Quality Control Strains for Antimicrobial Susceptibility Testing: Selection and Maintenance

Quality control (QC) strains for antimicrobial susceptibility testing (AST) are well-characterized bacterial reference strains, typically from the American Type Culture Collection (ATCC) or equivalent culture collections, used to verify that AST reagents, media, and procedures perform within established acceptable ranges. These strains are essential for ensuring the accuracy and reproducibility of disk diffusion and broth microdilution minimum inhibitory concentration (MIC) tests. QC strains are used routinely—daily, weekly, or with each new lot of media or antimicrobial disks—to monitor test performance, detect systematic errors, and validate results before reporting patient or surveillance isolate data. They are not substitutes for clinical isolates but serve as internal controls that confirm the testing system is functioning correctly.

At a Glance: QC Strains for AST

Scientific Principle: Why QC Strains Are Necessary for AST

Antimicrobial susceptibility testing relies on standardized conditions—inoculum density, medium composition, incubation temperature and atmosphere, and antimicrobial concentration—to produce reproducible results. Even minor deviations in any of these parameters can shift zone diameters or MIC values by one or more doubling dilutions, potentially causing false-susceptible or false-resistant results [5]. QC strains serve as biological calibrators that detect such deviations.

The principle underlying QC strain use is that a well-characterized reference organism, when tested under defined conditions, should yield results within a narrow, pre-established acceptable range. For disk diffusion, these ranges are zone diameter limits (in millimeters) published by the Clinical and Laboratory Standards Institute (CLSI) in tables M100. For broth microdilution, QC ranges are expressed as MIC values (in μg/mL). If the QC strain result falls outside the acceptable range, the entire test run is invalid, and results cannot be reported.

This approach is grounded in decades of multi-laboratory collaborative studies that established the expected performance of each QC strain against each antimicrobial agent. The CLSI M100 tables are updated annually to reflect new data, new antimicrobials, and revised breakpoints. Using QC strains is not optional—it is a fundamental requirement for any laboratory performing AST for clinical, surveillance, or research purposes.

Selecting QC Strains: Which Strains and Why

Core QC Strains for Routine AST

The most widely used QC strains for disk diffusion and broth microdilution are:

• Escherichia coli ATCC 25922: A broad-spectrum Gram-negative control. It is susceptible to most clinically relevant antibiotics and is used for β-lactams, aminoglycosides, fluoroquinolones, tetracyclines, and folate pathway inhibitors. It does not produce extended-spectrum β-lactamases (ESBLs) or carbapenemases, making it suitable for verifying that the test system correctly identifies susceptible phenotypes [1].

• Staphylococcus aureus ATCC 25923: The primary Gram-positive control for disk diffusion. It is methicillin-susceptible and produces β-lactamase. It is used for penicillins, cephalosporins, macrolides, lincosamides, aminoglycosides, and fluoroquinolones.

• Pseudomonas aeruginosa ATCC 27853: A non-fermenting Gram-negative control. It is intrinsically resistant to many antibiotics but susceptible to anti-pseudomonal agents. It is essential for testing aminoglycosides, carbapenems, cephalosporins, and fluoroquinolones against this organism group.

• Enterococcus faecalis ATCC 29212: Used primarily for broth microdilution MIC testing of enterococci. It is susceptible to ampicillin and vancomycin and is used to verify high-level aminoglycoside resistance screening tests.

• Staphylococcus aureus ATCC 29213: The MIC counterpart to ATCC 25923, used for broth microdilution testing of staphylococci.

Additional QC Strains for Specialized Testing

Depending on the laboratory's scope, additional QC strains may be required:

• Escherichia coli ATCC 35218: Produces TEM-1 β-lactamase and is used to test β-lactam/β-lactamase inhibitor combinations (e.g., amoxicillin-clavulanate, piperacillin-tazobactam). It should not be used for cephalosporin testing.

• Haemophilus influenzae ATCC 49247 and ATCC 49766: Used for testing fastidious organisms requiring supplemented media and CO₂ incubation.

• Streptococcus pneumoniae ATCC 49619: Used for testing pneumococci in cation-adjusted Mueller-Hinton broth with lysed horse blood.

• Neisseria gonorrhoeae ATCC 49226: Used for testing gonococci on GC agar base with defined supplements.

