How to Perform a Bacitracin Susceptibility Test: Principle and Protocol

The bacitracin susceptibility test is a disk-diffusion method used to presumptively identify Streptococcus pyogenes (Group A Streptococcus, GAS) based on its characteristic sensitivity to the antibiotic bacitracin. This test is most useful in clinical and teaching microbiology laboratories as a rapid, low-cost screening tool to differentiate S. pyogenes from other beta-hemolytic streptococci, particularly Groups B, C, and G, which are typically resistant to bacitracin. The test involves placing a filter-paper disk impregnated with a standardized concentration of bacitracin onto an agar plate inoculated with the test organism; after incubation, a zone of growth inhibition around the disk indicates susceptibility, supporting identification of S. pyogenes.

At a Glance

Scientific Principle

Bacitracin is a polypeptide antibiotic produced by Bacillus licheniformis that inhibits bacterial cell wall synthesis by interfering with the dephosphorylation of the lipid carrier (bactoprenol) that transports peptidoglycan precursors across the cytoplasmic membrane. This action prevents the incorporation of N-acetylmuramic acid and N-acetylglucosamine into the growing cell wall, leading to osmotic lysis of susceptible bacteria.

The differential susceptibility of Streptococcus pyogenes to bacitracin was first described in the 1940s and has since become a cornerstone of presumptive streptococcal identification. Approximately 95–99% of S. pyogenes isolates demonstrate susceptibility to low concentrations of bacitracin (0.04 units per disk), while most other beta-hemolytic streptococci—including Streptococcus agalactiae (Group B), Streptococcus equisimilis (Groups C and G), and Streptococcus anginosus group—are resistant at this concentration.

The mechanism underlying this differential susceptibility is not fully understood but is thought to relate to differences in cell wall composition or permeability among streptococcal species. Importantly, bacitracin resistance has been documented in some S. pyogenes isolates, and conversely, occasional susceptibility has been observed in non-Group A streptococci. Therefore, the bacitracin test is considered a presumptive screening tool and should always be confirmed with definitive serological grouping (Lancefield typing) or other confirmatory tests such as the PYR test.

Materials and Instrumentation Choices

Agar Medium

Sheep blood agar (SBA) is the standard medium for bacitracin susceptibility testing. The choice of blood source matters: sheep blood is preferred because it lacks the NADase activity present in horse blood that can inhibit the growth of some fastidious streptococci. Human blood should never be used due to the risk of bloodborne pathogen transmission and the presence of antibodies that may inhibit bacterial growth.

Commercial SBA plates are available from multiple manufacturers (e.g., Becton Dickinson, Hardy Diagnostics, bioMérieux). Plates should be stored at 2–8°C and used within their expiration date. Before inoculation, allow plates to warm to room temperature to prevent condensation that could interfere with disk diffusion.

Alternative media considerations: Mueller-Hinton agar supplemented with 5% sheep blood can be used for standardized disk diffusion testing, but for routine bacitracin screening, standard SBA is adequate and more commonly used in teaching laboratories.

Bacitracin Disks

Commercially prepared bacitracin disks contain 0.04 units of bacitracin per disk. This low concentration is critical for differential testing; higher concentrations (e.g., 10 units) are used for other applications and would not provide the same discriminatory power.

Disks should be stored at 2–8°C in a sealed desiccated container to maintain potency. Allow disks to reach room temperature before opening the container to prevent condensation. Never use expired disks, as antibiotic degradation can lead to false-resistant results.

Inoculation Tools

• Sterile cotton swabs or inoculating loops
• Sterile saline or broth for suspension preparation (if using direct colony suspension method)
• McFarland turbidity standard (0.5 McFarland) for inoculum standardization
• Sterile forceps or disk dispenser for placing disks

Incubation Conditions

Streptococci grow optimally at 35–37°C in an atmosphere enriched with 5–10% CO₂. A CO₂ incubator is ideal, but a candle jar (a sealed jar with a burning candle that consumes oxygen and produces CO₂) is an acceptable alternative for teaching laboratories. Incubation without CO₂ may result in poor growth and unreliable zone interpretation.

Quality Control

Quality control (QC) is essential to ensure that the bacitracin disks, medium, and test conditions are performing correctly. QC should be performed:

• With each new lot of disks or medium
• At least weekly when the test is performed routinely
• Whenever test results are unexpected or questionable

Recommended QC Strains

QC Procedure

1. Inoculate SBA plates with QC strains using the same method as for test organisms.
2. Apply bacitracin disks and incubate under the same conditions.
3. After 18–24 hours, verify that S. pyogenes shows a clear zone of inhibition and S. agalactiae shows no zone.
4. Document QC results in the laboratory records.

If QC strains fail to produce expected results, investigate potential causes: expired disks, contaminated medium, incorrect incubation conditions, or degraded QC strains. Do not report patient or teaching sample results until QC is acceptable.

