Optochin Susceptibility Test for Streptococcus pneumoniae Identification

The optochin susceptibility test is a disk diffusion method used to differentiate Streptococcus pneumoniae from other alpha-hemolytic (viridans group) streptococci based on the organism's susceptibility to ethylhydrocupreine hydrochloride (optochin). When an optochin-impregnated disk is placed on a lawn of S. pneumoniae on blood agar, a zone of inhibition ≥14 mm in diameter indicates susceptibility and supports identification as S. pneumoniae, while resistant isolates (zone <14 mm) are likely other viridans streptococci. This test is most useful as a primary identification tool in clinical and teaching laboratories when combined with colony morphology, Gram stain characteristics (Gram-positive cocci in pairs or chains), and alpha-hemolysis on blood agar. The optochin test should not be used as a standalone method; it is most reliable when interpreted alongside the bile solubility test, which provides confirmatory evidence for S. pneumoniae identification.

At a Glance

Scientific Principle

Optochin (ethylhydrocupreine hydrochloride) selectively inhibits S. pneumoniae by interfering with ATP synthase function. The compound binds to the F₀F₁ ATP synthase complex, specifically targeting the c subunit encoded by the atpC gene, and disrupts proton translocation across the bacterial membrane. This inhibition of ATP synthesis leads to bacterial growth arrest. Other alpha-hemolytic streptococci, such as Streptococcus mitis, Streptococcus oralis, and Streptococcus sanguinis, possess ATP synthase complexes with structural differences that render them naturally resistant to optochin at the standard disk concentration.

The genetic basis for optochin susceptibility and resistance has been characterized through whole-genome sequencing studies. Research examining paired optochin-susceptible and optochin-resistant S. pneumoniae isolates from patients with invasive pneumococcal disease identified point mutations in ATP synthase genes as the primary mechanism of acquired resistance [4]. Among ten resistant isolates studied, seven carried mutations in the atpC gene and one in the atpA gene, with one previously unreported mutation at position 143T>C resulting in a Val48Ala amino acid change [4]. These mutations alter the optochin binding site without affecting the organism's serotype or multilocus sequence type, meaning resistant variants remain true S. pneumoniae despite failing the optochin test.

The test exploits this differential susceptibility by measuring the zone of growth inhibition around an optochin-impregnated disk placed on a lawn of the test organism. The diameter of the inhibition zone correlates with the organism's susceptibility level and provides a standardized criterion for identification.

Materials and Instrumentation Choices

Optochin Disks

Commercially prepared optochin disks contain 5 µg of ethylhydrocupreine hydrochloride per disk. Disk selection requires attention to manufacturer specifications and expiration dates. Disks should be stored at 2–8°C in sealed containers with desiccant to maintain potency. Before use, allow disks to equilibrate to room temperature for approximately 30 minutes to prevent condensation that could affect drug diffusion. Never use disks beyond their expiration date, as degraded optochin may produce falsely small or absent zones of inhibition.

Culture Medium

Sheep blood agar (5% defibrinated sheep blood in tryptic soy agar base) is the standard medium for optochin testing. The medium must support adequate growth of alpha-hemolytic streptococci while allowing proper diffusion of optochin. Key considerations include:

• Agar depth: Pour plates to a uniform depth of 4 mm (approximately 25 mL per 100 mm plate). Deeper agar reduces drug diffusion and produces smaller zones; shallower agar increases diffusion and produces larger zones.
• Blood source: Sheep blood is preferred because it does not support the growth of Haemophilus species that might confuse interpretation. Avoid horse blood, which contains inhibitors that can affect streptococcal growth.
• Supplementation: Do not add supplements that alter the medium pH, as optochin activity is pH-dependent and most effective at neutral to slightly alkaline pH.

Inoculum Preparation

Prepare a bacterial suspension equivalent to a 0.5 McFarland standard. This turbidity standard corresponds to approximately 1.5 × 10⁸ colony-forming units per milliliter. Options for standardization include:

• Commercial turbidity standards: Pre-calibrated barium sulfate or latex particle standards provide reliable reference points.
• Spectrophotometric adjustment: Measure absorbance at 625 nm; a 0.5 McFarland standard typically gives an absorbance of 0.08–0.10.
• Visual comparison: For teaching laboratories without instrumentation, compare the suspension against a certified McFarland standard under good lighting against a white background with black lines.