Selection Criteria

When selecting QC strains for your laboratory, consider:

1. Test method: Disk diffusion and broth microdilution may require different QC strains (e.g., S. aureus ATCC 25923 for disk diffusion vs. ATCC 29213 for MIC).
2. Antimicrobial panel: Ensure the QC strain covers all antimicrobial agents in your testing panel.
3. Organism groups tested: Include QC strains representative of the major organism groups (Enterobacterales, staphylococci, enterococci, non-fermenters, fastidious organisms).
4. Regulatory requirements: Follow CLSI M100, EUCAST, or local regulatory standards for mandatory QC strains.

Materials and Instrumentation Choices

Culture Media

• Mueller-Hinton agar (MHA): Standard medium for disk diffusion. Use commercially prepared, quality-controlled plates. For fastidious organisms, use MHA with 5% defibrinated sheep blood (for streptococci) or Haemophilus test medium (for H. influenzae).
• Cation-adjusted Mueller-Hinton broth (CAMHB): Standard for broth microdilution. Cation adjustment (calcium and magnesium) is critical because these ions affect aminoglycoside and tetracycline activity.
• Media depth: For disk diffusion, pour MHA to a depth of 4 mm (approximately 25 mL in a 100 mm plate). Too-thin or too-thick agar alters zone diameters.

Antimicrobial Disks and Panels

• Disk potency: Use commercially prepared disks with specified drug concentrations. Store disks at 2–8°C in sealed containers with desiccant. Allow disks to warm to room temperature before opening to prevent condensation.
• MIC panels: Use commercially prepared frozen or dried panels, or prepare in-house using certified antimicrobial powders. In-house preparation requires analytical balance calibration, solubility verification, and sterility testing.

Inoculum Preparation

• Turbidity standard: 0.5 McFarland standard (equivalent to approximately 1.5 × 10⁸ CFU/mL for E. coli). Use a calibrated nephelometer or photometric device. Visual comparison to a barium sulfate standard is acceptable but less precise.
• Saline or broth: Use sterile 0.85% saline or Mueller-Hinton broth for suspension preparation. Do not use nutrient broth or other rich media that may alter growth characteristics.

Incubation

• Temperature: 35 ± 1°C for routine isolates. Some QC strains (e.g., P. aeruginosa ATCC 27853) may require 35°C; avoid 37°C for certain tests.
• Atmosphere: Ambient air for most non-fastidious organisms. 5% CO₂ for fastidious organisms (streptococci, H. influenzae, N. gonorrhoeae).
• Duration: 16–18 hours for disk diffusion; 16–20 hours for broth microdilution. Do not over-incubate, as this can cause zone shrinkage or trailing endpoints.

Controls: What to Include in Each Test Run

Internal Controls

Every AST run must include at least one QC strain appropriate for the organism group and antimicrobial panel being tested. For a typical run testing Enterobacterales, include E. coli ATCC 25922. For staphylococci, include S. aureus ATCC 25923 (disk diffusion) or ATCC 29213 (MIC).

Frequency of QC Testing

• Daily or per test batch: Test QC strains on each day that patient or surveillance isolates are tested. If multiple batches are run on the same day, test QC with each batch.
• New reagent lots: Test QC strains with each new lot of MHA, CAMHB, antimicrobial disks, or MIC panels before reporting results.
• After equipment maintenance: Test QC after recalibration of nephelometers, pipettes, or automated AST systems.

Acceptance Criteria

• Disk diffusion: Zone diameters for each QC strain/antimicrobial combination must fall within the CLSI-published acceptable range. If any value is out of range, the run is invalid.
• Broth microdilution: MIC values must fall within the acceptable range (typically ± one doubling dilution of the expected mode). Out-of-range results invalidate the run.

External Quality Assessment

Participation in external quality assessment (EQA) programs, such as those described in the WHO Tricycle-aligned Fleming Fund EQASIA program, provides independent verification of laboratory performance [1]. EQA samples are distributed as simulated clinical or food matrices containing well-characterized resistant strains. Laboratories perform AST and report results, which are compared to expected outcomes. EQA participation is recommended for all laboratories performing AST, particularly those involved in antimicrobial resistance surveillance.