Conceptual Workflow

Step 1: Specimen Preparation and Inoculum Standardization

The test requires a pure culture of the organism to be identified. For teaching laboratories, this is typically an 18–24 hour culture on SBA showing beta-hemolysis (complete clearing around colonies).

Inoculum preparation methods:

Method A – Direct colony suspension (preferred):

1. Using a sterile loop or swab, pick 3–5 well-isolated colonies of similar morphology.
2. Suspend the colonies in 2–3 mL of sterile saline or broth.
3. Adjust the turbidity to match a 0.5 McFarland standard (approximately 1–2 × 10⁸ CFU/mL).
4. Use within 15 minutes of preparation.

Method B – Direct swab from growth (acceptable for teaching labs):

1. Using a sterile swab, touch 3–5 colonies from the pure culture.
2. Dip the swab into sterile saline and rotate against the tube wall to release organisms.
3. Press the swab against the tube wall to remove excess fluid.

Why standardization matters: An overly heavy inoculum can produce false-resistant results (the bacteria overwhelm the antibiotic), while a light inoculum can produce false-susceptible results (the zone appears larger than it should). The 0.5 McFarland standard provides reproducible results.

Step 2: Inoculation of the Agar Plate

1. Dip a sterile cotton swab into the standardized inoculum suspension.
2. Rotate the swab against the tube wall to remove excess fluid.
3. Streak the swab evenly across the entire surface of the SBA plate in three directions (horizontal, vertical, diagonal) to ensure confluent growth.
4. Rotate the plate 60 degrees between each streaking direction.
5. Allow the plate surface to dry for 3–5 minutes (lid slightly ajar) before applying disks.

Why this technique matters: Confluent, even growth is necessary for accurate zone measurement. Patchy growth can produce irregular zones that are difficult to interpret.

Step 3: Application of the Bacitracin Disk

1. Using sterile forceps or a disk dispenser, aseptically place one bacitracin disk onto the inoculated agar surface.
2. Gently press the disk down with the forceps tip to ensure full contact with the agar.
3. Position the disk at least 15 mm from the edge of the plate and at least 20 mm from any other disks if performing multiple tests on the same plate.
4. For teaching purposes, a single bacitracin disk per plate is simplest, but multiple disks (e.g., bacitracin, optochin, novobiocin) can be placed on the same plate if they are sufficiently separated.

Disk placement considerations: If testing multiple antibiotics on one plate, ensure disks are spaced to prevent overlapping zones of inhibition. For streptococci, common combinations include bacitracin and optochin (for differentiating S. pyogenes from S. pneumoniae).

Step 4: Incubation

1. Invert the plate and place it in a CO₂ incubator (5% CO₂) or candle jar at 35–37°C.
2. Incubate for 18–24 hours.
3. Do not stack plates more than 4 high to ensure uniform temperature and gas exchange.

Why CO₂ is important: Streptococci are capnophilic (CO₂-loving) organisms. Incubation in ambient air may result in poor or no growth, making zone interpretation impossible.

Step 5: Reading and Interpreting Results

After incubation, examine the plate for a zone of inhibition around the bacitracin disk.

Interpretation criteria:

Important note: Unlike the Kirby-Bauer method for other antibiotics, there are no standardized zone diameter breakpoints for bacitracin. The test is interpreted qualitatively: any zone = susceptible; no zone = resistant. Some laboratories use a cutoff of ≥10 mm for susceptibility, but this is a laboratory-specific convention, not a universal standard.

Reading tips:

• Hold the plate against a dark background with transmitted light to visualize zones clearly.
• Measure the zone diameter (including the disk) using a ruler or caliper if desired for documentation.
• Ignore pinpoint colonies within the zone; these may represent resistant subpopulations or contaminants.
• If growth is not confluent, the test should be repeated.

Result Interpretation and Confirmatory Testing

Presumptive Identification

A bacitracin-susceptible, beta-hemolytic, catalase-negative, Gram-positive coccus in chains is presumptively identified as Streptococcus pyogenes (Group A Streptococcus). However, definitive identification requires additional testing.

Confirmatory Tests

The bacitracin test should be used in conjunction with other tests for accurate identification:

The PYR test is particularly useful because it is rapid (2–4 minutes), inexpensive, and highly specific for S. pyogenes among beta-hemolytic streptococci. A PYR-positive, bacitracin-susceptible isolate provides strong evidence for S. pyogenes identification.

Limitations of the Bacitracin Test

1. Not all S. pyogenes are susceptible: Approximately 1–5% of S. pyogenes isolates are bacitracin-resistant. These strains would be missed by the bacitracin test alone.

