# Building a Reference Strain Library for Microbial Identification ## Quick Answer - Build a reference strain library by selecting authenticated strains from a recognized culture collection, storing them under controlled conditions, and verifying identity before routine use. - Document every strain with source, passage history, storage location, and verification results so each identification run has a traceable control. - A reference library supports quality control but cannot replace method validation, staff training, or adherence to current standard procedures. ## Purpose of a Reference Strain Library Microbial identification systems produce results that laboratories use for clinical, food safety, environmental, and research decisions. A reference strain library provides authenticated organisms that serve as known controls when laboratories verify that their identification methods work correctly. Without such controls, a laboratory cannot distinguish between a system failure and a genuine identification result. The library functions as a collection of biological materials with documented provenance. Each strain has a known identity, a recorded source, and a defined storage condition. When a laboratory tests a reference strain alongside patient or product isolates, the result on the reference strain indicates whether the identification system is performing within expected parameters. Reference strains also support training. New laboratory personnel can practice identification techniques on organisms with known characteristics before working with clinical or food samples. The library becomes a teaching tool that reduces interpretive errors during the learning period. ## Core Principles of Reference Strain Management ### Authenticity and Provenance A reference strain is only useful if its identity is trustworthy. Laboratories should obtain strains from established culture collections that maintain authenticated biological materials and provide documentation of strain history. The American Type Culture Collection is one such source that supplies reference materials with known characteristics and quality documentation [1]. Strains from clinical samples, environmental sources, or other laboratories may carry uncertain identities. A strain labeled as a particular species in one laboratory may have been misidentified, contaminated, or mutated during storage. Using such a strain as a control introduces error into every identification run that relies on it. ### Stability and Viability Reference strains must remain viable and phenotypically stable throughout their storage period. Repeated subculture can select for mutants that no longer represent the original strain. Laboratories should minimize the number of passages between the original culture and the working cultures used in daily testing. Storage conditions determine how long a strain remains usable. Freeze-drying and ultra-low temperature storage preserve strains for extended periods, while refrigerated agar slants require more frequent transfer. Each storage method has a different risk profile for contamination, genetic drift, and viability loss. ### Traceability Every reference strain in the library should have a complete record that connects the current culture to its original source. The record should include the culture collection accession number, the date of receipt, the date of each subculture, the storage method, and the results of any verification tests performed. Traceability allows a laboratory to investigate problems. If a reference strain produces an unexpected result, the record shows whether the strain was handled correctly and whether the current culture is the same as the original. Without this record, the laboratory cannot distinguish between a strain problem and a system problem. ## At a Glance | Library Component | Purpose | Key Decision | Common Limitation | | --- | --- | --- | --- | | Strain selection | Choose organisms that match the identification system's expected range | Select strains from authenticated culture collections [1] | Limited panel may not cover all organisms in the laboratory's scope | | Storage method | Preserve viability and stability | Match storage to strain type and expected use frequency | Improper storage causes viability loss or contamination | | Documentation | Provide traceability for every culture | Record source, accession, passage, and verification | Incomplete records prevent error investigation | | Verification | Confirm identity before routine use | Test each strain with the identification system and a second method | Verification results may be misinterpreted without a reference method | | Periodic review | Detect drift or contamination over time | Schedule re-verification based on storage method and use | Long intervals allow silent strain deterioration | ## Strain Selection ### Defining the Panel The reference panel should reflect the organisms that the laboratory expects to identify. A clinical laboratory that processes urine cultures needs a different panel than a food microbiology laboratory that tests for pathogens. The panel should include organisms that are common in the laboratory's sample population and organisms that are difficult to identify or easily confused with other species. The panel should also include organisms that challenge the identification system. Some systems struggle with closely related species, with organisms that have unusual biochemical profiles, or with organisms that are rarely encountered. Including these in the library provides a way to test whether the system can handle the full range of samples the laboratory receives. ### Selecting Authenticated Strains Authenticated strains from a recognized culture collection provide the highest level of confidence in identity. The American Type Culture Collection [ATCC] maintains a large collection of reference strains with documented histories and quality control data [1]. Other national and international culture collections offer similar services. When selecting a strain, the laboratory should consider the following: - The species and subspecies designation - The source of the original isolate - The known characteristics of the strain - The availability of reference data for the strain - The cost and shipping requirements A strain that is well characterized and widely used in the field is preferable to a strain that is rarely studied. The more data available on a strain, the easier it is to verify that the laboratory's culture is the same as the original. ### Panel Size and Diversity A reference