Incubation Conditions

Incubate plates at 35 ± 2°C in 5% CO₂ for 18–24 hours. Carbon dioxide incubation is critical because S. pneumoniae is capnophilic (requires elevated CO₂ for optimal growth). Without CO₂ supplementation, growth may be insufficient to produce clear zone edges, leading to inaccurate measurements. Alternative methods include candle jars (which produce approximately 3–4% CO₂) or CO₂-generating sachets in sealed containers.

Controls

Positive Control

Use S. pneumoniae ATCC 49619, which is known to be optochin-susceptible and produces a zone of inhibition ≥14 mm. This control confirms that the optochin disk is potent and that the test conditions (medium, incubation, inoculum) are appropriate. If the positive control fails to produce an adequate zone, do not report results from test isolates until the issue is resolved.

Negative Control

Use an optochin-resistant alpha-hemolytic streptococcus such as S. mitis ATCC 49456 or S. oralis ATCC 35037. This control demonstrates that the test can correctly identify resistant organisms and that the medium does not contain substances that artificially inhibit growth. A negative control that shows a zone ≥14 mm indicates a problem with the disk, medium, or incubation conditions.

Sterility Control

Include an uninoculated blood agar plate incubated alongside test plates to verify that the medium and incubation environment are free from contaminating microorganisms.

Conceptual Workflow

Step 1: Colony Selection

Select 3–5 well-isolated colonies from an 18–24 hour culture on blood agar. Examine colony morphology: S. pneumoniae typically appears as small, gray, mucoid colonies with a central depression (draughtsman or "squashed" appearance) due to autolysis. Colonies are surrounded by a zone of alpha-hemolysis (greenish discoloration). Avoid selecting colonies from mixed cultures or colonies that appear contaminated.

Step 2: Inoculum Preparation

Emulsify selected colonies in 2–3 mL of sterile saline or tryptic soy broth to achieve a turbidity matching the 0.5 McFarland standard. Vortex the suspension thoroughly to ensure even distribution. If the suspension is too turbid, add more diluent; if too light, add more colonies. Overly heavy inocula produce smaller zones and may cause false resistance; overly light inocula produce larger zones and may cause false susceptibility.

Step 3: Inoculation

Within 15 minutes of preparing the suspension, dip a sterile cotton swab into the suspension and rotate it against the tube wall to remove excess liquid. Streak the swab evenly across the entire surface of a sheep blood agar plate in three directions (rotating the plate approximately 60° between each streaking) to ensure confluent growth. Allow the plate surface to dry for 3–5 minutes with the lid slightly ajar.

Step 4: Disk Application

Using sterile forceps or a disk dispenser, place an optochin disk firmly onto the inoculated agar surface. Press gently to ensure full contact with the agar. Do not move the disk once placed, as this creates a zone of disrupted growth that complicates measurement. Place the disk at least 15 mm from the plate edge and at least 24 mm from other disks if performing multiple tests on the same plate.

Step 5: Incubation

Invert the plate and incubate at 35 ± 2°C in 5% CO₂ for 18–24 hours. Do not stack plates more than four high, as uneven heating can affect growth and zone sizes.

Step 6: Measurement

After incubation, measure the diameter of the zone of complete inhibition (including the disk diameter) using a ruler, caliper, or automated zone reader. Hold the plate approximately 30 cm from the eye and measure across the widest diameter of the zone. For accurate measurement, view the plate against a dark background with reflected light. Record the measurement in millimeters.

Quality Checks

Zone Measurement Precision

Measure zones to the nearest whole millimeter. If the zone edge is irregular, measure the diameter in two perpendicular directions and record the average. For zones with faint growth within the inhibition area, measure to the point where growth is clearly reduced by 80% or more.

Timing of Reading

Read plates at exactly 18–24 hours. Premature reading (before 18 hours) may show incomplete growth and falsely large zones. Delayed reading (after 24 hours) may show secondary growth within the zone due to resistant subpopulations or contaminating organisms.