Conceptual Workflow: From Reference Stock to QC Testing

Step 1: Obtain and Verify Reference Strains

Acquire QC strains from a recognized culture collection (ATCC, NCTC, DSMZ). Upon receipt, verify the strain identity by Gram stain, colony morphology, and key biochemical tests. For E. coli ATCC 25922, confirm lactose fermentation on MacConkey agar and indole positivity. For S. aureus ATCC 25923, confirm catalase positivity, coagulase positivity, and mannitol fermentation.

Step 2: Prepare Reference Stocks

• Lyophilized cultures: Rehydrate according to the supplier's instructions. Subculture onto non-selective agar (e.g., Tryptic Soy Agar with 5% sheep blood) and incubate overnight.
• Frozen stocks: Prepare multiple vials of each QC strain in cryopreservative (e.g., Tryptic Soy Broth with 15% glycerol). Freeze at -70°C or colder. Label each vial with strain name, ATCC number, date, and passage number.
• Working stocks: From a frozen reference stock, subculture once onto agar to obtain isolated colonies. Use this plate for up to one week if stored at 2–8°C. Do not subculture more than five times from the original reference stock.

Step 3: Prepare Inoculum

• From an overnight agar plate (18–24 hours), pick 3–5 well-isolated colonies of identical morphology.
• Suspend in 3–5 mL of sterile saline or Mueller-Hinton broth.
• Vortex thoroughly and adjust turbidity to 0.5 McFarland using a nephelometer.
• For broth microdilution, dilute the adjusted suspension 1:100 in CAMHB to achieve approximately 5 × 10⁵ CFU/mL in the test well.

Step 4: Inoculate and Apply Disks (Disk Diffusion)

• Within 15 minutes of adjusting the inoculum, dip a sterile cotton swab into the suspension, rotate against the tube wall to remove excess liquid, and streak the entire MHA plate surface in three directions to ensure confluent growth.
• Allow the plate surface to dry for 3–5 minutes (no longer than 15 minutes).
• Apply antimicrobial disks using a disk dispenser or sterile forceps. Press each disk firmly onto the agar surface. Do not move a disk once applied.
• Invert plates and incubate at 35 ± 1°C for 16–18 hours.

Step 5: Read and Record Results

• Measure zone diameters (including the disk) to the nearest millimeter using calipers or a ruler on the back of the plate. Read at the point of complete inhibition as judged by the naked eye.
• For broth microdilution, read MIC as the lowest concentration showing no visible growth.
• Record results on a QC log sheet or in the laboratory information system.

Step 6: Evaluate Against Acceptable Ranges

• Compare each zone diameter or MIC value to the CLSI-published QC range.
• If all values are within range, the run is acceptable, and patient/surveillance results can be reported.
• If any value is out of range, investigate and repeat the QC test before reporting results.

Quality Checks and Troubleshooting

Common QC Failures and Corrective Actions

Systematic Approach to Out-of-Range Results

1. Repeat the QC test using a fresh inoculum from the same working stock.
2. If still out of range, prepare a new inoculum from a different colony.
3. If still out of range, subculture from the frozen reference stock (not the working stock).
4. If still out of range, open a new vial of the QC strain.
5. If still out of range, check all reagents (MHA lot, disk lot, saline, McFarland standard).
6. Document all corrective actions and results in the QC log.

Limitations of QC Strain Testing

QC Strains Do Not Represent All Resistance Mechanisms

Standard QC strains are susceptible to most antibiotics and do not carry acquired resistance genes such as ESBLs, AmpC β-lactamases, or carbapenemases [1]. They cannot verify that the test system correctly detects resistance. For this purpose, laboratories may use additional challenge strains (e.g., K. pneumoniae ATCC 700603 for ESBL detection, E. coli ATCC 35218 for inhibitor combinations). However, these are not substitutes for routine QC strains.

QC Ranges Are Method- and Medium-Specific

QC ranges published by CLSI are established using specific media (e.g., BD BBL Mueller-Hinton II agar). Different manufacturers' media may produce slightly different zone diameters, even if they meet CLSI specifications. Laboratories should verify that their medium supports acceptable QC performance before routine use.

Passage Number Affects Performance

Repeated subculture can select for genetic variants with altered susceptibility. CLSI recommends limiting subcultures to no more than five from the original reference stock. Laboratories must track passage numbers and replace working stocks regularly.

Environmental Factors Influence Results

Recent research demonstrates that environmental factors such as pH and temperature can significantly affect β-lactamase activity and MIC values [3]. For example, CTX-M-15 and CMY-2 β-lactamases show different activity at pH 5.5 versus pH 7.4. While standard AST is performed at neutral pH, these findings highlight that QC results may not fully predict test performance under all conditions.