panel does not need to include every species that the laboratory might encounter. A practical panel covers the most common organisms, the most difficult organisms, and a few organisms that test the system's limits. The panel should be large enough to provide meaningful QC but small enough to be manageable. A laboratory that identifies a wide range of organisms may need a larger panel than a laboratory that focuses on a narrow set of species. The panel should be reviewed periodically and updated when the laboratory changes its test menu or when new identification challenges emerge. ## Acquisition and Receipt ### Ordering from a Culture Collection When ordering from a culture collection, the laboratory should specify the exact strain and the desired format. Culture collections offer strains as freeze-dried cultures, frozen cultures, or growing cultures. The format affects the storage requirements and the time needed to prepare the strain for use. The laboratory should also consider the biosafety classification of the strain. Some reference strains are harmless, while others require specific containment conditions. The Biosafety in Microbiological and Biomedical Laboratories [BMBL] provides guidance on risk assessment and containment for work with biological materials [2]. The laboratory should review the biosafety level of each strain before ordering. ### Receiving and Opening When a strain arrives, the laboratory should inspect the packaging for damage and verify that the strain matches the order. The culture collection provides documentation with the strain, and the laboratory should file this documentation with the strain record. Opening a freeze-dried culture requires care. The ampoule may be under vacuum or contain a desiccant. The laboratory should follow the culture collection's instructions for reconstitution and should perform the work in a biosafety cabinet if the strain requires containment [2]. The first culture from the received material should be checked for purity and identity before it is added to the library. A contaminated or misidentified strain should not be used as a reference. ### Storage Methods The storage method determines how long the strain remains viable and stable. The most common methods are: - Freeze-drying, which preserves the strain for many years and requires no ongoing maintenance - Ultra-lowry storage, which preserves the strain for several years and requires a freezer - Refrigerated agar culture, which preserves the strain for a short period and requires periodic transfer The choice of storage method depends on the laboratory's resources and the expected frequency of use. A laboratory that uses a strain frequently may keep a working culture on agar and maintain a frozen stock for long-term preservation. A laboratory that uses a strain rarely may rely on freeze-dried cultures. The storage method should be recorded for each strain. The record should include the date of storage, the medium used, and the conditions of storage. ## Documentation and Records ### Strain Record Each strain in the library should have a record that includes the following: - The strain name and the species designation - The culture collection accession number - The source of the strain - The date of receipt - The storage method and location - The passage number or the number of times the strain has been subcultured - The results of verification tests - The date of the last verification The record should be updated every time the strain is used, subcultured, or verified. The record should be stored in a way that is accessible to all laboratory personnel who use the library. ### Verification Testing Verification is the process of confirming that the strain in the library is the same as the strain that was originally received. Verification should be performed when the strain is first added to the library and at regular intervals thereafter. The verification method should be appropriate for the strain and the identification system. A biochemical identification system can be used to verify the strain's identity, but the result should be compared with the expected result for the strain. If the system produces a different result, the strain may be contaminated, mislabeled, or the system may be failing. The laboratory should also consider using a second method for verification. A molecular method such as sequencing or a different biochemical system can provide independent confirmation of the strain's identity. The choice of the second method depends on the laboratory's resources and the strain's characteristics. ### Verification Schedule The verification schedule depends on the storage method and the frequency of use. A strain that is used frequently should be verified more often than a strain that is used rarely. A strain that is stored in a frozen state may be verified less often than a strain that is stored on agar. The laboratory should establish a schedule and document the schedule in the library's procedures. The schedule should be reviewed periodically and adjusted if the laboratory's needs change. ## Practical Implementation Steps ### Step 1: Define the Library Scope The laboratory should list the organisms that it identifies and the identification systems that it uses. The list should include the organisms that are common in the laboratory's sample population and the organisms that are difficult to identify. The laboratory should also consider the organisms that are required by any applicable standards or regulations. ### Step 2: Select the Strains The laboratory should select strains from authenticated culture collections. The selection should cover the organisms in the scope and should include strains that test the limits of the identification system. The laboratory should order the strains and document the order. ### Step 3: Receive and Prepare the Strains The laboratory should receive the strains, inspect them for damage, and prepare them according to the culture collection's instructions. The laboratory should verify the purity and viability of each strain before adding it to the library. ### Step 4: Store the Strains The laboratory should store each strain using the method that best preserves its viability and stability. The storage method should be documented in the strain record. ### Step 5: Create the Strain Records The laboratory should create a record for each strain that includes all the information needed for traceability. The record should be stored in a location that is accessible to all laboratory personnel. ### Step 6: Verify the Strains The