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.

Some pneumococci are optochin resistant and others are bile resistant and others are resistant. No one test is 100% accurate, and the combination of these two tests will prevent erroneous reporting.

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

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 mg/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 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 are lysed owing to changes in the surface tension, and a zone of inhibition is produced.

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.


Optochin (OP or P) disks (6 mm, 5 µg) can be obtained from a commercial vendor. If a commercial source of optochin disks is not available, a 1:4000 solution of ethylhydrocupreine hydrochloride can be applied to sterile 6 mm 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% CO2 (or in a candle jar) for  18 to 24 hours. (Culture does not grow well in ambient air, and larger zones of inhibition occur).

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 that 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 blood agar plate and observe the zone of inhibition around S. pneumoniae. 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.


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

Optochin Sensitivity test for S. pneumoniae

References and further readings

  1. Koneman’s Color Atlas And Textbook of Diagnostic Microbiology (check the latest edition)
  2. Clinical Microbiology Procedures Handbook, Fourth Edition. (2016). American Society of Microbiology.

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

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