Positive and Negative Controls in Antibiotic Susceptibility Testing (Kirby-Bauer)

The Kirby-Bauer disk diffusion method is a standardized, qualitative antimicrobial susceptibility test (AST) that uses antibiotic-impregnated paper disks on an agar plate inoculated with a bacterial lawn. Positive and negative control strains—such as Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923—are essential to validate that the test system is functioning correctly, that the disks contain the stated antibiotic concentration, and that the measured zone diameters can be interpreted against established clinical breakpoints. This method is useful for clinical microbiology laboratories, teaching laboratories, and research settings where rapid, cost-effective screening of bacterial resistance patterns is needed before confirmatory minimum inhibitory concentration (MIC) testing.

At a Glance

Scientific Principle of Disk Diffusion

The Kirby-Bauer method relies on the diffusion of an antibiotic from a paper disk into solid agar medium. As the antibiotic concentration decreases with distance from the disk, a gradient is established. Bacterial growth is inhibited where the antibiotic concentration exceeds the minimum inhibitory concentration for that organism. The resulting zone of inhibition—measured in millimeters—correlates inversely with the organism's susceptibility: larger zones indicate greater susceptibility, while smaller or absent zones indicate resistance.

The test is performed on Mueller-Hinton agar (MHA), which provides consistent cation concentrations (calcium and magnesium) and low levels of thymidine and thymine, ensuring reproducible diffusion and growth conditions. The inoculum is standardized to a 0.5 McFarland turbidity standard (approximately 1–2 × 10⁸ CFU/mL for E. coli), and plates are incubated at 35 ± 2°C for 16–18 hours in ambient air.

Control strains serve as internal calibrators. Their known susceptibility patterns confirm that the medium, disks, incubation conditions, and operator technique are all within acceptable limits. Without these controls, a zone diameter that appears "large" or "small" cannot be reliably interpreted—it might reflect true susceptibility, disk deterioration, or an inoculum error.

Materials and Instrumentation Choices

Agar Medium

Mueller-Hinton agar is the standard medium for disk diffusion. It must be prepared according to the manufacturer's instructions, with a depth of 4 mm (approximately 25 mL per 100 mm plate). The pH should be 7.2–7.4 at room temperature. Commercial dehydrated MHA is acceptable, but each lot should be tested for performance with control strains before routine use. Some fastidious organisms (e.g., Streptococcus pneumoniae, Haemophilus influenzae) require supplemented MHA (e.g., with 5% sheep blood or 1% hemoglobin and 1% IsoVitaleX), but these are beyond the BSL-1 scope of this article.

Antibiotic Disks

Commercially prepared disks are preferred over in-house prepared disks because they have validated potency and stability. Disks should be stored at 2–8°C in sealed containers with desiccant. Disks for beta-lactam antibiotics (e.g., penicillins, cephalosporins) are particularly sensitive to moisture and should be brought to room temperature before opening to prevent condensation. Each disk is labeled with the antibiotic name and concentration (e.g., "CIP 5 µg" for ciprofloxacin). Expired disks must not be used.

Inoculum Standardization

A 0.5 McFarland turbidity standard is required. Commercial standards are available, or a barium sulfate standard can be prepared (0.5 mL of 1% BaCl₂ added to 99.5 mL of 1% H₂SO₄). The standard must be vortexed before each use and replaced every 6 months. A spectrophotometer can also be used to adjust the inoculum to an optical density of 0.08–0.1 at 625 nm.

Incubator

A standard microbiological incubator set to 35 ± 2°C is sufficient. Temperature should be monitored daily with a calibrated thermometer. For most non-fastidious organisms, ambient air is used; S. pneumoniae and H. influenzae require 5% CO₂.