laboratory should verify each strain when it is added to the library and at regular intervals thereafter. The verification results should be recorded in the strain record. ### Step 7: Use the Strains for QC The laboratory should use the reference strains in the identification system according to the system's instructions. The results should be compared with the expected results for the strain. Any discrepancy should be investigated. ### Step 8: Review and Update the Library The laboratory should review the library periodically to ensure that it still meets the laboratory's needs. The review should consider changes in the laboratory's scope, changes in the identification system, and the results of the verification testing. ## Records and Measurements ### What to Record The laboratory should record the following for each strain: - The strain name and accession number - The source of the strain - The date of receipt - The storage method and location - The passage number - The verification results - The date of the last verification - The results of any QC tests that used the strain The records should be complete and legible. The records should be stored in a way that prevents loss or damage. ### How to Measure Viability Viability is the ability of the strain to grow and produce the expected results. The laboratory can measure viability by culturing the strain on the appropriate medium and observing the growth. The laboratory should record the date of the culture and the result. A strain that fails to grow or that grows poorly may have lost viability. The laboratory should investigate the cause and decide whether to replace the strain. ### How to Measure Stability Stability is the ability of the strain to maintain its characteristics over time. The laboratory can measure stability by comparing the results of the verification tests over time. If the strain produces different results at different times, the strain may be unstable. The laboratory should also observe the strain's morphology and biochemical profile. A change in the morphology or the profile may indicate that the strain has mutated or become contaminated. ## Common Failure Patterns ### Contamination Contamination is the most common failure in a reference strain library. A strain can become contaminated during storage, during subculture, or during use. The contamination may be visible as a mixed culture or may be subtle and only detected by verification testing. The laboratory should use aseptic technique when handling strains and should inspect the cultures for purity. The laboratory should also verify the strains periodically to detect contamination that is not visible. ### Viability Loss Viability loss occurs when the strain no longer grows or grows poorly. The loss can be caused by improper storage, by too many subcultures, or by the age of the culture. The laboratory should monitor the viability of the strains and replace the strains that lose viability. ### Genetic Drift Genetic drift occurs when the strain changes over time. The change can be caused by repeated subculture or by the accumulation of mutations. The change can alter the strain's characteristics and make it less useful as a control. The laboratory should minimize the number of subcultures and should use the storage method that preserves the strain's stability. The laboratory should also verify the strain's characteristics periodically to detect drift. ### Mislabeling Mislabeling occurs when a strain is labeled with the wrong identity. The mislabeling can occur during the receipt, during the subculture, or during the storage. The mislabeling can be detected by verification testing. The laboratory should verify the identity of each strain when it is added to the library and should verify the identity of the strain when it is used for QC. The laboratory should also label the cultures clearly and should check the labels before use. ## Quality Control and Verification ### QC Testing with Reference Strains The reference strains are used in QC testing to verify that the identification system is working correctly. The QC test should be performed according to the system's instructions and should include the reference strains that are appropriate for the system. The QC test should be performed at the frequency that is recommended by the system's manufacturer or by the laboratory's procedures. The results of the QC test should be recorded and reviewed. ### Verification of the Identification System The reference strains can also be used to verify the identification system's performance. The laboratory can test a panel of reference strains and compare the results with the expected results. The comparison can identify the system's limitations and the organisms that the system cannot identify correctly. The verification should be performed when the system is first installed and when the system is changed. The verification should also be performed periodically to ensure that the system continues to work correctly. ### Handling Discrepancies A discrepancy occurs when the reference strain produces a result that is different from the expected result. The discrepancy can be caused by a problem with the strain, a problem with the system, or a problem with the test. The laboratory should investigate the discrepancy before using the system for routine testing. The investigation should include a review of the strain record, a repeat of the test, and a check of the system's performance. ## Biosafety and Containment ### Risk Assessment The laboratory should perform a risk assessment for each reference strain before it is added to the library. The risk assessment should consider the strain's pathogenicity, the route of transmission, and the containment requirements. The Biosafety in Microbiological and Biomedical Laboratories [BMBL] provides guidance on risk assessment and containment [2]. The risk assessment should be documented and reviewed periodically. The laboratory should ensure that the containment level is appropriate for the strain. ### Containment The laboratory should use the containment level that is appropriate for the strain. The containment level may require a biosafety cabinet, a separate room, or other controls. The laboratory should follow the containment requirements when handling the strain. ### Decontamination The laboratory should decontaminate the cultures and the materials that come into contact with the strain. The decontamination method should be appropriate for the strain and the material. The laboratory should follow the decontamination procedures