Confirmation of Alpha-Hemolysis

Before interpreting the optochin result, confirm that the test organism produces alpha-hemolysis on blood agar. Beta-hemolytic streptococci and non-hemolytic organisms should not be tested with optochin, as the test is validated only for alpha-hemolytic isolates.

Bile Solubility Confirmation

For isolates showing optochin susceptibility (zone ≥14 mm), perform a bile solubility test as confirmatory identification. The bile solubility test uses sodium desoxycholate to lyse S. pneumoniae cells by activating autolytic enzymes. Published evaluations of modified bile solubility testing report rapid performance in selected clinical-laboratory settings, but the result should be interpreted within the validated workflow and confirmed according to local identification policy [1].

Result Interpretation

Susceptible (Positive for S. pneumoniae)

A zone of inhibition ≥14 mm in diameter indicates optochin susceptibility and supports identification as S. pneumoniae. The likelihood that an optochin-susceptible alpha-hemolytic streptococcus is S. pneumoniae exceeds 95% when combined with typical colony morphology and Gram stain characteristics.

Resistant (Negative for S. pneumoniae)

A zone of inhibition <14 mm indicates optochin resistance. While most optochin-resistant alpha-hemolytic streptococci are not S. pneumoniae, rare optochin-resistant S. pneumoniae strains exist due to mutations in ATP synthase genes [4]. These resistant variants retain their pathogenic potential and serotype characteristics despite failing the optochin test. Therefore, any isolate with strong clinical suspicion for S. pneumoniae (e.g., from cerebrospinal fluid or blood in a patient with pneumonia) that is optochin-resistant should undergo bile solubility testing or molecular identification.

Borderline Results

Zones measuring 12–13 mm are considered borderline and require repeat testing and confirmatory methods. Possible causes include:

• Suboptimal inoculum density
• Degraded optochin disk
• Mixed culture containing both susceptible and resistant organisms
• Atypical S. pneumoniae strain with partial resistance

Troubleshooting

Limitations

False Susceptibility

Some non-pneumococcal alpha-hemolytic streptococci, particularly S. mitis and S. oralis, can occasionally show optochin susceptibility with zones ≥14 mm. This cross-reactivity occurs because these species share genetic relatedness with S. pneumoniae and may possess similar ATP synthase structures. The bile solubility test is essential to distinguish true S. pneumoniae from these optochin-susceptible viridans streptococci.

False Resistance

Optochin-resistant S. pneumoniae strains exist and are increasingly recognized through genomic surveillance. A study of invasive pneumococcal disease isolates found that 8 of 10 optochin-resistant S. pneumoniae isolates carried point mutations in atpC or atpA genes [4]. These resistant strains are not detected by the optochin test alone and require alternative identification methods.

Medium and Condition Dependence

Zone sizes vary with agar depth, blood source, incubation atmosphere, and inoculum density. Laboratories must establish their own zone diameter breakpoints if using non-standard conditions. The 14 mm breakpoint is validated for 5% sheep blood agar incubated in 5% CO₂ for 18–24 hours.

Not for Direct Specimen Testing

The optochin test is designed for pure cultures of alpha-hemolytic streptococci. Direct testing on clinical specimens (sputum, blood culture broth, cerebrospinal fluid) is not validated and may produce unreliable results due to mixed flora or inhibitory substances.

Not for Antimicrobial Susceptibility Testing

The optochin disk is an identification tool, not an antimicrobial susceptibility test. The zone diameter does not predict clinical response to optochin or any therapeutic agent. Do not report optochin zone sizes as part of antimicrobial susceptibility panels.

Documentation

Record the following information for each optochin test:

• Patient/sample identifier: Unique laboratory accession number
• Organism source: Specimen type (e.g., blood, sputum, CSF)
• Colony morphology: Description of colony appearance and hemolysis pattern
• Gram stain result: Gram-positive cocci in pairs or chains
• Inoculum standardization method: McFarland standard or spectrophotometric adjustment
• Medium type and lot number: Sheep blood agar, manufacturer, and expiration date
• Optochin disk manufacturer and lot number: Including expiration date
• Incubation conditions: Temperature, CO₂ concentration, duration
• Zone diameter: Measured in millimeters
• Interpretation: Susceptible (≥14 mm) or resistant (<14 mm)
• Confirmatory test results: Bile solubility result if performed
• Final identification: Report as S. pneumoniae or "alpha-hemolytic streptococcus, not S. pneumoniae"
• Technician initials and date

Documentation should follow laboratory standard operating procedures and comply with regulatory requirements for diagnostic testing.

