Novobiocin Susceptibility Test


What is the purpose of the test?


Novobiocin is an antibiotic interfering with the unpackaging and repackaging of DNA during DNA replication and the bacterial cell cycle. Different types of bacteria have different degrees of susceptibility to novobiocin. This test determines whether the bacterium is either sensitive (susceptible) to novobiocin or resistant to the drug. Knowledge about novobiocin susceptibility is valuable in identification of Gram positive cocci, some of which are susceptible and others of which are resistant.




How is novobiocin susceptibility determined? 


Susceptibility to novobiocin is determined by placing a novobiocin-impregnated paper 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 antibiotic diffusing into the agar from the paper disk. If the bacteria are susceptible to novobiocin, there will be a visible zone of inhibition forming around the disk, representing an area where the antibiotic 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.




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. An antibiotic-impregnated disk containing novobiocin 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 antibiotic. An obvious clear zone around the disk, termed the zone of inhibition, indicates the bacterium to be susceptible to the antibiotic.



What reagents are needed?

None.  The antibiotic disk is added to the plate after being seeded with bacteria and before being incubated.



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 novobiocin - 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.