Optochin Susceptibility Test: Principle, Objective, Procedure And Results

  • Streptococcus pneumoniae can be distinguished from other viridans streptococci by showing that it is sensitive to optochin, dissolves in bile, reacts with a specific DNA probe, or has species-specific capsular polysaccharides. 
  • The optochin susceptibility test is used in almost all clinical microbiology labs today.
  • Even though the optochin susceptibility test was first described in 1915 as a way to tell pneumococci apart from other -hemolytic streptococci, laboratories almost never used it until the mid-1950s.
  • The chemical that makes up optochin is called ethylhydrocupreine hydrochloride, and it dissolves completely in water. 
  • It can be used to find Streptococcus pneumoniae, which is the alpha-hemolytic Streptococcus that is most often affected by this chemical. 
  • Other types of alpha-hemolytic streptococci are resistant to optochin. This test finds out if the bacterium is sensitive to the chemical optochin or if it is resistant to it.
  • The optochin test is often done with ethylhydrocupreine hydrochloride-soaked filter paper discs that are placed directly on plates that have been inoculated before they are put in an incubator. 
  • The optochin test takes less time than the test to see how well something dissolves in bile.

Objective of Optochin Susceptibility Test

  • To see what effect Optochin (ethyl hydrocupreine hydrochloride) has on an organism.
  • To distinguish between alpha-hemolytic Streptococcus pneumoniae and other alpha-hemolytic viridans Streptococci.

Principle of Optochin Susceptibility Test

Optochin is a substance that is entirely soluble in water (ethylhydrocupreine hydrochloride). Antibiotic optochin blocks the ATPase and prevents bacteria from producing adenosine triphosphate (ATP). Presumptive detection of alpha-hemolytic Streptococcus pneumoniae is facilitated by optochin. The chemical induces surface tension changes in S. pneumoniae, which ultimately leads to cell lysis, providing a measure of the membrane’s permeability. Gram-positive lanceolate diplococcus, S. pneumoniae can also exist as unicellular or multicellular forms. When it comes to temperature and CO2 concentration, S. pneumoniae is a picky bacteria that thrives between 35 and 37 degrees. Pneumococcal colonies in their early stages seem similar to viridans streptococcal colonies in their elongated, rounded shape, making it difficult to tell them apart. Although certain pneumococcal strains are resistant to the antibiotic, optochin sensitivity allows for the presumed identification of alpha-hemolytic streptococci as S. pneumoniae. Optochin is not effective against other types of alpha-hemolytic streptococci. In order to determine whether or not an organism is resistant to the antibiotic optochin, a disc impregnated with the antibiotic is placed on a lawn of the organism on a sheep blood agar plate and allowed to permeate into the medium. A clearing, or zone of inhibition, forms surrounding the disc as a result of the antibiotic’s effect on a susceptible bacterium. In order to be considered susceptible and for Streptococcus pneumoniae to be identified presumptively, the zone of detection must be 14 mm or larger.

Materials required

  • Test organism-Alpha hemolytic streptococci
  • 5% sheep blood agar
  • Optochin disk(6 mm diameter in size)-5μg
  • Forceps
  • 5% CO2 incubator

Quality Control

Each batch of optochin discs should be evaluated alongside a positive and a negative control.

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

Procedure of Optochin Susceptibility Test

  1. Pick a healthy colony of the alpha-hemolytic organism to test using a sterile inoculating loop.
  2. Distribute the isolates on a plate of sheep blood agar (5%).
  3. The inoculated agar plate should have an optochin disc placed on it using sterile forceps.
  4. Gently press the disc down onto the agar surfaces using the sterile forceps to ensure a good bond.
  5. Put the plate in a 5-10% CO2 incubator at 35-37°C for 18-24 hours.
  6. After 18 to 24 hours of incubation, check the plate to see if the zone of inhibition has formed around the disc, and take a measurement.
Procedure of Optochin Susceptibility Test
Procedure of Optochin Susceptibility Test

Result and Interpretation of Optochin Susceptibility Test

  • Optochin Sensitive: If the zone of inhibition produced by optochin is less than 14 millimetres in diameter around a 6-millimeter disc, then Streptococcus pneumoniae can be confidently identified.
  • Optochin Resistant:  No zone of inhibition around the disk.
Result and Interpretation of Optochin Susceptibility Test
Result and Interpretation of Optochin Susceptibility Test

To exclude the possibility that other strains of Streptococcus pneumoniae are present, further tests (bile solubility or serology) should be conducted if the zone of inhibition is less than 14 mm. 

Result and Interpretation of Optochin Susceptibility Test
Result and Interpretation of Optochin Susceptibility Test

Limitations of Optochin Susceptibility Test

  • Some Streptococcus pneumoniae isolates will grow poorly or not at all unless they are incubated in a CO2enriched atmosphere.
  • The susceptibility test for optochin is just indicative. Additional biochemical testing are suggested for definitive identification.
  • Pneumococci with an inhibitory zone of less than 14 mm are likely to be false positives; a positive bile-solubility test or serology can establish the strain’s identity as pneumococcus.

Keynote on Optochin Susceptibility Test 

  • If the alpha-hemolytic colony from gram-positive cocci in pairs is catalase-negative and sensitive to optochin, then report an identification of S. pneumoniae.
  • Spot bile solubility testing should be done to confirm the identification of any alpha-hemolytic colonies from gram-positive cocci in pairs that are catalase-negative but yield intermediate zone sizes. If testing positive, identify as Streptococcus pneumoniae.
  • Gram-positive cocci in pairs that are resistant to optochin and exhibit catalase negativity and alpha-hemolysis should be reported as a viridans group streptococcus.
  • The susceptibility to optochin is a highly accurate method of identifying S. pneumoniae, with a sensitivity of 99% for encapsulated strains and a specificity of 98 to 99%.


  • Because alpha-streptococci are resistant to optochin (a negative test), it can be used to help identify pneumococci (a positive test).


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