Biosafety Considerations

Risk Assessment

Streptococcus pneumoniae is classified as a Risk Group 2 pathogen capable of causing serious human disease, particularly pneumonia, meningitis, and bacteremia. Clinical specimens and cultures should be handled at Biosafety Level 2 (BSL-2) according to guidelines from the CDC and NIH [6]. Teaching laboratories using well-characterized, non-pathogenic strains (e.g., ATCC strains) may operate at BSL-1 with appropriate institutional approval.

Personal Protective Equipment

• Laboratory coat or gown
• Disposable gloves (nitrile or latex)
• Eye protection when performing procedures that may generate aerosols (e.g., vortexing, streaking)
• Closed-toe shoes

Work Practices

• Perform all manipulations of clinical isolates in a biological safety cabinet (Class II)
• Minimize aerosol generation by avoiding vigorous vortexing of open tubes
• Decontaminate work surfaces before and after procedures with 10% bleach or appropriate disinfectant
• Dispose of contaminated materials in biohazard waste containers
• Wash hands thoroughly after removing gloves

Spill Management

For small spills (drops on work surface): Cover with absorbent material, apply disinfectant, allow 20-minute contact time, then clean and dispose of waste as biohazardous.

For large spills (culture plates broken, significant liquid release): Evacuate area, restrict access, allow aerosols to settle for 30 minutes, then clean wearing appropriate PPE with disinfectant.

Waste Disposal

All cultures, contaminated disposables, and used optochin disks must be autoclaved before disposal. Follow institutional guidelines for biohazardous waste management.

Frequently Asked Questions

Can the optochin test be used on beta-hemolytic streptococci?

No. The optochin test is validated only for alpha-hemolytic (viridans group) streptococci. Beta-hemolytic streptococci, including Streptococcus pyogenes and Streptococcus agalactiae, are not reliably differentiated by optochin susceptibility. Use bacitracin susceptibility testing for S. pyogenes identification and other specific tests for beta-hemolytic groups.

What should I do if my isolate shows a 13 mm zone?

A 13 mm zone is borderline and requires repeat testing. First, verify that the inoculum was correctly adjusted to 0.5 McFarland and that the optochin disk is within its expiration date. Repeat the test in duplicate. If the zone remains borderline, perform a bile solubility test. A bile-soluble isolate with a borderline optochin zone should be reported as S. pneumoniae; a bile-insoluble isolate should be reported as an alpha-hemolytic streptococcus.

How long can I store optochin disks?

Optochin disks should be stored at 2–8°C in their original sealed container with desiccant. Under proper storage conditions, disks remain stable until the manufacturer's expiration date. Once opened, the container should be resealed immediately after each use. Discard any disks that show discoloration, physical damage, or that fail quality control testing with the positive control strain.

Is the optochin test sufficient for definitive identification of S. pneumoniae?

No. The optochin test should not be used as a standalone definitive identification method. While a zone ≥14 mm in an alpha-hemolytic, Gram-positive coccus with typical colony morphology is highly suggestive, confirmatory testing with the bile solubility test is recommended. Published studies report strong performance for bile solubility testing in defined settings, but no single phenotypic test should be treated as universally definitive. Molecular methods such as PCR targeting the lytA gene or matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) provide additional confirmation when available.

References and Further Reading

1. Evaluation of a new protocol for rapid identification of Streptococcus pneumoniae in blood cultures using the modified bile solubility test
2. Preliminary genomic assessment of invasive Streptococcus pneumoniae serotype 3 isolates in Malaysia
3. Anthocyanin-Incorporated Chromogenic Agar for Rapid, Selective Detection of Streptococcus pneumoniae
4. Comparison of Streptococcus pneumoniae isolates occurring in optochin-susceptible and optochin-resistant variants by analyzing whole-genome sequencing data
5. Impact of prophylactic oral azithromycin during labor on Azithromycin Resistance in nasal Staphylococcus aureus and Streptococcus pneumoniae
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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