Optochin test

Optochin also known as ethylhydrocupreine hydrochloride, is currently widely used as an inexpensive and reliable means to presumptively identify Streptococcus pneumoniae 1). It has been known, since the turn of the century, when optochin (ethylhydrocupreine hydrochloride) was first synthesized, to inhibit the growth of Streptococcus pneumoniae 2). The early studies of Moore in 1915 3) showed that while optochin was ineffective as a therapeutic agent, it did inhibit the growth of Streptococcus pneumoniae in broth culture. In 1955, Bowers and Jeffries 4) showed that optochin-saturated filter paper placed on the surface of unspecified horse blood agar medium onto which Streptococcus pneumoniae had been plated reliably produced a zone of inhibition. This simple test made it possible to distinguish Streptococcus pneumoniae from Streptococcus viridans, which was consistently optochin resistant 5). Bowen et al. 6) subsequently showed that optochin inhibition was independent of capsular type when inhibition tests were performed on Trypticase soy agar (TSA) supplemented with human blood.

The current National Committee for Clinical Laboratory Standards (NCCLS) guidelines regarding optochin inhibition recommend using a “blood agar plate,” specifying neither the type of blood nor the type of agar 7). There are currently several choices of sheep blood agar media which support the growth of Streptococcus pneumoniae. Current guidelines also recommend incubation in 5% CO2, and this has been previously shown to improve recovery of the organism 8) and result in smaller zones when Trypticase soy agar-sheep blood agar plates are used 9).

Procedure of Optochin sensitivity test

  1. Using an inoculating loop, select three to four well-isolated colonies of the alpha-hemolytic organism to be tested. An 18-24 hour culture of isolated organism can also be used for testing.
  2. Streak the isolate onto one-half of a Trypticase soy agar (TSA)-5% sheep blood agar plate so as to obtain confluent growth.
    • Important: Use of media other than Trypticase soy agar (TSA)-5% sheep blood agar is not recommended, as false identification may result.
  3. Using sterile forceps, place an optochin disk onto the inoculated surface of the agar.
  4. Press disk gently with the sterile forceps or loop so that the disk adheres firmly to the agar surface.
  5. Incubate the plate at 35 +/- 2.0 degrees Celsius for 18-24 hours in 5-10% CO2 enriched environment.
  6. If zone of inhibition is present, measure the diameter with a millimeter ruler or caliper.

Result interpretaion of Optochin sensitivity test

  • Positive Optochin sensitivity test: Zone of inhibition is 14 mm or greater in diameter with 6 mm optochin disc.
  • Negative Optochin sensitivity test: No zone of inhibition or a zone of inhibition of <14 mm diameter.

Note: Optochin lyses Streptococcus pneumoniae (positive test), but alpha-streptococci are resistant (negative test), thus aiding in its identification.

A presumptive identification for Streptococcus pneumoniae can be made if the alpha-hemolytic colony produces a zone of inhibition of 14 mm or greater around the disk. Organisms producing smaller zone sizes should be tested for bile solubility. Organisms with questionable zone sizes (6-14 mm) around the disk should be presumptively identified as a pneumococci only if it is bile soluble.

Limitations of Optochin susceptibility test

  1. It has been shown that other strains of alpha-hemolytic streptococci may show a slight susceptibility to optochin.
  2. Optochin susceptibility is a presumptive test only. It is recommended that further biochemical tests be performed for complete identification.
  3. Many isolates will show indeterminate zones on media other than Trypticase soy agar (TSA)-sheep blood agar. Isolates showing the indeterminate zones would require further testing to identify them as Streptococcus pneumoniae.
  4. Streptococcus pneumoniae isolates should be incubated in a CO2 enriched environment, as some isolates will grow poorly or not at all aerobically.

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