Optochin Susceptibility Test: Introduction, Principle, Test Requirements, Procedure, Result-Interpretation, and Keynotes

Optochin Susceptibility Test

Optochin Susceptibility Test uses for presumptive identification of Streptococcus pneumoniae from viridans streptococci.

 Principle of Optochin Susceptibility Test

Ethylhydrocupreine hydrochloride (Optochin), a quinine derivative, selectively inhibits the growth of S. pneumoniae at a very low concentration (5ug/ml). Optochin may also inhibit other viridians streptococci, but only at much higher concentrations. The test has a sensitivity of more than 95%. Optochin is water-soluble and diffuses readily into an agar medium. Therefore, filter paper impregnated with Optochin is used in a disk diffusion test format to determine 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.

Requirements for Optochin Susceptibility Test

1.  Test organism-Alpha hemolytic streptococci
2. 5% sheep blood agar
3. Optochin disk(6 mm diameter in size)-5μg
4. Forceps
5. 5% CO₂ incubator

Procedure for Optochin Susceptibility Test

1. Select and streak using a straight wire, three to four well-isolated colonies of the suspected organism over a blood agar.
2.  Place an Optochin disk was over it with firmly.
3. Incubate the plate at 35°C for 18-24 hours in a CO₂ incubator (5-10% CO₂).
4. Observe for the zone of inhibition around the disk.  was ≥ 14 mm around the disk, the organism was considered as S. pneumoniae.

Result and Interpretation of Optochin Test

Zone of inhibition of optochin disk  14 mm or greater: Optochin sensitive: Positive

No Zone of inhibition of optochin disk: Optochin resistance: Negative

Use of Optochin disk on blood agar during Sputum culture

Keynotes on Optochin Susceptibility Test 

• Report an identification of Streptococcus pneumoniae, if the alpha-hemolytic colony from gram-positive cocci in pairs is catalase-negative and susceptible to optochin.
• Optochin is the commercial name whereas the generic name is Ethylhydrocupreine hydrochloride, a quinine derivative.
• For any alpha-hemolytic colonies from gram-positive cocci in pairs that are catalase-negative but produce zone sizes with intermediate results, perform spot bile solubility test for confirmation of identification. Report as Streptococcus pneumoniae if positive.
• If the organism is optochin resistant and is a catalase-negative, alpha-hemolytic colony from gram-positive cocci in pairs, report as a viridans group streptococcus.
• Optochin susceptibility is an excellent test to identify S. pneumoniae, with a 99% sensitivity for encapsulated strains and 98 to 99% specificity.
• Use of optochin sensitivity test in blood agar at the time of inoculating sputum specimen; this is doing so because of sputum carrying numerous normal flora and the optochin disk is put on a secondary zone of streaking that inhibits normal flora. The optochin is also called ethylhydrocupreine hydrochloride and Streptococcus pneumoniae grows far from the optochin disk while Enterocococcus, Streptococcus pyogenes and Streptococcus agalactiae may grow towards the disk.

Further Readings

1. Cowan & Steel’s Manual for identification of Medical Bacteria. Editors: G.I. Barron & R.K. Felthani, 3rd ed 1993, Publisher Cambridge University press.
2. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
3. Clinical Microbiology Procedure Hand book, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
4. Colour Atlas and Text book of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr and Sommers H.M.
5. Jawetz, Melnick and Adelberg’s Medical Microbiology. Editors: Geo. F. Brook, Janet S. Butel & Stephen A. Morse, 21st ed 1998, Publisher Appleton & Lance, Co Stamford Connecticut.
6. Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.
7.  Text book of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.