that are established in the laboratory's biosafety manual. ## Limitations and Escalation Criteria ### Limitations of the Library A reference library is a control tool, not a substitute for the standard methods. The library cannot correct a system that is not working correctly, and it cannot identify an organism that the system cannot identify. The library is also limited by the strains that it contains. A library that does not contain a particular species cannot be used to test the system's ability to identify that species. The laboratory should review the library periodically and add strains as needed. ### When to Escalate The laboratory should escalate a problem when it cannot resolve the problem internally. The escalation should be to a supervisor, a manager, or an external expert. The escalation should include the documentation of the problem and the steps that have been taken to resolve it. The laboratory should also escalate when the problem affects the safety of the laboratory or the quality of the results. The escalation should be prompt and should include the relevant information. ## Professional Escalation Criteria ### When to Seek External Help The laboratory should seek external help when the problem is beyond the laboratory's expertise. The external help may include the culture collection, the manufacturer of the identification system, or a reference laboratory. The laboratory should seek external help when the problem is a safety concern. The laboratory should also seek external help when the problem cannot be resolved with the laboratory's resources. ### When to Replace a Strain The laboratory should replace a strain when the strain is contaminated, when the strain has lost its viability, or when the strain has changed its characteristics. The laboratory should also replace a strain when the strain is no longer needed. The replacement should be documented in the strain record. The laboratory should verify the replacement strain before it is added to the library. ## Building a Risk-Based Verification Schedule for Reference Strain Libraries A reference strain library loses its value when verification becomes a calendar ritual instead of a risk-based decision. Many laboratories verify every strain on the same schedule, treating a lyophilized stock that has never been opened the same as an agar working culture that is subcultured weekly. This uniform approach wastes resources on strains that are unlikely to change and leaves high-risk strains with insufficient oversight. A risk-based verification schedule assigns each strain a risk category based on how it is stored, how often it is used, and how critical it is to the laboratory's identification decisions. This section provides a practical framework for building such a schedule, including the risk scoring system, the verification intervals, the record fields needed to support the schedule, and the troubleshooting steps when a strain fails verification. ### Why Uniform Verification Schedules Fail The common practice of verifying all reference strains at a fixed interval, such as every six months or every year, creates two distinct problems. The first problem is over-verification of stable strains. A freeze-dried culture that remains sealed in its original ampoule and is only opened once to create a master stock does not need the same verification frequency as a working culture that is transferred weekly. Each time a laboratory opens a freeze-dried ampoule, it consumes a portion of the original material. If the laboratory verifies a strain that has not been used, it spends time and materials without reducing any actual risk. The second problem is under-verification of high-risk strains. A strain that is used daily for quality control, that is stored on agar slants at refrigerator temperature, and that has a history of phenotypic drift requires more frequent verification than a strain that is used quarterly. When all strains are verified on the same schedule, the high-risk strains may go for months without a check while the low-risk strains are checked unnecessarily. The risk-based approach corrects both problems by making the verification interval proportional to the strain's actual risk of change. The risk is not a fixed property of the strain itself. It is a function of three factors: the storage method, the frequency of use, and the consequence of a failure. Each factor contributes to the overall risk score that determines the verification interval. ### The Three-Factor Risk Scoring System The risk scoring system uses three factors that are easy to assess from existing records. Each factor receives a score from one to three, and the total score determines the verification interval. The three factors are storage stability, use frequency, and failure consequence. #### Storage Stability Score The storage stability score reflects how likely the strain is to change while in storage. The score is based on the storage method and the time since the strain was last prepared. A score of one applies to strains stored in freeze-dried form or in ultra-low temperature freezers below minus 70 degrees Celsius. These methods preserve the strain in a metabolically inactive state and are the most stable. A strain that has been stored in this way for less than five years and has not been opened since the original preparation receives a score of one. A score of two applies to strains stored in mechanical freezers at minus 20 to minus 70 degrees Celsius, or to freeze-dried strains that have been opened and reconstituted. These methods are stable but carry a higher risk of temperature fluctuation or contamination during handling. A score of three applies to strains stored on agar slants, in broth, or on other solid media at refrigerator temperature. These strains are metabolically active and require periodic transfer. Each transfer introduces the risk of contamination, selection of mutants, and loss of viability. The longer the strain has been in continuous culture, the higher the risk. #### Use Frequency Score The use frequency score reflects how often the strain is removed from storage and handled. Each handling event is an opportunity for contamination, mislabeling, or damage. A score of one is a strain that is used less than once per month. A score of two is a strain that is used one to four times per month. A score of three is a strain that is used more than four times per month, which includes strains used in daily quality control runs. The use frequency score is independent of the storage score. A freeze-dried strain that is