QC Testing Does Not Replace Proficiency Testing

Routine QC testing verifies day-to-day consistency but does not assess the laboratory's ability to correctly identify resistance phenotypes in unknown isolates. External quality assessment programs, such as the EQASIA scheme, provide independent evaluation of laboratory performance and should be used in conjunction with internal QC [1].

Documentation and Record Keeping

Essential Records

• QC strain log: Strain name, ATCC number, source, date received, date of reference stock preparation, passage number, and expiration date.
• Daily QC results: Date, test method, QC strain, antimicrobial agent, observed zone diameter or MIC, acceptable range, and pass/fail status.
• Corrective action records: Date of failure, investigation findings, corrective action taken, and resolution.
• Reagent lot records: Lot numbers and expiration dates for MHA, CAMHB, disks, and MIC panels. QC results for each new lot.
• Equipment maintenance records: Calibration dates for nephelometers, pipettes, refrigerators, freezers, and incubators.

Retention Period

Retain QC records for at least the period specified by your accreditation body (typically 2–5 years). For research laboratories, retain records for the duration of the study plus any required archival period.

Biosafety Considerations

Standard QC strains (E. coli ATCC 25922, S. aureus ATCC 25923, P. aeruginosa ATCC 27853, E. faecalis ATCC 29212) are classified as Biosafety Level 1 (BSL-1) agents. They pose minimal risk to healthy adults and can be handled on the open bench using standard microbiological practices [6].

However, laboratories that also handle clinical isolates must follow BSL-2 practices when processing patient specimens. QC strains should be handled separately from clinical isolates to avoid cross-contamination. Use dedicated media, pipettes, and work surfaces for QC testing when possible.

Key biosafety practices for QC strain work include:

• Perform all manipulations in a biosafety cabinet if clinical isolates are processed in the same laboratory.
• Decontaminate work surfaces before and after QC testing with 10% bleach or an appropriate disinfectant.
• Autoclave all waste (plates, swabs, pipette tips) before disposal.
• Wear laboratory coats and gloves. Wash hands after removing gloves.
• Do not eat, drink, or apply cosmetics in the laboratory.

For laboratories working with recombinant or synthetic nucleic acid molecules (e.g., engineered QC strains), follow the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules [7].

Frequently Asked Questions

1. Can I use the same QC strain for both disk diffusion and broth microdilution? Yes, but you may need different ATCC numbers for the same species. For example, S. aureus ATCC 25923 is the standard for disk diffusion, while S. aureus ATCC 29213 is used for broth microdilution MIC testing. Check CLSI M100 tables for the correct strain for each method.

2. How often should I replace my QC strain working stock? Replace working stocks weekly if stored at 2–8°C. Limit subcultures to no more than five from the original reference stock. Replace frozen reference stocks every 2–3 years or according to the culture collection's recommendations.

3. What should I do if my QC strain results are consistently at the edge of the acceptable range? Consistently borderline results may indicate a systematic issue with media, disks, or technique. Check the age and storage conditions of your reagents. Verify your McFarland standard calibration. Consider testing a different lot of MHA or disks. Document all findings and consult CLSI M100 for guidance.

4. Can I prepare my own QC strains from clinical isolates? No. QC strains must be well-characterized reference strains from a recognized culture collection. Clinical isolates have unknown genetic backgrounds and may carry resistance genes that compromise their utility as controls. Using uncharacterized isolates invalidates QC testing and may lead to erroneous patient results.

References and Further Reading

1. Insights from the WHO Tricycle-aligned Fleming Fund EQASIA External Quality Assessment (EQA) programme
2. Next generation sequencing as a panacea for antibiotic susceptibility testing: yea or nay?
3. Contrasting pH optima of β-lactamases CTX-M and CMY influence Escherichia coli fitness and resistance ecology
4. Genomic and phenotypic characterization of plasmid-mediated extensively drug-resistant Salmonella Typhi from Lahore Pakistan
5. Evaluation of a Novel Rapid Phenotypic Antimicrobial Susceptibility Testing System
6. Biosafety in Microbiological and Biomedical Laboratories (BMBL), 6th Edition
7. NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules
8. NCBI Bookshelf: Molecular Biology and Laboratory Methods

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