Deoxyribonuclease (DNase) Test

 

What is the purpose of the test? 

 

The purpose is to see if the microbe can use DNA as a source of carbon and energy for growth. Use of DNA is accomplished by an enzyme called DNase.

 

 

How is DNase activity determined? 

 

A medium containing DNA and an indicator is used. The indicator complexes with intact DNA, making the medium appear mint green. When DNA is digested, the indicator changes from mint green to clear. Halos surrounding colonies is indicative of their ability to digest the DNA in the medium due to the presence of DNase.

 

 

What medium is used?  

 

The medium used is DNase agar with methyl green. The medium is a nutrient agar to which DNA is added. The indicator methyl green produces a mint green medium.

 

 

How is the test performed?  

 

An inoculum from a pure culture is streaked on a sterile plate of DNase agar. The inoculated plate is incubated at 25 C for 24 hours. Presence of clear halos surrounding colonies is positive for their ability to digest the DNA and thus indicates presence of DNase.

 

 

What reagents are added?

 

None.

 

 

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

 

Inoculation of Medium

 

1. Select the DNase agar with methyl green plate medium.  

2. Start your Bunsen burner.  

3. Select the inoculating loop tool.  

4. Flame your inoculating loop to sterilize it.  

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

6. Flame the mouth of your inoculum tube.  

7. Use the sterile inoculating tool to pick up an inoculum from the culture tube of the unknown bacterium.

8. 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 streak marks appear on the agar surface.  

9. Flame the mouth of your inoculum tube once again.  

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

11. Re-flame the inoculating tool.  

 

Incubation of the Inoculated Medium

 

12. Place the inoculated plate into the 25 C incubator.  

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

 

Determination of Test Results

 

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

15. Retrieve desired incubated culture from the incubator.  

16. Observe the color of the medium surrounding colonies in the plate. The culture showing clear halos around colonies is capable of breaking down DNA. The medium surrounding colonies without DNase remains unchanged.  

17. Record test result.  

18. Dispose of the culture.