Optochin Susceptibility Test- Principle, Procedure and Results

Objectives of Optochin Susceptibility Test

  • To determine the effect of Optochin (ethyl hydrocupreine hydrochloride) on an organism.
  • To differentiate between alpha-hemolytic Streptococcus pneumoniae from other alpha-hemolytic viridans Streptococci.

Principle of Optochin Susceptibility Test

Optochin (ethylhydrocupreine hydrochloride) is a chemical and is completely soluble in water. Optochin is an antibiotic that interferes with the ATPase and production of adenosine triphosphate (ATP) in microorganisms. Optochin is used in the presumptive identification of alpha-hemolytic Streptococcus pneumoniae. The chemical tests the fragility of the bacterial cell membrane and causes S. pneumoniae to lyse due to changes in surface tension. S. pneumoniae is a gram-positive lanceolate diplococcus, but can also occur as single cocci or in short chains of cocci. S. pneumoniae is a fastidious bacterium, growing best at 35-37°C with ~5% CO2. Differentiating pneumococci from viridans streptococci is difficult as young pneumococcal colonies appear raised, similar to viridans streptococci. 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. For the optochin susceptibility test, the Optochin impregnated disk is placed on a lawn of the organism on a sheep blood agar plate, allowing the antibiotic to diffuse into the medium. The antibiotic inhibits the growth of a susceptible organism, creating a clearing, or zone of inhibition, around the disk. A zone of 14mm or greater is considered susceptible and presumptive identification for Streptococcus pneumoniae.

Procedure of Optochin Susceptibility Test

  1. With a sterile inoculating loop, select a well-isolated colony of the alpha-hemolytic organism to be tested.
  2. Streak the isolates onto a 5% sheep blood agar plate.
  3. Using sterile forceps, place an optochin disk onto the inoculated surface of the agar.
  4. Press disk gently with the sterile forceps so that the disk adheres firmly to the agar surfaces.
  5. Incubate the plate at 35-37°C for 18-24 hours in 5 to 10% CO2.
  6. Examine the plate after 18 – 24 hours of incubation, observe for the zone of inhibition around the disk, and measure the zone of inhibition.  

Result Interpretation of Optochin Susceptibility Test

Optochin Susceptibility Test- Principle, Procedure and Results

Optochin Sensitive: The zone of inhibition is ≥14 mm, around a 6-mm disk, then identify the organism as Streptococcus pneumoniae.

Optochin Resistant:  No zone of inhibition around the disk.

If the zone of inhibition is less than 14 mm, further testing (bile solubility or serology) should be done for the identification of other strains of Streptococcus pneumoniae.

Limitations of Optochin Susceptibility Test

  1. Streptococcus pneumoniae isolates should be incubated in a CO2enriched environment, as some isolates will grow poorly or not at all.
  2. Optochin susceptibility is a presumptive test only. It is recommended that further biochemical tests be performed for complete identification.
  3. Any zone of inhibition less than 14 mm is questionable for pneumococci; the strain is identified as pneumococcus with confirmation by a positive bile-solubility test or serology can be performed.

Quality Control of Optochin Susceptibility Test

Streptococcus pneumonia (ATCC49619): Optochin sensitive
Streptococcus mitis (ATCC 49456): Optochin resistant
Streptococcus pyogenes (ATCC12384): Optochin resistant


  1. Tille P.M. 2014. Bailey and Scott’s diagnostic microbiology. Thirteen edition. Mosby, Inc., an affiliate of Elsevier Inc. 3251 Riverport Lane. St. Louis. Missouri 63043
  2. Uk standards for Microbiology Investigations. 2015. Optochin test.
  3. Centre for Disease Control and Prevention. Identification and Characterization of Streptococcus pneumonia. https://www.cdc.gov/meningitis/lab-manual/chpt08-id-characterization-streppneumo.pdf

About Author

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Sagar Aryal

Sagar Aryal is a microbiologist and a scientific blogger. He attended St. Xavier’s College, Maitighar, Kathmandu, Nepal, to complete his Master of Science in Microbiology. He worked as a Lecturer at St. Xavier’s College, Maitighar, Kathmandu, Nepal, from Feb 2015 to June 2019. After teaching microbiology for more than four years, he joined the Central Department of Microbiology, Tribhuvan University, to pursue his Ph.D. in collaboration with Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Saarbrucken, Germany. He is interested in research on actinobacteria, myxobacteria, and natural products. He has published more than 15 research articles and book chapters in international journals and well-renowned publishers.

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