How to Perform an Optochin Susceptibility Test: Principle and Protocol

The optochin susceptibility test is a biochemical method used to differentiate Streptococcus pneumoniae from other alpha-hemolytic streptococci (viridans group streptococci) based on the organism's sensitivity to ethylhydrocupreine hydrochloride (optochin). This test is performed by placing an optochin-impregnated disk on a lawn of the test organism on a blood agar plate; after incubation, a zone of inhibition around the disk indicates susceptibility. The test is most useful in routine microbiology laboratories for the presumptive identification of S. pneumoniae from clinical or environmental samples, serving as a rapid and cost-effective alternative to more complex molecular or serological methods. While optochin susceptibility is a cornerstone of pneumococcal identification, it must be interpreted alongside colony morphology, Gram stain results, and bile solubility testing for definitive identification.

At a Glance

Scientific Principle

Optochin (ethylhydrocupreine hydrochloride) is a quinine derivative that selectively inhibits Streptococcus pneumoniae by interfering with the organism's ATP synthase and membrane potential. The compound binds to the F₀F₁ ATPase complex, disrupting proton translocation and energy production. This mechanism is particularly effective against S. pneumoniae due to specific structural features of its ATP synthase that are not shared by most other alpha-hemolytic streptococci. The result is a clear zone of growth inhibition surrounding the optochin disk on solid media.

The test exploits the differential susceptibility of alpha-hemolytic streptococci: S. pneumoniae is uniformly susceptible to optochin, while viridans group streptococci (e.g., S. mitis, S. oralis, S. sanguinis) are typically resistant. This differential susceptibility has been validated through decades of clinical microbiology practice and is supported by external quality assurance programs that monitor laboratory performance in pneumococcal identification [1][2]. The SIREVA project, a Pan American Health Organization initiative, demonstrated that standardized optochin testing, when combined with serotyping and antibiotic susceptibility testing, achieves high accuracy in pneumococcal identification across international laboratory networks [1].

Materials and Instrumentation

Essential Materials

• Optochin disks: Commercially available disks impregnated with 5 µg of optochin (ethylhydrocupreine hydrochloride). Disks should be stored at 2–8°C in a sealed desiccated container to maintain potency. Allow disks to reach room temperature before opening to prevent condensation.
• Blood agar plates: Tryptic soy agar supplemented with 5% defibrinated sheep blood. Plates should be fresh (prepared within 7–14 days) and free from contamination. The quality of the blood agar is critical; hemolyzed or aged plates may produce inconsistent zone sizes.
• Inoculating loop: Sterile, disposable plastic loops (1 µL or 10 µL) or calibrated nichrome loops.
• Sterile saline or broth: For preparing a standardized bacterial suspension (0.5 McFarland standard).
• McFarland turbidity standard: 0.5 McFarland standard (approximately 1.5 × 10⁸ CFU/mL) or a nephelometer for precise standardization.
• Forceps: Sterile forceps for placing disks onto inoculated plates.
• Incubator: Capable of maintaining 35–37°C with 5–10% CO₂ (candle jar or CO₂ incubator).
• Ruler or caliper: For measuring zone diameters to the nearest millimeter.

Quality Control Strains

• Positive control (susceptible): Streptococcus pneumoniae ATCC 49619. This strain should produce a zone of inhibition ≥14 mm with a 5 µg optochin disk.
• Negative control (resistant): Streptococcus mitis ATCC 49456 or Streptococcus oralis ATCC 35037. These strains should produce no zone or a zone <14 mm.

Instrumentation Considerations

The choice of incubator affects test results. S. pneumoniae is capnophilic (requires increased CO₂), and incubation in ambient air may yield smaller or absent zones of inhibition. A CO₂ incubator set to 5–10% CO₂ is ideal, but a candle jar (which produces approximately 3–5% CO₂) is acceptable for teaching laboratories. The incubation temperature must be tightly controlled; temperatures above 37°C may inhibit growth of S. pneumoniae, while temperatures below 35°C may slow growth and produce falsely small zones.

Controls

Positive Control

The positive control strain (S. pneumoniae ATCC 49619) must be tested with each batch of optochin disks and whenever a new lot number is introduced. The expected zone diameter is ≥14 mm. If the zone is smaller than expected, the disks may have degraded, the inoculum may be too heavy, or the incubation conditions may be suboptimal. Document the zone diameter for each control run.

Negative Control

The negative control strain (e.g., S. mitis ATCC 49456) should demonstrate resistance (zone <14 mm or no zone). This control confirms that the optochin disk is not nonspecifically inhibiting all alpha-hemolytic streptococci. If the negative control shows a zone ≥14 mm, the disks may be contaminated with another antimicrobial agent, or the strain may have been misidentified.