Control Strains: Selection and Rationale

Control strains are well-characterized bacterial isolates with known, reproducible susceptibility patterns. They are obtained from national culture collections such as the American Type Culture Collection (ATCC) or equivalent. The most commonly used strains for routine disk diffusion are:

• Escherichia coli ATCC 25922: A broad-spectrum control for Gram-negative antibiotics. It is susceptible to most agents, including ampicillin, ciprofloxacin, gentamicin, and ceftriaxone. Its zone diameters are published in CLSI M100.
• Staphylococcus aureus ATCC 25923: A control for Gram-positive antibiotics. It is susceptible to oxacillin, vancomycin, and linezolid, but resistant to penicillin G (due to beta-lactamase production). This strain is also used to test cefoxitin as a surrogate for methicillin resistance.
• Pseudomonas aeruginosa ATCC 27853: A control for anti-pseudomonal agents such as ceftazidime, ciprofloxacin, and meropenem. It is intrinsically resistant to many antibiotics, so its zone diameters are narrower than those of E. coli ATCC 25922.
• Enterococcus faecalis ATCC 29212: Used for testing vancomycin and high-level aminoglycoside resistance.

For teaching laboratories operating at BSL-1, only non-pathogenic control strains should be used. ATCC 25922 and ATCC 25923 are classified as BSL-1 organisms when handled by trained personnel using standard aseptic technique. Clinical isolates from patient specimens are BSL-2 and require additional containment.

Positive Control

A positive control is a strain that is known to be susceptible to the antibiotics being tested. It confirms that the disk contains active antibiotic, that the medium supports growth, and that the incubation conditions are appropriate. For example, E. coli ATCC 25922 should produce a zone of 30–36 mm for ciprofloxacin (5 µg disk). If the zone is smaller than 30 mm, the disk may have lost potency, the inoculum may be too heavy, or the agar depth may be excessive.

Negative Control

A negative control consists of a sterile blank disk (no antibiotic) placed on the inoculated plate. No zone of inhibition should appear around the blank disk. If a zone is observed, it indicates contamination of the disk or the agar, or the presence of an inhibitory substance (e.g., residual disinfectant on the forceps). The negative control also verifies that the bacterial lawn is confluent and that any zones seen around antibiotic disks are due to the antibiotic, not to physical inhibition by the disk itself.

Quality Control Frequency

CLSI recommends that QC testing be performed each day the test is performed, or at least weekly if the laboratory can document consistent performance. If a new lot of MHA, disks, or a new batch of saline is introduced, QC must be performed before routine use. Results should be recorded in a QC log and reviewed for trends.

Conceptual Workflow

1. Prepare inoculum: Using a sterile loop, pick 3–5 isolated colonies of the control strain from an 18–24 hour agar plate. Suspend in 3–5 mL of sterile saline or Mueller-Hinton broth. Vortex and adjust to 0.5 McFarland.
2. Inoculate plate: Within 15 minutes of adjusting the inoculum, dip a sterile cotton swab into the suspension, rotate against the tube wall to remove excess fluid, and streak the entire surface of the MHA plate in three directions to ensure confluent growth.
3. Apply disks: Using sterile forceps or a disk dispenser, place antibiotic disks onto the inoculated agar. Press gently to ensure contact. For the negative control, place a blank disk. Space disks at least 24 mm apart (center to center) to prevent overlapping zones.
4. Incubate: Invert plates and incubate at 35 ± 2°C for 16–18 hours. Do not stack more than 5 plates high to ensure uniform temperature.
5. Measure zones: Using a ruler or caliper, measure the diameter of each zone of inhibition to the nearest millimeter. Measure from the edge of the disk to the edge of visible growth. For swarming organisms (e.g., Proteus), measure the inner zone where growth is completely inhibited.
6. Interpret: Compare measured zones to CLSI QC ranges. If all zones fall within the acceptable range, the test is valid. If any zone is outside the range, investigate and repeat.

