Optochin Sensitivity Test: Principle, Procedure, Results

Streptococcus pneumoniae strains are sensitive to the chemical optochin (ethylhydrocupreine hydrochloride). Optochin sensitivity allows for the presumptive identification of alpha-hemolytic streptococci as S. pneumoniae, although some pneumococcal strains are optochin-resistant.

Other alpha-hemolytic streptococcal species are optochin-resistant. Optochin sensitivity test is simple to perform, inexpensive, and has a sensitivity of more than 95%.

Some pneumococci are optochin-resistant, and some are not bile-soluble. No single test is 100% accurate, so combining optochin sensitivity with the bile solubility test prevents erroneous reporting.

Principle of Optochin Sensitivity Test

Ethylhydrocupreine hydrochloride (optochin) is a quinine derivative. Optochin selectively inhibits the growth of Streptococcus pneumoniae at a very low concentration (5 μg/mL or less). S. pneumoniae should have zones around optochin disks but viridans group streptococci have no zone around the optochin disk. Optochin may also inhibit viridans streptococci, but only at much higher concentrations.

Optochin's molecular target is the bacterial F0F1 ATP synthase, the enzyme that generates the cell's energy currency. S. pneumoniae's version of this enzyme is exquisitely sensitive to optochin at low concentrations, while most other alpha-hemolytic streptococci are not, this is the actual basis of the test's selectivity. Resistant strains typically carry point mutations in the atpC gene (more common) or atpA gene (less common), which alter the enzyme enough to block optochin binding.

Optochin is water-soluble and diffuses readily into an agar medium. Filter paper disks impregnated with optochin can be used in a disk diffusion test format to determine the susceptibility of suspected pneumococci and, thereby, confirm their identity as such. S. pneumoniae cells surrounding the disk fail to grow because their disrupted energy metabolism can't sustain replication, producing a zone of inhibition.

How to Remember

Optochin jams the engine room, it disables the cell's energy-generating machinery, the ATP synthase already described in Principle above. That's why a susceptible organism simply can't grow near the disk. When resistance shows up, you're looking at a mutation in that same engine, not a separate or unrelated failure mode.

This connects directly to the BOSS mnemonic from the S. pneumoniae article: the "OS" in BOSS is this test. Knowing why it works, rather than just that it works, makes the resistance exceptions make sense instead of feeling like an arbitrary caveat.

Materials required

1. Optochin disks Purchase optochin disks from vendors. Each disk is impregnated with 5 μg optochin. Store stock at 2-8°C. Protect from light, excessive heat, and moisture. Check the expiration date and use the disk before expiry.
2. 5% sheep blood agar.
3. Standard microbiological loops
4. Co2 incubator at 35-37°C.
5. Sterile forceps.

Quality Control

Each new lot of optochin disks should be tested with positive and negative controls.

1. Positive: Growth of S. pneumoniae strain ATCC 49619 is inhibited by optochin
2. Negative: Growth of S. mitis strain ATCC 49456 is not inhibited by optochin.

Procedure

Optochin (OP or P) disks (6mm, 5 µg) can be obtained from a commercial vendor. If a commercial source of optochin disks is unavailable, a 1:4000 solution of ethylhydrocupreine hydrochloride can be applied to sterile 6mm filter paper disks.

From Pure Culture

1. Using an inoculating loop, streak two or three suspect colonies of a pure culture to be tested on 5% sheep blood agar plate.
2. Place an optochin disk within the streaked area of the plate.
3. Incubate the blood agar plate at 35-37°C with 5-10% CO2 (or in a candle jar) for 18 to 24 hours. Incubation in CO2 is required: in ambient air, S. pneumoniae grows poorly and produces smaller, less reliable zones, which can cause a false equivocal or false-resistant reading.

Note: Two different isolates can be tested on the same plate (by streaking onto one half of the blood agar plate), but care must be taken to ensure the cultures do not overlap.

For Direct Detection

For direct detection of S. pneumoniae on primary plates, add an optochin disk to the second quadrant of a blood agar plate and observe the zone of inhibition. Adding an optochin disk is not recommended routinely but may be helpful for laboratories with a large number of specimens from patients with community-acquired pneumonia.