opened and reconstituted daily has a high use frequency even though its storage method is stable. The daily handling introduces risk that the storage method does not eliminate. #### Failure Consequence Score The failure consequence score reflects the impact of a strain failure on the laboratory's work. A strain that is used to verify the identification of a critical pathogen has a higher consequence than a strain that is used to verify a common environmental organism. A score of one applies to strains that are used for training, for demonstration, or for identification of organisms that are rarely encountered and have low clinical or regulatory impact. A score of two applies to strains that are used for routine quality control of common organisms. A score of three applies to strains that are used to verify the identification of organisms with high clinical, regulatory, or public health significance, including organisms that are part of antimicrobial resistance surveillance or that are reportable to public health authorities. The World Health Organization has identified antimicrobial resistance as a global public health concern that requires surveillance and stewardship [3]. A reference strain used to verify the identification of a resistant organism carries a high failure consequence because an incorrect identification could lead to a missed resistance detection. ### Calculating the Verification Interval The three scores are added to produce a total risk score from three to nine. The total score determines the verification interval. | Total Risk Score | Verification Interval | Example Strain Profile | | --- | --- | --- | | 3 to 4 | Every 24 months | Freeze-dried, used quarterly, low consequence | | 5 to 6 | Every 12 months | Frozen, used monthly, moderate consequence | | 7 to 9 | Every 6 months | Agar culture, used daily, high consequence | The intervals in the table are starting points. A laboratory can adjust the intervals based on its own experience with the strains and the identification systems. The key is that the interval is tied to the risk score and is documented in the strain record. A strain with a total score of three is a freeze-dried strain that is used rarely and has low failure consequence. This strain can be verified every two years. A strain with a total score of nine is an agar culture that is used daily and has high failure consequence. This strain should be verified every six months. The laboratory should review the risk score for each strain whenever the strain is used or verified. A change in the storage method, the use frequency, or the laboratory's test menu can change the risk score and the verification interval. ### The Verification Decision Record The risk-based schedule requires a record that captures the risk factors and the verification decisions. The record should include the following fields for each strain: - The strain name and accession number - The storage method and the date of the last preparation - The use frequency, which is the number of times the strain was used in the last 90 days - The failure consequence score, which is assigned by the laboratory supervisor - The total risk score and the resulting verification interval - The date of the last verification and the date of the next scheduled verification - The verification method and the result - The name of the person who performed the verification The record should be updated at least quarterly. The use frequency is calculated from the usage log, which is described in the next section. The failure consequence score is reviewed when the laboratory changes its test menu or when the strain is used for a new purpose. The verification decision log serves as the basis for the verification schedule. The laboratory can generate a list of strains that are due for verification each month by sorting the log by the next scheduled verification date. This list becomes the monthly verification workload. ### The Usage Log and Its Role in the Schedule The risk-based schedule depends on accurate use frequency data. The laboratory should maintain a usage log for each strain that records the date of each use, the purpose of the use, and the result of the identification test. The usage log is not the same as the verification record. The usage log captures every time the strain is used in a quality control test, a training exercise, or a method verification. The verification record captures the periodic checks of the strain's identity. The usage log is reviewed quarterly to update the use frequency score. A strain that was used 20 times in the last 90 days has a use frequency score of three. A strain that was used once in the last 90 days has a use frequency score of one. The usage log also provides the data needed to detect problems. If a strain is used frequently and produces consistent results, the risk of a hidden failure is lower. If a strain is used frequently and produces variable results, the risk is higher and the verification interval should be shortened. ### Verification Methods for the Risk-Based Schedule The verification method should be matched to the risk score. A low-risk strain can be verified with a single method, such as the identification system that the laboratory uses for routine testing. A high-risk strain should be verified with two independent methods. The first method is the identification system that the strain is used to control. The strain is tested with the system and the result is compared with the expected result. This method verifies both the strain and the system. The second method is an independent method that does not rely on the same biochemical or physiological principles as the first method. The second method can be a different biochemical identification system, a molecular method such as sequencing, or a serological method. The choice of the second method depends on the laboratory's resources and the strain's characteristics. The second method is not required for every verification. A strain with a risk score of three or four can be verified with the identification system alone. A strain with a risk score of seven or higher should be verified with two methods. The laboratory should document the verification method in the strain record. ### Handling Verification Failures A verification failure occurs when the strain produces a result that does not match the expected result. The failure can be caused by a problem with the strain, a problem with the identification system, or a problem with the test procedure. The laboratory should follow a structured troubleshooting