Quality Checks and Acceptance Criteria

The following quality checks must be performed before reporting results from clinical isolates:

• Inoculum check: The 0.5 McFarland standard should be verified periodically by comparing to a spectrophotometric reading or by subculturing a loopful of the suspension to confirm colony count.
• Medium check: Each new lot of MHA should be tested for pH, depth, and performance with control strains. Plates with visible cracks, bubbles, or uneven surfaces should be discarded.
• Disk check: Disks should be inspected for discoloration, brittleness, or moisture. Expired disks must not be used.
• Incubation check: Temperature logs should show 35 ± 2°C throughout the incubation period.
• Control strain check: Control strains should be subcultured weekly from frozen stocks to maintain purity. Stock cultures should be stored at -70°C or in lyophilized form.

Acceptance criteria for control strains are published in CLSI M100 (Table 4A for E. coli ATCC 25922, Table 4B for S. aureus ATCC 25923, etc.). For example, for E. coli ATCC 25922 tested with ampicillin (10 µg disk), the acceptable zone range is 16–22 mm. If the measured zone is 15 mm or 23 mm, the test is out of control and must be investigated.

Result Interpretation

Once the control strains produce zones within the acceptable ranges, the test results for clinical isolates can be interpreted. Zone diameters are compared to CLSI breakpoints, which categorize isolates as Susceptible (S), Intermediate (I), or Resistant (R). For example, for ciprofloxacin (5 µg disk) against Enterobacteriaceae, a zone ≥ 26 mm is susceptible, 22–25 mm is intermediate, and ≤ 21 mm is resistant.

It is critical to note that zone diameters are not directly equivalent to MIC values. A large zone does not guarantee a low MIC, and a small zone does not guarantee a high MIC. However, the correlation is generally good for most organism-drug combinations. When discrepancies arise (e.g., a zone suggests susceptibility but the patient fails therapy), MIC testing by broth microdilution or gradient strip should be performed.

Troubleshooting

Limitations

The Kirby-Bauer method has several important limitations:

• Qualitative only: It provides S/I/R categorization but not an exact MIC. For drugs with narrow therapeutic windows (e.g., vancomycin), MIC testing is preferred.
• Not suitable for all organisms: Fastidious organisms (e.g., Neisseria gonorrhoeae, H. influenzae) require specialized media and incubation conditions. Anaerobes cannot be tested by disk diffusion.
• Disk potency: Disks have a fixed concentration, which may not reflect achievable serum levels. For example, a 30 µg disk of vancomycin may overestimate susceptibility compared to MIC testing.
• Subjectivity: Zone measurement can vary between operators. Automated zone readers improve reproducibility but are not universally available.
• No detection of heteroresistance: Subpopulations of resistant cells within a susceptible population may not be detected.
• No detection of inducible resistance: For example, inducible clindamycin resistance in S. aureus requires a D-zone test, not standard disk diffusion.

Documentation and Record Keeping

Each QC test must be documented in a permanent log that includes:

• Date of test
• Control strain name and ATCC number
• Antibiotic disks tested (name and concentration)
• Measured zone diameters (mm)
• Acceptance range from CLSI
• Pass/fail status
• Operator initials
• Corrective action if out of range

For clinical isolates, the following should be recorded:

• Patient identifier
• Specimen type and source
• Organism identification
• Antibiotic tested and zone diameter
• Interpretation (S/I/R)
• Date and operator

Records should be retained according to institutional policy, typically for at least 2–3 years for teaching laboratories and longer for clinical or research settings.

Biosafety Considerations

All work with bacterial cultures should be performed in a BSL-1 or BSL-2 laboratory, depending on the organisms used. Control strains such as E. coli ATCC 25922 and S. aureus ATCC 25923 are BSL-1 and can be handled on an open bench with standard aseptic technique. However, clinical isolates from patient specimens are BSL-2 and require:

• Use of a biological safety cabinet (BSC) for all manipulations that may generate aerosols (e.g., vortexing, pipetting)
• Personal protective equipment (PPE): lab coat, gloves, eye protection
• Decontamination of all waste by autoclaving before disposal
• Restricted access to the laboratory during work

The CDC and NIH Biosafety in Microbiological and Biomedical Laboratories (BMBL), 6th Edition provides comprehensive guidance on risk assessment and containment for microbiological work [6]. For laboratories using recombinant or synthetic nucleic acid molecules (e.g., engineered control strains), the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules must be followed [7].

