Optochin Susceptibility Test

Optochin susceptibility test is usually done for identifying the species of Streptococcus pneumoniae.

Differentiating Streptococcus pneumoniae from the other species of Streptococci variants depends on demonstrating optochin susceptibility, bile solubility, reaction with a specific DNA probe, or detection of species-specific capsular polysaccharides.

Most of the clinical microbiology laboratories depends on the optochin susceptibility test.

Initially Optochin susceptibility was first described for differentiating pneumococci, from other α-hemolytic streptococci in the year 1915, but this test is being virtually unused in the laboratories in the mid-1950s.

Optochin is a chemical named as ethylhydrocupreine hydrochloride which is completely soluble in water.

Optochin is used to identify Streptococcus pneumoniae, and alpha hemolytic Streptococcus which is mostly susceptible to the chemical, Optochin.

Where as the species of alpha-hemolytic Streptococcal species are resistant to optochin.

This test helps in determining whether bacterium is either sensitive or resistant to the chemical.

This test is also widely used in the form of filter paper discs or impregnated with ethylhydrocupreine hydrochloride, and they are applied directly on the inoculated plates before incubating.

Optochin test is one of the tests which can be performed in shorter time than the other tests like bile solubility test.

Features of Optochin Susceptibility Test

Streptococcus pneumonia is most commonly found in the respiratory tract of the humans, as other pathogens like Streptococci, and it has a hemolytic pattern which helps in distinguishing it from the other alpha-Streptococci and the Lacto cocci.

Optochin is a chemical that is used in the presumptive identification of alpha-hemolytic Streptococcus pneumoniae, which is sensitive to optochin.

Where as the other species like alpha-hemolytic Streptococcus species are resistant to optochin.

Optochin is completely soluble in water.

This test is widely used in the form of filter paper disks.

Bowen and Jeffries in the year 1955 impregnated the disks with the reagent ethylhydrocupreine hydrochloride that are applied directly to inoculated plates before incubation to demonstrate the susceptibility of pneumococcus for identification.

Optochin Susceptibility Test Principle

Streptococcus pneumoniae is generally found in the respiratory tract and it also has its hemolytic pattern which is identical to other alpha-hemolytic Lacto bacilli and streptococci.

Streptococcus pneumoniae is sensitive to the chemical optochin, which is not able to form colonies in its presence and as and its change in surface tension and causes the cell membrane of Streptococcus pneumonia to lyse.

Optochin is a chemical which is quinine derivative and it has a capacity to soluble in water where all the alpha-hemolytic streptococcal species are resistant to this chemical.

Optochin susceptibility test is usually employing in identifying the streptococcus which is sensitive to the chemical optochin.

For determining the susceptibility of the organism, Streptococcus pneumonia, filter paper disks are impregnated with the chemical optochin and it is used in a disk diffusion test.

This test is usually performed on blood agar, which is a zone for creating inhibition of the lysis of the cell membranes in the Streptococcus pneumonia cells surrounding the disk which helps in determining the positive test.

This test is very easy to perform and it also economic and has a sensitivity of more than 95%.

Optochin Susceptibility Test Requirements

• 5% of sheep blood agar.

• Optochin disks- each disk should be impregnated with 5µg of optochin

• Incubator

• Sterile forceps

• Sterile inoculating loops

Optochin Susceptibility Test Procedure

 With a help of an inoculating loop, 2 to 3 streaks are drawn on a well isolated colonies of a pure culture medium, which is tested on 5% of blood agar.

 Optochin disks are placed on the inoculated surface of agar with the help of sterile forceps.

 Ensure whether the disks are adhered firmly to the surface of the agar by using a sterile forceps or loops.

 Then the plates are placed in an incubator at temperature of about 35 to 37ºC.

 Observe the growth on the surface of the blood agar plates and finally the inhibition of the diameter zone is measured including the diameter of the disk plate.

Optochin Susceptibility Test Results

Positive Results:

 If the zone of inhibition is detected as 14mm or greater than that and a diameter of 6mm disk, it results in the positive condition.

 If the zone of inhibition is formed lesser than 14mm the strain is identified as pneumococcus only if it is soluble in bile.

Negative Results:

 If there is no detection of any formation of zones, the test is negative and it indicates the absence of Streptococcus pneumoniae.

Optochin Susceptibility Test Limitations

 Usually, Streptococcus pneumoniae are isolated and they must be incubated in a carbon-dioxide enriched environment, as some isolates will grow poorly or it does not grow at all.

 Optochin susceptibility is one of the presumptive tests which is recommended as one of the important biochemical tests which is used to perform the complete identification process.

 Any zone of inhibition which is less than 14mm is not enough for performing the test of pneumococci, as the strain is important for identifying the pneumococcus with confirmation by a positive solubility test of bile or serology is performed.