Sterility Control

Uninoculated blood agar plates should be incubated alongside test plates to verify that the medium is sterile. Any growth on sterility controls invalidates the test results.

Inoculum Density Control

The inoculum density must be standardized to 0.5 McFarland. An overly dense inoculum can produce falsely small zones, while a light inoculum can produce falsely large zones. Use a nephelometer or visual comparison against a McFarland standard. For teaching laboratories, a visual approximation is acceptable but should be verified periodically against a calibrated standard.

Conceptual Workflow

Step 1: Specimen Preparation and Inoculum Standardization

1. Select a well-isolated colony of the test organism from a pure culture (18–24 hours old) on blood agar.
2. Emulsify the colony in 2–3 mL of sterile saline or broth to achieve a turbidity equivalent to 0.5 McFarland standard.
3. Vortex the suspension thoroughly to ensure homogeneity.

Step 2: Inoculation of Blood Agar Plate

1. Using a sterile swab, dip into the standardized suspension and rotate against the tube wall to remove excess liquid.
2. Streak the swab evenly across the entire surface of a blood agar plate in three directions (rotating the plate approximately 60° each time) to ensure confluent growth.
3. Allow the plate surface to dry for 3–5 minutes at room temperature. Do not leave the plate open longer than necessary to avoid contamination.

Step 3: Application of Optochin Disk

1. Using sterile forceps, aseptically remove an optochin disk from the container.
2. Place the disk firmly onto the inoculated agar surface, pressing gently to ensure full contact.
3. Do not move the disk once placed; repositioning can create false zones of inhibition.
4. For multiple isolates on one plate, place disks at least 24 mm apart (center to center) to prevent overlapping zones.

Step 4: Incubation

1. Invert the plate and incubate at 35–37°C in 5–10% CO₂ for 18–24 hours.
2. Do not stack plates more than four high to ensure uniform temperature and gas exchange.
3. Record the incubation start time and temperature.

Step 5: Reading and Interpretation

1. After incubation, examine the plate for a zone of inhibition around the optochin disk.
2. Measure the zone diameter (including the disk) to the nearest millimeter using a ruler or caliper held against the bottom of the plate.
3. Hold the plate up to transmitted light to visualize the zone edge clearly. The zone edge is defined as the point where there is no visible growth; do not include faint haze or satellite colonies.
4. Record the zone diameter and interpret according to established criteria.

Quality Checks

Pre-Test Quality Checks

• Verify the expiration date of optochin disks. Expired disks may have reduced potency.
• Confirm that blood agar plates are free from contamination, cracks, or excessive moisture.
• Check that the incubator temperature and CO₂ levels are within acceptable ranges (35–37°C, 5–10% CO₂).
• Ensure that control strains are viable and have been subcultured within the past 24–48 hours.

During-Test Quality Checks

• Monitor the inoculum density; if the suspension appears too turbid or too light, adjust and re-standardize.
• Verify that the disk adheres to the agar surface; a loose disk may not release optochin properly.
• Document the time of inoculation and incubation start.

Post-Test Quality Checks

• Compare control strain results to expected values. If controls fail, repeat the test with fresh disks and media.
• Examine the plate for contamination (colonies with different morphology or hemolysis patterns).
• Confirm that the test organism is alpha-hemolytic and Gram-positive before interpreting the optochin result.

Result Interpretation

Zone Diameter Criteria

Using a 5 µg optochin disk on blood agar incubated in 5–10% CO₂ at 35–37°C for 18–24 hours:

Interpretation Notes

• Susceptible result (≥14 mm): The organism is presumptively identified as Streptococcus pneumoniae. However, rare optochin-susceptible viridans group streptococci (e.g., some strains of S. mitis and S. oralis) have been reported. Confirmatory testing (bile solubility, bile esculin, or molecular methods) should be performed for definitive identification, especially in clinical settings.
• Resistant result (<14 mm): The organism is likely a viridans group streptococcus. However, optochin-resistant S. pneumoniae strains have been documented, particularly in regions with high antibiotic use. If colony morphology and Gram stain strongly suggest S. pneumoniae (draughtsman-like colonies, Gram-positive diplococci), perform bile solubility testing or molecular confirmation.
• No zone: Complete resistance. The organism is not S. pneumoniae unless confirmed by other methods.

Edge Cases

• Hazy zone: A faint haze of growth within the zone may indicate partial inhibition. Measure from the edge of confluent growth. If the haze extends to the disk, interpret as resistant.
• Satellite colonies: Small colonies within the zone may indicate contamination or a mixed culture. Subculture and retest.
• Double zone: A clear inner zone with a hazy outer zone may occur with some strains. Measure the inner clear zone; if ≥14 mm, interpret as susceptible.