Results

Observe the growth on the blood agar plate near the optochin disk and measure the zone of inhibition, if applicable.

1. Using a 6 mm, 5 µg disk, a zone of inhibition of 14 mm or greater indicates sensitivity and allows for presumptive identification of pneumococci.
2. Measure zones of inhibition in millimeters, including the diameter of a disk. In the case of an isolate completely resistant to optochin, the diameter of the disk (6 mm) should be recorded.

Figure: Identification chart for Gram Positive cocci Source: sigmaaldrich.com

Expected result in Optochin test:

1. Positive: Zone of inhibition is 14 mm or greater in diameter with 6 mm disk
2. Negative: No zone of inhibition
3. Equivocal: Any zone of inhibition less than 14 mm is questionable for pneumococci; the strain is identified as pneumococcus only if it is bile soluble.

Note: A smaller zone of inhibition (< 14 mm) or no zone of inhibition indicates that the bile solubility test is required. It is important to remember that pneumococci are sometimes optochin-resistant.

Figure: Optochin Sensitivity test for S. pneumoniae

Where optochin fits: the Gram-positive coccus disk tests

Optochin is one of three disk-diffusion tests used to separate look-alike Gram-positive cocci. Each answers a different question, and which one you reach for depends on the hemolysis and the Gram/catalase result you already have:

Read the sequence like a decision tree:

• Catalase-negative + alpha-hemolytic → optochin (this test). Sensitive = S. pneumoniae; resistant → confirm viridans, or confirm pneumococcus by bile solubility if equivocal.
• Catalase-negative + beta-hemolytic → bacitracin (and PYR). Sensitive = presumptive Group A strep.
• Catalase-positive + coagulase-negative → novobiocin. Resistant = S. saprophyticus (the key positive), the cause of young-women UTIs.

Note the catalase result routes you first: optochin and bacitracin are for the catalase-negative streptococci, novobiocin is for the catalase-positive staphylococci. The full method for each disk test is in its own article; this grid is the map.

Where students actually get confused

• A resistant or equivocal result on an invasive isolate is not the end of the workup. From meningitis, blood, or other sterile-site specimens, an optochin-resistant alpha-hemolytic coccus should be confirmed with bile solubility (or molecular testing) before being called viridans, misidentification here has real clinical stakes.
• Colonies growing inside an otherwise-qualifying zone aren't a labeling mistake. Some resistant isolates are heterogeneous populations, susceptible and resistant cells from the same culture. A ≥14mm zone with colonies visible within it can still represent a resistant strain and shouldn't be dismissed as a measurement error.
• "Equivocal" has no special lower bound, treat everything under 14mm the same way. Whether the zone is 10mm or completely absent, the next step is identical: bile solubility. Don't try to split sub-14mm results into "probably fine" versus "definitely concerning" without that confirmatory test.
• Ambient air incubation isn't a shortcut, it's a source of error. Skipping CO2 enrichment can shrink the zone enough to manufacture a false equivocal or false-resistant reading on a truly susceptible organism.

Key exam facts in one table

References

1. Procop GW, Church DL, Hall GS, Janda WM, Koneman EW, Schreckenberger PC, Woods GL. Koneman's Color Atlas and Textbook of Diagnostic Microbiology. 7th ed. Philadelphia: Wolters Kluwer; 2017.
2. Leber AL, editor. Clinical Microbiology Procedures Handbook. 4th ed. Washington, DC: ASM Press; 2016. doi:10.1128/9781555818814
3. Souza ARV, de Pina SECM, Costa NS, Neves FPG, Merquior VLC, Peralta JM, Pinto TCA, Teixeira LM. Description of optochin-resistant Streptococcus pneumoniae due to an uncommon mutation in the atpA gene and comparison with previously identified atpC mutants from Brazil. Sci Rep. 2021;11:7936. doi:10.1038/s41598-021-87071-8
4. Tille PM. Bailey and Scott's Diagnostic Microbiology. 15th ed. St. Louis: Elsevier; 2022.