process before deciding whether to replace the strain. #### Step 1: Repeat the Test The first step is to repeat the verification test using a fresh culture from the strain. The repeat test should use the same method and the same conditions as the original test. If the repeat test produces the expected result, the original failure was likely caused by a technical error, such as a contaminated inoculum or an incorrect reagent. #### Step 2: Check the Strain Record If the repeat test produces the same unexpected result, the laboratory should review the strain record. The record should show the storage method, the passage number, and the date of the last verification. A strain that has been subcultured many times or that has been stored for a long period may have changed. #### Step 3: Test the Identification System The laboratory should test the identification system with a different reference strain that is known to be stable. If the system produces the expected result with the other strain, the problem is likely with the strain. If the system produces an unexpected result with the other strain, the problem is likely with the system. #### Step 4: Test the Strain with a Second Method The laboratory should test the strain with a second method that is independent of the identification system. If the second method confirms the expected identity, the strain is likely correct and the identification system has a problem. If the second method does not confirm the expected identity, the strain is likely contaminated, mislabeled, or changed. #### Step 5: Decide Whether to Replace the Strain If the troubleshooting confirms that the strain has changed, the laboratory should replace the strain. The replacement should be ordered from the original culture collection or from another authenticated source. The replacement strain should be verified before it is added to the library. The laboratory should document the verification failure and the troubleshooting steps in the strain record. The documentation should include the date of the failure, the unexpected result, the steps taken to investigate, and the final decision. ### Adjusting the Schedule After a Failure A verification failure is a signal that the risk score for the strain was too low. The laboratory should increase the risk score for the strain and shorten the verification interval. The strain should be moved to a higher risk category and verified more frequently until the laboratory has confidence that the strain is stable. The laboratory should also review the risk scores for other strains that are stored under the same conditions or used at the same frequency. A failure in one strain may indicate a systematic problem with the storage method or the handling procedure. ### The Role of the Laboratory Supervisor The risk-based verification schedule requires a designated person to assign the failure consequence scores and to review the verification decision log. The laboratory supervisor or the quality manager should perform this role. The supervisor should review the log quarterly and should approve any changes to the verification intervals. The supervisor should also review the verification failures and the troubleshooting records. The supervisor should decide whether the failure indicates a problem with the strain, the system, or the procedure. The supervisor should document the decision and the rationale. The supervisor should ensure that the verification schedule is followed. The monthly verification work should be assigned to the appropriate personnel and the results should be reviewed. A strain that is overdue for verification should not be used for quality control until the verification is completed. ### The Relationship Between Verification and Quality Control The risk-based verification schedule is separate from the quality control testing that uses the reference strains. Quality control testing is the routine use of the strain to verify that the identification system is working correctly. Verification is the periodic check of the strain itself. The two activities are related. A strain that is used in quality control testing on a daily basis is also a strain that is at higher risk of change. The quality control results provide data that can be used to assess the strain's stability. If the quality control results are consistent over time, the strain is likely stable. If the quality control results vary, the strain may be changing. The laboratory should review the quality control results as part of the verification decision. The quality control results are a source of data for the risk assessment. A strain that has produced consistent quality control results for six months is a lower risk than a strain that has produced variable results. ### The Role of the Culture Collection in Verification The culture collection that supplied the strain can provide information that supports the verification schedule. The American Type Culture Collection provides documentation of strain history and quality control data for its reference materials [1]. The laboratory should use this documentation as the baseline for the strain's expected characteristics. When a strain fails verification, the laboratory should contact the culture collection to confirm the expected characteristics and to obtain guidance on the troubleshooting. The culture collection may also provide a replacement strain if the original strain is found to be defective. The laboratory should not attempt to repair a strain that has failed verification. A strain that has changed cannot be restored to its original state. The laboratory should replace the strain and should document the replacement in the strain record. ### The Verification Schedule as a Living Document The risk-based verification schedule is not a static document. The schedule should be reviewed and updated whenever the laboratory changes its test menu, its identification system, or its storage methods. The schedule should also be updated when the laboratory adds new strains to the library. The review should include the following: - The list of strains in the library and their risk scores - The verification intervals and the dates of the last verification - The verification failures and the troubleshooting records - The quality control results for each strain - The changes in the laboratory's scope or methods The review should be documented and should be signed by the laboratory director. The review should be conducted at least annually. ### The Practical Implementation of the Risk-Based Schedule The laboratory should implement the risk-based verification schedule in the following steps: 1. Assign a risk score to each strain in the library using the three-factor system. 