Frequently Asked Questions

1. Can I use a single control strain for all antibiotics? No. Different control strains are needed for Gram-positive and Gram-negative antibiotics. E. coli ATCC 25922 is suitable for most Gram-negative agents, but S. aureus ATCC 25923 is required for Gram-positive agents such as oxacillin, vancomycin, and linezolid. P. aeruginosa ATCC 27853 is needed for anti-pseudomonal agents. Using the wrong control strain may produce zones that are out of range even when the test is valid.

2. What should I do if my control strain zone is out of range? First, check for obvious errors: disk expiration, incorrect incubation temperature, or wrong inoculum density. Repeat the test with fresh materials. If the zone remains out of range, test a new lot of MHA and new disks. If the problem persists, contact the manufacturer of the disks or medium. Document all corrective actions in the QC log.

3. How often should I replace my 0.5 McFarland standard? Commercial turbidity standards should be replaced according to the manufacturer's instructions, typically every 6–12 months. Homemade barium sulfate standards should be replaced every 6 months. The standard should be vortexed before each use and checked periodically against a spectrophotometric reading.

4. Can I use the same control strain for both disk diffusion and MIC testing? Yes, but the control strain requirements may differ. For broth microdilution MIC testing, E. coli ATCC 25922 and S. aureus ATCC 29213 are commonly used. S. aureus ATCC 29213 is preferred over ATCC 25923 for MIC testing because its MIC values are more reproducible. Always consult the relevant CLSI standard (M07 for MIC, M02 for disk diffusion) for the appropriate control strains.

References and Further Reading

1. Garedew A, Ayele L. Bacteria isolates and antimicrobial resistance patterns in burn wound infections: a cross-sectional study at two hospitals. 2026. PubMed ID: 42035017. Link – Demonstrates use of Kirby-Bauer method following CLSI guidelines in a clinical study.
2. Dipa RA, Khalil I, Hossen MS, et al. Cross-sectoral antimicrobial resistance patterns of Escherichia coli across human, animal, and environmental interfaces in southern Bangladesh. 2026. PubMed ID: 42344343. Link – Illustrates disk diffusion testing of E. coli from multiple sources.
3. Chhetri AK, Dhakal HP, Gurung B, et al. Detection of hypervirulence genes in carbapenem resistant Klebsiella pneumoniae from cancer patients at a tertiary referral hospital in Nepal. 2026. PubMed ID: 42026492. Link – Uses Kirby-Bauer method for carbapenem-resistant K. pneumoniae.
4. Shakhatreh MAK, Atawneh FH, Swedan SF, et al. Vaginal Colonization by Streptococcus agalactiae Among Pregnant Women in Jordan: Antimicrobial Resistance, Virulence Genes, and Biofilm Formation. 2026. PubMed ID: 42164746. Link – Applies Kirby-Bauer method to S. agalactiae.
5. Singh U, Jain P, Singhai A, et al. Exploring the Genetic and Morphological Basis of Biofilm-Linked Drug Resistance in Clinical Isolates of Acinetobacter baumannii. 2026. PubMed ID: 42317929. Link – Uses Kirby-Bauer method for A. baumannii.
6. CDC and NIH. Biosafety in Microbiological and Biomedical Laboratories (BMBL), 6th Edition. U.S. Department of Health and Human Services, 2020. Link – Authoritative biosafety guidance.
7. National Institutes of Health. NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules. Link – Biosafety framework for recombinant work.
8. National Center for Biotechnology Information. NCBI Bookshelf: Molecular Biology and Laboratory Methods. Link – Searchable collection of methods references.

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