Optochin Sensitivity Test: Principle, Procedure, expected results and quality control

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.

Performing the optochin test

Optochin (P) disks (6 mm, 5 µg) can be obtained from a commercial vendor. Optochin disks are often called “P disks” and many commercial versions are labeled with a capital “P”. If a commercial source of P disks is not available, a 1:4000 solution of ethylhydrocupreine hydrochloride can be applied to sterile 6 mm filter paper disks.

  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 a P 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 do 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 blood agar plate) but care must be taken to ensure that the cultures do not overlap.

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

Reading the optochin test results

  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 diameter of 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

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 a 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.

The Organism on the right side is susceptible to Optochin: So it is Streptococcus pneumoniae 

Quality control of Optochin Sensitivity test

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.