Optochin Susceptibility Test


What is the purpose of the test?


Optochin is a chemical that is toxic to some bacteria but harmless to others. It is useful in the identification of Streptococcus pneumoniae, the alpha-hemolytic Streptococcus most commonly susceptible to this chemical. This test determines whether the bacterium is either sensitive (susceptible) to optochin or resistant to the chemical.



How is optochin susceptibility determined? 


Susceptibility to optochin is determined by placing an optochin-impregnated disk on a nutrient agar plate seeded with the microbe under investigation. As the microbe multiplies during incubation to produce a lawn of confluent growth, cells are exposed to the chemical diffusing into the agar from the paper disk. If the bacteria are susceptible to optochin, there will be a visible zone of inhibition forming around the disk, representing an area where the chemical concentration has prevented bacterial growth. Should the microbe be resistant, the lawn of cells will form visible growth up to the margin of the disk.


Note: Our version of the test is clearly "susceptibility" or "resistance". In actuality, interpretation of this test requires measurement of the diameter of the zone of inhibition and comparison to standards to determine susceptibility or resistance.



What medium is used? 


The medium used for growing the bacterial lawn is typically either blood agar or a nutrient rich, general-purpose medium like nutrient agar. In VirtualUnknown:Microbiology™, we use nutrient agar.




How is the test performed?  


An inoculum from a pure culture is transferred aseptically to a sterile plate of nutrient agar, typically by use of cotton-tipped applicator (swab). In a clinical setting, the applicator often contains other fibers than cotton to prevent any toxic residue found in cotton after processing from interfering with the growth of delicate pathogens. The inoculum is spread over the plate to distribute the bacteria as evenly and thoroughly as possible over the entire agar surface. A chemical-impregnated disk containing optochin is then aseptically placed in the center of the agar surface. The inoculated plate is incubated at 35-37 C for 24 hours and the results are determined. Growth of the lawn up to the margin of the disk indicates the bacterium is resistant to the chemical. An obvious clear zone around the disk, termed the zone of inhibition, indicates the bacterium to be susceptible to the chemical.



What reagents are needed?


None. The disk is added before incubation. No additions are made following incubation.




To perform this test in VirtualUnknown™ Microbiology, complete the following steps:


Inoculation of Medium


1. Select the nutrient agar plate medium.  

2. Start your Bunsen burner.  

3. Select the cotton-tipped applicator tool.  

4. Remove the cap from your inoculum and your lid from your sample plate.  

5. Flame the mouth of your inoculum tube.  

6. Use the sterile cotton tipped applicator to pick up an inoculum from the culture tube of the unknown bacterium.  

7. Immediately transfer the inoculum into the fresh, sterile medium. NOTE: you must streak the inoculum back and forth across the plate for several seconds to inoculate the plate. You will know it has been successfully done when the appearance of the plate changes.  

8. Flame the mouth of the tube.  


Addition of the Antibiotic Sensitivity Disk


9. Click the right mouse button, as you would to replace the caps and lids.  Instead you will be prompted to add an antibiotic disk.  Select optochin - the disk will be added to the plate.

10. Replace the cap on the inoculum tube and the plate lid.  


Incubation of the Inoculated Medium


11. Place the inoculated plate into the 35-37 C incubator.  

12. Press the New Day button to move forward 24 hours.  

13. Incubate for the appropriate length of time. For this test 24 hours is sufficient.  

14. Retrieve desired incubated culture from the incubator.  


Interpretation of Results


15. Observe the growth on the surface of the plate, especially in the vicinity of the antibiotic disk. If there is a visible zone of inhibition surrounding the disk, the microbe is susceptible to the antibiotic. If no zone is present, the microbe is resistant to the antibiotic.

16. Record test result.  

17. Dispose of the culture.