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33: Deoxyribonuclease (DNAse) Test

Objectives

  • Identify the presence of DNAse enzyme

DEOXYRIBONUCLEASE 

Deoxyribonuclease (DNAse) agar actually has DNA in it. Deoxyribonuclease is an exoenzyme excreted from the cell which will break the DNA down into smaller molecules. There are 2 kinds of DNAse agar that can be used.:

  1. The first medium has no indicator, but is does require HCl reagent  to be added after growth has occurred on the plate.  Without DNAse enzyme, the HCL precipitates out the undestroyed DNA. The rest of the agar with the remaining DNA, which binds to HCL (hydrochloric acid)  will have turned cloudy. Around the growth area, if the bacterium has made DNAse and destroyed the DNA, there will be clear zones seen.
  2. DNAse agar with the indicator methyl green can be used. In this medium, when the DNA in the medium is hydrolyzed by the bacterial DNAse enzyme, the pieces of cut DNA release from binding to methyl green, and you will see a yellowish/ orange zone around the growth area.

MATERIALS NEEDED

  • No reagent for methyl green DNAse--HCl (AFTER incubation) if using the other type 1
  • DNAse agar per table
  • A positive control bacterium (e.g. Serratia marcescens)

THE PROCEDURE

  1. YOUR INSTRUCTOR will run a known DNAse + organism with which your class can compare results.
  2. You will make a single line down the center of the plate (if running 2 Staph species, you can divide the plate in half and run each organism on a side).
  3. Incubate at 30º C or 37º C.
  4. AFTER INCUBATION: Flood the plate with HCl and wait for 1 minute.

INTERPRETATION

For the regular DNAse agar, the interpretation of the reaction requires a reagent added to the culture after it grows, HCl. Within a minute after addition of the reagent, a clear zone (large or small is +) around the growth area. Be sure that the plate is sitting on the black table top: The black will enhance the zone identification.

 

 

IF USING THE METHYL GREEN AGAR, plate the agar plate on a white background for good contrast. Look for yellowish/ tan zones around the growth areas. No reagent is added.

QUESTIONS

  1. When HCl is added to this agar, what happens to remaining DNA in the agar?
  2. Why look at the DNAse agar against a different background?
  3. What is the advantage of using DNAse agar medium with methyl green?

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