Optochin Test: Principle, Requirements, Procedure, Result Interpretation and Limitations

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  • Optochin test is used to determine the effect of Optochin (ethylhydrocupreine hydrochloride) on an organism.
  • Optochin is a chemical used as an antibiotic that interferes with the activity of enzyme ATPase and production of ATP in microorganisms.
  • It helps in the presumptive identification of alpha-hemolytic Streptococcus pneumoniae (sensitive to Optochin) from other alpha-hemolytic streptococcal species (resistant to Optochin), although some pneumococcal strains are Optochin resistant.
  • Optochin changes the surface tension of cell membrane of pneumoniae and makes it more fragile causing the lysis of the cell.
  • Differentiating pneumococci from viridans streptococci is difficult as young pneumococcal colonies appear raised, similar to viridans streptococci and therefore Optochin sensitivity test is performed for their differentiation.
  • For the Optochin susceptibility test, Optochin impregnated disc is placed on a lawn of the inoculated organism on a sheep blood agar plate. This allows the antibiotic to diffuse into the medium.
  • The antibiotic inhibits the growth of a susceptible organism forming a zone of inhibition around the disc. A zone of 14mm or greater is considered susceptible and presumptive identification for Streptococcus pneumoniae. No zone of inhibition around the disc indicates resistant organism.


  • Test organism (alpha hemolytic)
  • 5% sheep blood agar plate
  • Inoculating loops
  • Sterile forceps
  • Optochin impregnated discs
  • Control organisms
    • Positive or susceptible: Streptococcus pneumoniae
    • Negative or resistant: Streptococcus mitis


  • An isolated colony of the alpha-hemolytic organism to be tested was selected with a sterile inoculating loop.
  • It was then streaked onto a 5% sheep blood agar plate in order to make a lawn culture.
  • Using sterile forceps, an Optochin impregnated disc was placed on the inoculated surface of the agar.
  • The disc was then gently pressed so that it would adhere firmly to the agar surface.
  • The plate was incubated at 35-37°C for 18-24 hours in 5 to 10% CO2.
  • After complete incubation, the plate was examined and observed for the zone of inhibition around the disc. The zone of inhibition (if any) was then measured .

Result Interpretation:

  • Positive test or Optochin Sensitive: The zone of inhibition is 14 mm or more around a 6mm disc. The organism is Streptococcus pneumoniae.
  • Negative test or Optochin Resistant: No zone of inhibition around the Optochin impregnated disc.


  • Streptococcus pneumoniae isolates should be incubated in an environment enriched with CO2 because some isolates will grow poorly or not at all.
  • It is a presumptive test only and further biochemical tests are recommended for complete identification.
  • Any zone of inhibition less than 14 mm is questionable for pneumococci and the organism is identified as pneumococcus only if it is bile soluble.

Optochin Test: Principle, Requirements, Procedure, Result Interpretation and Limitations