2. Some non-Group A streptococci are susceptible: Occasional isolates of Groups B, C, G, and other streptococci may show bacitracin susceptibility, leading to false-positive identification.

3. Test conditions affect results: Variations in medium, inoculum size, incubation atmosphere, and disk potency can all influence results.

4. Not a substitute for serological grouping: The bacitracin test is a screening tool, not a definitive identification method. Lancefield grouping (by latex agglutination or other methods) remains the gold standard.

Troubleshooting

Limitations

Specificity and Sensitivity

The bacitracin test has reported sensitivity of 95–99% and specificity of 80–95% for identifying S. pyogenes, depending on the population studied and the test conditions. These values mean that approximately 1–5% of true S. pyogenes isolates will be missed (false negatives), and 5–20% of non-Group A streptococci will be incorrectly identified as S. pyogenes (false positives).

Geographic and Temporal Variation

Bacitracin susceptibility patterns can vary geographically and over time. Some regions have reported higher rates of bacitracin resistance among S. pyogenes isolates, potentially due to antibiotic pressure or clonal spread of resistant strains. Laboratories should be aware of local epidemiology when interpreting results.

Alternative Identification Methods

Several alternative or supplementary methods exist for identifying S. pyogenes:

• PYR test: Rapid (2–4 minutes), highly specific, and can be performed directly from colony growth.
• Lancefield serogrouping: The gold standard, using latex agglutination or coagglutination to detect Group A carbohydrate antigen.
• MALDI-TOF MS: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry provides rapid, accurate species-level identification.
• Molecular methods: PCR-based detection of S. pyogenes-specific genes (e.g., speB, emm) offers definitive identification.

When Not to Use the Bacitracin Test

• For non-hemolytic or alpha-hemolytic streptococci (the test is validated only for beta-hemolytic isolates)
• For isolates from sterile sites where definitive identification is critical (e.g., blood cultures, CSF)
• When the isolate shows ambiguous hemolysis or colony morphology
• In outbreak investigations where strain-level typing is needed

Documentation

Proper documentation is essential for reproducibility, quality assurance, and educational purposes. For each bacitracin test performed, record the following information:

Required Documentation Elements

1. Sample information:

   • Sample identifier (e.g., culture number, student ID)
   • Source of isolate (if known)
   • Date of testing

2. Test conditions:

   • Medium type and lot number
   • Bacitracin disk lot number and expiration date
   • Incubation temperature and atmosphere
   • Incubation duration

3. Results:

   • Zone diameter (if measured)
   • Interpretation (susceptible/resistant)
   • Photograph of plate (recommended for teaching records)

4. Quality control:

   • QC strains used and their results
   • Date of QC testing
   • Any QC failures and corrective actions taken

5. Interpretation and follow-up:

   • Presumptive identification
   • Confirmatory tests performed and results
   • Final identification

Example Documentation Template

Bacitracin Susceptibility Test Record
Date: _______________
Technician: _______________

Sample ID: _______________
Organism description: Gram-positive cocci, beta-hemolytic, catalase-negative

Medium: TSA + 5% sheep blood, Lot #: _______________
Bacitracin disk: 0.04 units, Lot #: _______________, Exp: _______________
Incubation: 35°C, 5% CO₂, 24 hours

Zone diameter: _______________ mm
Interpretation: [ ] Susceptible [ ] Resistant

QC results:
S. pyogenes ATCC 19615: [ ] Susceptible [ ] Resistant
S. agalactiae ATCC 13813: [ ] Susceptible [ ] Resistant

Presumptive identification: _______________
Confirmatory tests: PYR [ ] Positive [ ] Negative
Final identification: _______________

Biosafety Considerations

Risk Assessment

The bacitracin susceptibility test is routinely performed with beta-hemolytic streptococci, which are classified as Biosafety Level 2 (BSL-2) agents when handling clinical isolates. However, for teaching laboratories using well-characterized, non-pathogenic strains (e.g., ATCC strains), BSL-1 practices are appropriate.

BSL-1 practices (teaching laboratory with ATCC strains):

• Standard microbiological practices
• Hand washing after handling cultures
• No eating, drinking, or applying cosmetics in the laboratory
• Decontamination of work surfaces daily and after spills
• Proper waste disposal (autoclaving of all contaminated materials)

BSL-2 practices (clinical isolates or unknown samples):

• All BSL-1 practices, plus:
• Restricted access to the laboratory
• Use of personal protective equipment (lab coat, gloves, eye protection)
• Biosafety cabinet for procedures that may generate aerosols
• Sharps disposal for any glass or sharp items
• Specific training for personnel

Safe Work Practices

1. Work in a biosafety cabinet when handling clinical isolates or performing procedures that may generate aerosols (e.g., opening culture tubes, preparing suspensions).

