antimicrobial Resistance](https://www.who.int/health-topics/antimicrobial-resistance) | official guidance | Check the linked page for current local requirements | ## Related Bacteriology Guides - [Bacterial Pathogens in Poultry: Comprehensive Clinical Reference](/knowledge/bacteria/avian-bacteria/bacterial-pathogens-in-poultry-clinical-reference) - [Duck Disease: Comprehensive Veterinary Reference on Common Pathogens and Clinical Management](/knowledge/bacteria/avian-bacteria/duck-disease-comprehensive-veterinary-reference) - [Haemophilus parasuis (Glaesserella parasuis) and Glässer's Disease in Swine: A Comprehensive Clinical Reference](/knowledge/bacteria/livestock-bacteria/haemophilus-parasuis-glaessers-disease-pigs) - [Histophilus somni: A Comprehensive Veterinary Reference on Pathogenesis, Clinical Syndromes, Diagnostics, and Control](/knowledge/bacteria/livestock-bacteria/histophilus-somni) - [Fowl Cholera: Etiology, Clinical Manifestations, and Control in Poultry](/knowledge/bacteria/avian-bacteria/fowl-cholera-etiology-clinical-manifestations-control-poultry) ## 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. - [Broth Microdilution and Gradient Diffusion Strips vs. Reference Agar Dilution Method: First Evaluation for Clostridiales Species Antimicrobial Susceptibility Testing.](https://pubmed.ncbi.nlm.nih.gov/34439025). Antibiotics (Basel, Switzerland), 2021. - [Comparison of Clostridium difficile minimum inhibitory concentrations obtained using agar dilution vs broth microdilution methods.](https://pubmed.ncbi.nlm.nih.gov/28188880). Anaerobe, 2017. - [Fosfomycin-based combinations against carbapenemase-producing Klebsiella pneumoniae bloodstream infections: Correlating in vitro synergy with clinical outcomes.](https://pubmed.ncbi.nlm.nih.gov/41352396). International journal of antimicrobial agents, 2026. - [Evaluation of the Impact of Methicillin-Resistant Staphylococcus pseudintermedius Biofilm Formation on Antimicrobial Susceptibility.](https://pubmed.ncbi.nlm.nih.gov/27500604). Veterinary surgery : VS, 2016. > This article is educational and does not replace validated laboratory procedures, clinical diagnosis, institutional biosafety review, or public-health guidance.