Use of Optochin in Blood Agar during Sputum Inoculation: Introduction, Principle and Procedure

Optochin sensitivity test

 Introduction  of Optochin Sensitivity Test

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. The optochin is also called ethylhydrocupreine hydrochloride.

Principle of Optochin Sensitivity Test

The technique applying so for the presumptive identification of Streptococcus pneumoniae , which is optochin-sensitive, from other alpha -haemolytic streptococci, which are resistant since, in the presence of optochin, colonies of Streptococcus pneumoniae are selectively lysed indicated by a zone of inhibition after incubation under increased CO2 while other alpha-hemolytic streptococci are resistant to optochin.

Requirements for optochin Sensitivity Test

  • Sputum ( test specimen)
  • 5% sheep blood agar
  • Inoculating loop
  • Bunsen burner
  • Optochin disc(6 mm)
  • Forceps for putting a disk
  • CO2 incubator

Quality control

To maintain quality control of the test, it is mandatory to check quality control in each new lot of optochin discs using the following quality control strains.

Positive control: Streptococcus pneumoniae ATCC 49619

Negative control: Streptococcus mitis ATCC 49456

Procedure of Optochin Sensitivity Test

  1. Take a loopful sputum specimen and streak on a 5% sheep blood agar plate.
  2. Make primary, secondary and tertiary streaking zones.
  3. Apply  5 µg optochin disc on secondary streaking zone.
  4. Incubate the blood agar plate for 24 hours in a CO2  incubator.

Observations 

Look for the growth of organisms whether sensitive or resistant towards the optochin disk.

Measure the zone of inhibition using a clipper or scale.

Result and interpretation of Optochin Sensitivity Test

Test positive: Zone of inhibition of organism is equal or greater than 14 mm

Test negative:  No zone of inhibition

Equivocal: any zone of inhibition less than 14 mm is suspicious. Therefore confirm it by performing a bile solubility test.

Test result:  From the above figure, alpha-haemolytic bacteria are sensitive to optochin, therefore the organism is  presumptively Streptococcus pneumoniae

Keynotes on Optochin Sensitivity Test

  • The commercial name is optochin whereas the generic name is Ethylhydrocupreine hydrochloride, a quinine derivative.
  • Optochin may inhibit viridans streptococci only at much higher concentrations whereas Streptococcus pneumoniae is selectively inhibited at very low concentrations i.e. 5 mg/ml or less.
  • The optochin sensitivity test is simple to perform and inexpensive
  • It has a sensitivity of more than 95%.
  • If there are colony characteristics of the organism similar to Streptococcus pneumoniae, but a smaller zone of inhibition (< 14 mm) or no zone of inhibition in this condition bile solubility test is required. It is important to remember that Streptococcus pneumoniae is sometimes optochin-resistant.

Bibliography

  • Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
  • Clinical Microbiology Procedure Handbook Vol. I & II, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
  • Colour Atlas and Textbook of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr, and Sommers H.M.
  • Cowan & Steel’s Manual for identification of Medical Bacteria. Editors: G.I. Barron & R.K. Felthani, 3rd ed 1993, Publisher Cambridge University Press.
  • 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.
  • 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.
  •  Manual of Clinical Microbiology. Editors: P.R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover and R. H. Yolken, 7th ed 2005, Publisher ASM, USA
  •  Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.
  • Medical Microbiology-The Practice of Medical Microbiology Vol-2-12th Edn. –Robert Cruickshank
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