2. Use proper personal protective equipment: Lab coat, gloves, and safety glasses are minimum requirements.

3. Decontaminate work surfaces before and after each procedure with an appropriate disinfectant (e.g., 10% bleach solution, 70% ethanol, or a commercial disinfectant validated for the organisms in use).

4. Dispose of all contaminated materials by autoclaving. This includes used agar plates, swabs, pipette tips, and any other materials that have contacted the culture.

5. Never recap needles if used; place sharps directly into a puncture-resistant sharps container.

6. Report all spills and exposures immediately to the laboratory supervisor.

Decontamination

All cultures and contaminated materials must be autoclaved at 121°C for at least 30 minutes before disposal. Liquid cultures should be autoclaved in containers that can withstand the pressure and temperature. Agar plates should be placed in biohazard bags and autoclaved.

For surface decontamination, 10% bleach (0.5% sodium hypochlorite) with a 10-minute contact time is effective against streptococci. Alternatively, 70% ethanol can be used for surfaces that may be damaged by bleach.

Frequently Asked Questions

1. Can I use the bacitracin test for identifying streptococci from throat cultures?

Yes, the bacitracin test is commonly used for presumptive identification of S. pyogenes from throat cultures. However, it should be performed only on beta-hemolytic, catalase-negative, Gram-positive cocci that have been isolated in pure culture. Direct testing of mixed cultures from throat swabs is not recommended because other bacteria may interfere with zone interpretation. Always subculture to obtain isolated colonies before testing.

2. Why do some S. pyogenes isolates show resistance to bacitracin?

Bacitracin resistance in S. pyogenes can occur through several mechanisms, including mutations in genes involved in cell wall synthesis (e.g., mprF or bacA), acquisition of resistance genes, or changes in cell wall permeability. The prevalence of bacitracin-resistant S. pyogenes varies geographically but is generally low (1–5%). Some studies have reported higher resistance rates in certain regions or among specific strain types. For example, research on Staphylococcus aureus has documented bacitracin resistance in animal isolates, though the mechanisms may differ from those in streptococci [1].

3. How does the bacitracin test compare to the PYR test for identifying S. pyogenes?

The PYR test (pyrrolidonyl arylamidase) is generally considered superior to the bacitracin test for identifying S. pyogenes. The PYR test is rapid (2–4 minutes), highly sensitive (>99%), and highly specific (>99%) for S. pyogenes among beta-hemolytic streptococci. In contrast, the bacitracin test requires 18–24 hours of incubation and has lower specificity (80–95%). Many laboratories use the PYR test as the primary screening method and reserve the bacitracin test for confirmation or for teaching purposes. However, the bacitracin test remains useful because it is inexpensive, does not require special reagents, and provides a visual demonstration of antibiotic susceptibility.

4. What should I do if my bacitracin test gives an ambiguous result (e.g., a very small or hazy zone)?

An ambiguous result should be investigated before reporting. First, check the quality control results to ensure the test system is functioning correctly. If QC is acceptable, repeat the test with careful attention to inoculum standardization (0.5 McFarland) and incubation conditions (CO₂ atmosphere). If the ambiguous result persists, consider performing a PYR test or Lancefield grouping for definitive identification. A small zone may indicate weak susceptibility, which could be a true property of the isolate or an artifact of test conditions. In teaching laboratories, ambiguous results provide excellent learning opportunities for discussing test limitations and the importance of confirmatory testing.

References and Further Reading

1. Marked Presence of Methicillin-Resistant Staphylococcus aureus in Wild Lagomorphs in Valencia, Spain. Moreno-Grúa E, Pérez-Fuentes S, Viana D, et al. (2020). This study documents bacitracin resistance in S. aureus isolates from wild rabbits, illustrating that bacitracin resistance can occur in Gram-positive cocci and may be more common in animal reservoirs than previously recognized. PubMed

2. Rapid bacterial identification and resistance detection using a low complexity molecular diagnostic platform in Zimbabwe. Mwaturura T, Olaru ID, Chimhini G, et al. (2025). This validation study compares molecular diagnostic methods to conventional phenotypic testing for blood culture isolates, demonstrating the ongoing importance of culture-based methods alongside molecular approaches. PubMed

3. Biosafety in Microbiological and Biomedical Laboratories (BMBL), 6th Edition. CDC and NIH (2020). The authoritative U.S. reference for biosafety practices in microbiological laboratories, including risk assessment, containment levels, and decontamination procedures relevant to handling streptococci. CDC

4. NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules. National Institutes of Health. Provides the institutional framework for biosafety and biosecurity in research settings, including guidance on risk assessment for microbiological procedures. NIH

5. NCBI Bookshelf: Molecular Biology and Laboratory Methods. National Center for Biotechnology Information. A searchable collection of authoritative biomedical references and methods protocols, including background on streptococcal identification and antibiotic susceptibility testing. NCBI

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