Troubleshooting

Limitations

False Susceptibility (False Positive for S. pneumoniae)

Some viridans group streptococci, particularly Streptococcus mitis and Streptococcus oralis, can exhibit optochin susceptibility, producing zones ≥14 mm. This cross-reactivity is well-documented and can lead to misidentification if optochin susceptibility is used as the sole criterion. The frequency of optochin-susceptible viridans streptococci varies by geographic region and patient population. In a study of European reference laboratories, discrepancies in pneumococcal identification were observed in 7.1% of isolates tested across 22 EQA panels, highlighting the need for confirmatory testing [2].

False Resistance (False Negative for S. pneumoniae)

Optochin-resistant S. pneumoniae strains have been reported globally, with prevalence rates ranging from 1–5% in some regions. These strains typically have mutations in the ATP synthase genes that confer optochin resistance while retaining other pneumococcal characteristics. Resistance can also develop through laboratory passage or storage. If a strain is suspected to be S. pneumoniae based on colony morphology (draughtsman-like, mucoid colonies) and Gram stain (Gram-positive diplococci), but is optochin-resistant, perform bile solubility testing or bile esculin testing.

Technical Limitations

• Disk potency: Optochin disks lose potency over time, especially if stored improperly. Always check expiration dates and store disks at 2–8°C in a desiccated container.
• Medium composition: Blood agar from different manufacturers may produce slightly different zone sizes. Laboratories should establish their own zone diameter criteria if using non-standard media.
• CO₂ concentration: Incubation in ambient air (without supplemental CO₂) can reduce zone sizes and increase false resistance rates. Always incubate in 5–10% CO₂.
• Inoculum density: Failure to standardize the inoculum to 0.5 McFarland is the most common cause of inconsistent results.

Confirmatory Testing

Optochin susceptibility should never be used alone for definitive identification of S. pneumoniae. The following confirmatory tests are recommended:

• Bile solubility test: S. pneumoniae is bile soluble (lysed by bile salts), while viridans group streptococci are resistant.
• Bile esculin test: S. pneumoniae is bile esculin negative; enterococci and group D streptococci are positive.
• Molecular methods: 16S rRNA gene sequencing, PCR targeting the lytA gene, or whole-genome sequencing provide definitive identification [4].
• Serotyping: Capsular typing using the Quellung reaction or latex agglutination confirms S. pneumoniae and provides epidemiological information [1][2].

Documentation

Required Records

For each optochin susceptibility test performed, document the following:

1. Test date and time
2. Technician initials
3. Specimen or isolate identifier
4. Source of isolate (if applicable)
5. Optochin disk lot number and expiration date
6. Blood agar plate lot number and expiration date
7. Incubation conditions (temperature, CO₂ concentration, duration)
8. Inoculum density (McFarland standard value)
9. Zone diameter (to nearest millimeter)
10. Interpretation (susceptible or resistant)
11. Control results (positive and negative control zone diameters)
12. Any deviations from standard protocol

Quality Assurance Records

Maintain a log of all quality control results, including:

• Weekly positive and negative control results
• New lot verification results (test each new lot of disks against controls)
• Incubator temperature and CO₂ monitoring records
• Corrective actions taken when controls fail

Reporting

Results should be reported as:

• "Presumptive Streptococcus pneumoniae" (if susceptible and colony morphology consistent)
• "Not Streptococcus pneumoniae" (if resistant)
• "Optochin-susceptible alpha-hemolytic streptococcus" (if susceptible but colony morphology atypical; recommend confirmatory testing)

Biosafety Considerations

Risk Assessment

Streptococcus pneumoniae is classified as a Risk Group 2 (RG2) pathogen by the CDC and NIH [5]. While the optochin test itself is performed on isolated colonies from pure culture, the original clinical specimens may contain viable S. pneumoniae and other potential pathogens. All work with clinical specimens should be performed at Biosafety Level 2 (BSL-2).

BSL-1 Teaching Laboratory Scope

For teaching laboratories using known, non-pathogenic strains (e.g., ATCC strains), the optochin test can be performed at BSL-1 with the following precautions:

• Use only well-characterized, non-pathogenic control strains.
• Perform all work in a clean, uncluttered laboratory area.
• Wear laboratory coats and gloves.
• Decontaminate work surfaces before and after use with 10% bleach or 70% ethanol.
• Dispose of all contaminated materials in biohazard waste containers.
• Do not use sharps (e.g., glass Pasteur pipettes) unless necessary.