2. Calculate the verification interval for each strain using the total risk score. 3. Create a verification decision log that lists each strain, its risk score, and its next verification date. 4. Maintain a usage log for each strain that records the date and purpose of each use. 5. Review the usage log quarterly and update the use frequency score. 6. Perform the verification tests according to the schedule. 7. Document the verification results in the strain record. 8. Investigate any verification failure using the structured troubleshooting procedure. 9. Adjust the risk score and the verification interval after a failure. 10. Review the entire schedule at least once per year. The implementation should be documented in the laboratory's quality manual. The laboratory should train all personnel on the risk-based schedule and the troubleshooting procedure. ### Common Mistakes in Risk-Based Verification The most common mistake is assigning the same risk score to all strains. The laboratory should resist the temptation to simplify the schedule by using a single verification interval. The risk-based schedule is designed to allocate the verification effort to the strains that need it most. The second common mistake is failing to update the risk score when the strain's use changes. A strain that was used rarely and is now used daily should have its risk score updated. The laboratory should review the usage log quarterly and update the risk scores accordingly. The third common mistake is ignoring the verification failures. A verification failure is a signal that the strain or the system has a problem. The laboratory should investigate the failure and should not continue to use the strain until the problem is resolved. The fourth common mistake is failing to document the verification schedule and the verification results. The documentation is the basis for the traceability of the library. Without the documentation, the laboratory cannot demonstrate that the library is under control. ### The Public Health Context for Verification The verification of reference strains has a public health context that extends beyond the individual laboratory. The World Health Organization has identified antimicrobial resistance as a global health threat that requires coordinated surveillance and stewardship [3]. The reference strains that are used to verify the identification of resistant organisms are part of this surveillance. A failure in the reference strain can lead to a missed resistance case or a false resistance report. The laboratory should consider the public health context when assigning the failure consequence score. A strain that is used to verify the identification of a resistant organism should receive a high failure consequence score. The verification interval for such a strain should be shorter than the interval for a strain that is used to verify a common environmental organism. The laboratory should also consider the regulatory context. The FDA's Bacteriological Analytical Manual provides official methods for food microbiology [4]. The reference strains that are used to verify these methods are part of the quality system. The laboratory should ensure that the verification schedule is consistent with the requirements of the methods. ### The Verification Schedule and the Identification System The verification schedule should be coordinated with the quality control requirements of the identification system. The Clinical and Laboratory Standards Institute provides standards for antimicrobial susceptibility testing and other microbiology methods [5]. The laboratory should follow the quality control requirements of the system and should use the reference strains to meet those requirements. The verification schedule should not be confused with the quality control schedule. The quality control schedule is the frequency at which the identification system is tested with the reference strains. The verification schedule is the frequency at which the reference strains themselves are checked. The two schedules are related but distinct. The laboratory should document both schedules and should ensure that the personnel understand the difference. The quality control schedule is determined by the system's requirements. The verification schedule is determined by the risk assessment. ### The Verification Schedule and the Laboratory's Scope The verification schedule should be reviewed when the laboratory changes its scope. A laboratory that adds a new identification test or a new organism to its test menu should review the reference strains that are needed for the new test. The new strains should be added to the library and the risk scores should be assigned. The laboratory should also review the verification schedule when the laboratory changes its identification system. A new system may have different quality control requirements and may require different reference strains. The laboratory should verify the new system with the reference strains before using it for routine testing. The verification schedule should be a living document that reflects the current state of the laboratory. The schedule should be reviewed at least annually and should be updated whenever the laboratory's needs change. ### The Verification Schedule and the Staff The verification schedule should be communicated to the laboratory staff. The staff should know which strains are due for verification and when the verification is scheduled. The staff should also know the troubleshooting procedure for verification failures. The laboratory should train the staff on the risk-based verification schedule. The training should include the risk scoring system, the verification intervals, and the troubleshooting procedure. The training should be documented and should be repeated when the schedule is changed. The staff should be encouraged to report any unexpected results with the reference strains. The unexpected result may be the first sign of a problem with the strain or the system. The laboratory should investigate the unexpected result promptly. ### The Verification Schedule and the External The laboratory should consider the external requirements that apply to its work. The laboratory may be accredited by a body that requires a documented verification schedule. The laboratory may also be subject to regulatory requirements that specify the verification of reference strains. The laboratory