Optochin susceptibility test by 5 μg disk
S. pneumoniae can be identified using Gram stain, catalase, and optochin tests simultaneously, with bile solubility as a confirmation
test. If these tests indicate that the isolate is
S. pneumoniae, serological tests to identify the serotype can be performed.
REFERENCES
1. Robert A. Whiley and Jeremy M. Hardie, 2009. Genus I. Streptococcus Rosenbach 1884, 22AL. In: (Eds.) P.D. Vos, G. Garrity, D.
Jones, N.R. Krieg, W. Ludwig, F.A. Rainey, K.-H. Schleifer, W.B. Whitman. Bergey’s Manual of Systematic Bacteriology, Volume 3: The
Firmicutes, Springer, 655-711.
2. National Center for Immunization and Respiratory Diseases. Bacterial Meningitis Chapter 8: Identification and Characterization of
Streptococcus pneumoniae.
3. Dumitru Buiuc, Marian Negut. 41.54. Sensibilitatea la optochin; in Tratat de microbiologie clinica. Ed. medicala, 2009, p 1162.
Optochin Susceptibility Test
DESCRIPTION
Streptococcus pneumoniae can be distinguished from other alpha-hemolytic streptococci on the basis of its sensitivity to optochin
(ethylhydrocupreine hydrochloride). The substance lyses the cell membrane. Other alpha-hemolytic streptococcal species are usually
optochin-resistant.
PROCEDURE
Steps are similar to diffusimetric antibiogram:
1. Select a pure culture of alpha-haemolytic Streptococcus strain.
2. Use a disposable loop to remove 2-3 colonies from the overnight culture and
streak onto a sheep-blood agar plate.
3. Place the 5 μg optochin paper disk  in the middle of the plate.
4. Incubate at 35 ± 1 ºC for 18 ± 2h in 5-10% CO
2 enriched environment. During
the incubation the water-soluble ethylhydrocupreine will diffuse in the area
surrounding the disk, and a round clear area (where the strain's growth is  
inhibited) will appear.
5. Reading: sensitivity is indicated by an inhibition zone of >14 mm with a 6 mm  
disk or >16 mm with a 10 mm disk.
QUALITY CONTROLS
Streptococcus pneumoniae ATCC 49619  - susceptible;
Steptococcus mitis ATCC 4945 - resistant.

NOTES
Pneumococci are sometimes optochin-resistant, also other strains of alpha-hemolytic streptococci may show a slight susceptibility to
optochin.
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