BSL-2 Clinical Laboratory Requirements

When testing clinical isolates or potentially pathogenic strains:

• Perform all manipulations in a Class II biological safety cabinet (BSC).
• Wear appropriate personal protective equipment (PPE): laboratory coat, gloves, and eye protection.
• Use aerosol-resistant centrifuge tubes if centrifugation is required.
• Decontaminate all waste by autoclaving before disposal.
• Follow institutional biosafety committee (IBC) guidelines for RG2 work [5][6].

Decontamination

• All plates, swabs, and disposable loops must be autoclaved at 121°C for 30 minutes before disposal.
• Reusable equipment (forceps, glassware) should be autoclaved or disinfected with 10% bleach for 30 minutes.
• Work surfaces should be decontaminated daily and after any spill.

Spill Procedure

In case of a spill:

1. Alert others in the area.
2. Cover the spill with absorbent material (paper towels).
3. Apply 10% bleach or 1% sodium hypochlorite solution around the edges, then over the spill.
4. Allow 30 minutes contact time.
5. Clean up using fresh absorbent material and dispose in biohazard waste.
6. Decontaminate the area again with fresh disinfectant.

Frequently Asked Questions

1. Can I use optochin disks that have been stored at room temperature?

No. Optochin disks must be stored at 2–8°C in a sealed, desiccated container to maintain potency. Room temperature storage accelerates degradation, leading to reduced zone sizes and false resistance. If disks have been accidentally left at room temperature for more than 24 hours, they should be discarded and replaced with a fresh lot. Always allow refrigerated disks to reach room temperature (approximately 30 minutes) before opening the container to prevent condensation, which can also degrade the disks.

2. Why do I need to incubate in CO₂? Can I use a regular incubator?

Streptococcus pneumoniae is capnophilic and requires elevated CO₂ for optimal growth. Incubation in ambient air (approximately 0.04% CO₂) results in poor growth, smaller colonies, and reduced zone sizes. This can lead to false resistance (zone <14 mm) even for true S. pneumoniae isolates. If a CO₂ incubator is unavailable, use a candle jar (which produces 3–5% CO₂) or a CO₂-generating sachet in a sealed container. Do not use a regular incubator without supplemental CO₂ for optochin testing.

3. My test organism shows a zone of 13 mm. Is it resistant or susceptible?

A zone of 13 mm is below the 14 mm cutoff and should be interpreted as resistant (not S. pneumoniae). However, this result should be interpreted with caution. First, verify that the inoculum was standardized to 0.5 McFarland and that incubation conditions were correct (35–37°C, 5–10% CO₂). If the colony morphology is typical of S. pneumoniae (draughtsman-like, mucoid, Gram-positive diplococci), perform a bile solubility test. Bile-soluble organisms are S. pneumoniae even if optochin-resistant. Some laboratories use a 12 mm cutoff for equivocal results, but the CLSI-recommended cutoff is 14 mm.

4. Can I use the optochin test for identification of S. pneumoniae from mixed cultures?

No. The optochin test requires a pure culture of the test organism. Mixed cultures can produce confusing results, including satellite colonies within the zone of inhibition or overlapping zones from different species. Always subculture to purity before performing the optochin test. If the original specimen shows alpha-hemolytic colonies on blood agar, pick a single, well-isolated colony and streak for isolation before testing. This ensures that the optochin result reflects the susceptibility of a single organism.

References and Further Reading

1. Lovgren M, Talbot JA, Brandileone MC, et al. Evolution of an international external quality assurance model to support laboratory investigation of Streptococcus pneumoniae, developed for the SIREVA project in Latin America, from 1993 to 2005. J Clin Microbiol. 2007. PubMed

2. Slotved HC, Sheppard CL, Dalby T, et al. External Quality Assurance for Laboratory Identification and Capsular Typing of Streptococcus pneumoniae. J Clin Microbiol. 2017. PubMed

3. Mwaturura T, Olaru ID, Chimhini G, et al. Rapid bacterial identification and resistance detection using a low complexity molecular diagnostic platform in Zimbabwe. J Clin Microbiol. 2025. PubMed

4. Gajic I, Jovicevic M, Kekic D, et al. Evolving Approaches to Bacterial Identification: A Review of Classical and Modern Techniques. Microorganisms. 2026. PubMed

5. CDC and NIH. Biosafety in Microbiological and Biomedical Laboratories (BMBL), 6th Edition. U.S. Department of Health and Human Services, 2020. CDC

6. National Institutes of Health. NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules. NIH Office of Science Policy

7. National Center for Biotechnology Information. NCBI Bookshelf: Molecular Biology and Laboratory Methods. NCBI Bookshelf

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