should ensure that the verification schedule meets the external requirements. The schedule should be documented and the verification results should be recorded. The laboratory should be able to demonstrate the verification schedule to an external auditor. The laboratory should also consider the requirements of the culture collection. The culture collection may provide guidance on the storage and verification of the strains. The laboratory should follow the guidance and should document the compliance. ### The Verification Schedule as a Continuous Improvement Tool The risk-based verification schedule is a continuous improvement tool. The laboratory should use the verification results to improve the library and the identification system. The verification failures should be investigated and the root causes should be addressed. The laboratory should also use the verification results to improve the risk scoring system. If a strain fails verification despite a low risk score, the risk scoring system should be reviewed. The laboratory should adjust the scoring system to better reflect the actual risk. The verification schedule should be reviewed as part of the laboratory's quality management system. The review should include the verification results, the failures, and the improvements. The review should be documented and the findings should be used to improve the laboratory's processes. ### The Verification Schedule and the Future The risk-based verification schedule is a practical approach that can be adapted to the laboratory's needs. The schedule should be reviewed and updated as the laboratory's methods and the identification system evolve. The schedule should also be updated as new reference strains are added to the library. The laboratory should consider the future needs of the library. The laboratory may need to add new reference strains for new identification tests or for new organisms. The laboratory should plan for the addition of the new strains and should assign the risk scores and the verification intervals. The laboratory should also consider the future of the identification system. The system may be updated or replaced. The laboratory should verify the new system with the reference strains and should update the verification schedule accordingly. The risk-based verification schedule is a living document that supports the laboratory's quality system. The schedule should be maintained and updated to ensure that the reference strain library continues to provide reliable controls for the identification system. ## Frequently Asked Questions ### What is a reference strain library? A reference strain library is a collection of authenticated microbial strains that a laboratory uses as controls for its identification systems. Each strain has a documented source, storage condition, and verification history. ### How do I choose which strains to include in my library? Choose strains that represent the organisms your laboratory identifies, including common organisms and difficult ones. Select strains from an authenticated culture collection such as the American Type Culture Collection [1]. ### How should I store reference strains? The storage method depends on the strain and the expected use. Freeze-drying and ultra-lowry storage preserve strains for long periods, while agar culture requires more frequent transfer. Record the storage method for each strain. ### How often should I verify reference strains? Verify each strain when it is added to the library and at regular intervals thereafter. The interval depends on the storage method and the frequency of use. A strain that is used frequently should be verified more often. ### What should I do if a reference strain produces an unexpected result? Investigate the discrepancy before using the result. Review the strain record, repeat the test, and check the system's performance. If the problem cannot be resolved, escalate it to a supervisor or an external expert. ### Can I use a reference strain from another laboratory? A strain from another laboratory may not have a verified identity. Use a strain from an authenticated culture collection to ensure that the strain is correctly identified and documented. ### What is the difference between a reference strain and a quality control strain? A reference strain is a strain with a documented identity and source. A quality control strain is a reference strain that is used to test the performance of an identification system. The same strain can serve both purposes. ### How do I document a reference strain? Create a record for each strain that includes the accession number, the source, the date of receipt, the storage method, the passage number, and the verification results. Update the record whenever the strain is used or verified. ## Related Diagnostic Guides - [Clinical Microbiology: From Specimen Collection to Pathogen Identification](/knowledge/diagnostics/microbiology/clinical-microbiology-from-specimen-collection-to-pathogen-identification) - [MALDI-TOF Mass Spectrometry for Veterinary Microbial Identification](/knowledge/diagnostics/microbiology/maldi-tof-mass-spectrometry-for-veterinary-microbial-identification) - [Method Validation in Clinical Laboratories: A Practical Protocol](/knowledge/diagnostics/emerging-tech/method-validation-in-clinical-laboratories-a-practical-protocol) - [Laboratory Quality Management Systems: A Practical Implementation Framework for Diagnostic Laboratories](/knowledge/diagnostics/emerging-tech/laboratory-quality-management-systems-a-practical-implementation-framework-for-diagnostic-labora) - [Biochemical Tests in Microbiology: A Practical Overview](/knowledge/diagnostics/microbiology/biochemical-tests-in-microbiology-a-practical-overview) ## References and Further Reading [1] [Culture Collections](https://www.atcc.org/). American Type Culture Collection. [2] [Biosafety in Microbiological and Biomedical Laboratories](https://www.cdc.gov/labs/bmbl/index.html). CDC and NIH. [3] [Antimicrobial Resistance](https://www.who.int/health-topics/antimicrobial-resistance). World Health Organization. [4] [Bacteriological Analytical Manual](https://www.fda.gov/food/laboratory-methods-food/bacteriological-analytical-manual-bam). U.S. Food and Drug Administration. [5] [Antimicrobial Susceptibility Testing](https://clsi.org/standards/products/microbiology). Clinical and Laboratory Standards Institute. > This article is educational and does not replace validated laboratory procedures, institutional biosafety review, manufacturer instructions, or professional interpretation.