Optochin Susceptibility Test- Principle, Test Procedure, Results and Interpretation

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This test is used to determine an organism’s susceptibility to the chemical optochin (ethylhydrocupreine hydrochloride) for presumptive identification of Streptococcus pneumonia.

Principle

Ethylhydrocupreine hydrochloride (optochin), a quinine derivative, selectively inhibits the growth of Streptococcus pneumoniae at very low concentrations (5 mg/mL or less). Optochin may also inhibit other viridans streptococci, but only at much higher concentrations. The test has a sensitivity of more than 95%, is simple to perform, and is inexpensive. Optochin is water-soluble and diffuses readily into agar medium. Therefore, filter paper disks impregnated with optochin can be 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.

Media and Reagents

1. Well-isolated colonies of the organism to be tested on sheep blood agar

2. Sheep blood agar plate

3. Optochin disks (5 μg)

4. Disks should be stored at 4°C when not in use.

Quality Control

Positive control: Streptococcus pneumoniae

Negative control: Viridans streptococcus or Enterococcus faecalis

Test Procedure

1. Using an inoculating loop or wire, select three to four well-isolated colonies of the organism to be tested and streak onto one-half to a one-third of a blood agar plate. The inoculated area should be about 3 cm2.

2. Place an optochin disk in the upper third of the streaked area. Tap down the disk with flamed or otherwise sterilized forceps so that the disk adheres firmly to the agar surface.

3. Incubate the plate at 35°C for 18–24 hours in a candle jar or in 5%–7% CO2.

Results and Interpretation

A viridans streptococcus can be presumptively identified as S. pneumoniae if it shows a zone of inhibition of 14 mm or more around a 6-mm disk, or a zone of 16 mm or more around a 10-mm disk. Organisms showing zones smaller than these should be tested for bile solubility.


Optochin Susceptibility Test

Limitations

Occasionally, viridans streptococci will show small zones of inhibition around the optochin disk. These organisms, however, will not be bile-soluble.

Reference

Forbes BA, Sahm DF, Weissfeld AS. Bailey & Scott’s Diagnostic Microbiology, 12th Ed., Mosby Elsevier.

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