DNase Test
Deoxyribonucleic acid (DNA) is a large polymer of nucleotides that is way too large to enter the cell membrane.  In order to utilize external DNA, bacteria cells secrete exoenzymes (DNases) outside of the cell that hydrolyze (digestion by the addition of water) DNA into nucleotides.  The nucleotides can then move across the cell membrane via transport proteins to be utilized.  The cell can use nucleotides to make nucleic acids and to use as a source of nitrogen, phosphate and carbon.  Extracellular DNases have been reported in a relatively small subset of prokaryotes, including a number of human pathogens.  DNA hydrolysis is tested by growing an organism on a DNase Test Agar plate (providing nutrients and DNA) and then checking the plate for hydrolysis.  Plates without any hydrolysis will have no clearing on the plate, whereas those exhibiting DNA hydrolysis will have zones of clearing around the growth.  Two procedures are described here, using DNase Test Agar with Methyl Green, and using DNase Test Agar with HCL.  In using DNase Test Agar with HCL, if the DNA remains intact and undigested, adding HCl to the DNA will make the plate appear cloudy and opaque.  If hydrolysis of the DNA has occurred, the addition of HCl will create a clear zone around the growth.  If using DNase Test Agar with Methyl Green, the Methyl Green complexes directly with intact DNA making the plate have a diffuse green color.  Because DNase will cut the DNA into nucleotides, it will break the Methyl Green-DNA complex, creating colorless zones around the growth.  With either proceedure, a clear zone around the growth indicates that the culture secreted DNase and hydrolysed the DNA in the medium.

Purpose: to test for the digestion of DNA by DNase exoenzymes that are secreted outside the cell.  Helpful in identifying and differentiating among Staphylococcaceae (particularly Staphylococcocus aureus), Enterobacteriaceae, Moraxellaceae, Aeromonadaceae, Campylobacteraceae, Streptococcaceae, Bacillaceae, Pseudomonadaceae, and a few other families.

Day 1 Procedure:
  1. Obtain a DNase Test Agar plate (either with or without Methyl Green) and your culture or cultures of interest.  If testing several cultures, obtain 1 plate/3 cultures, and divide the plate bottom into thirds with a heavy marker.  (Use a heavy marker because the plates are opaque or semi-opaque.)  Appropriately label the plate including what the culture is.
  2. Using the wire loop and a heavy inoculum, aseptically spot transfer a culture to the plate.  Inoculations are best done by streaking a single line 1 inch (2.4 cm) long in an area on the plate.  (Round spot inoculations of 1/4th an inch or more, about the width of a pencil, are also okay.) 
  3. Remind the instructor to set up a positive and negative control.
  4. Invert the plates and incubate them at 35 degrees C for 1-2 days.  (Enterobacteriaceae and Bacillaceae only need a day, but 2 days work better for most slower growing species.)
Day 2 Using DNase Test Agar with Methyl Green:
  1. Obtain and examine the plate(s) with Methyl Green.  The Methyl Green and DNA should make the agar look green and opaque.  Look for areas of clearing around the bacterial growth, comparing the results to the positive and negative controls.  
  2. If an organism produces a clear zone, a zone of DNA hydrolysis, score it as positive.  Otherwise, score it as negative.

Day 2 Using DNase Test Agar and HCL:

  1. Obtain the plate(s) and some 1N HCl.
  2. Flood the plates with 1N HCl and wait a few minutes for a reaction that will produce a clear zone where DNA hydrolysis has occurred.  The HCl treatment of the DNA should make the agar look cloudy and opaque from precipitating the DNA.
  3. If an organism produces a clear zone around its growth, the DNA has been hydrolysed, so score it as positive.  Otherwise, if the area is remains cloudy, score it as negative.

References:

Cappuccino J.G. and Sherman N.  2008.  Microbiology: A Laboratory Manual, 8th ed. Pearson Benjamin Cummings, San Francisco, CA, USA.

Harley J.P. and Prescott L.M. 2002.  Laboratory Exercises in Microbiology, 5th ed. McGraw-Hill Higher Education, New York, NY, USA.

Leboffe M.J. and Pierce B.E.  2011.  Exercises for the Microbiology Laboratory, 4th ed. Morton Publishing Company, Englewood, CO, USA.

MacFaddin J.F. 2000.  Biochemical Tests for the Identification of Medical Bacteria, 3rd ed.  Lippincott Williams & Wilkins, Philadelphia, PA, USA.

Zimbro M.J., Power D.A., Miller S.M., Wilson G.E., and Johnson J.A.  2009.  Difco & BBL Manual Manual of Microbiological Culture Media, 2nd ed.  Becton, Dickinson and Company, Sparks, MD, USA.

Instructor Notes:

In this test, it is best to use an unknown being tested, a positive control, and a negative control all on the same plate.  DNase Agar with Toluidine Blue may be used for the Enterobacteriaceae following the same proceedure as DNase Test Agar with Methyl Green, but the color is blue instead of green, and the hydrolysed DNA zone will appear redish instead of clear.  Bacillus subtilis ATCC 6051, Serratioa marcescens ATCC 13880, Pseudomonas aeruginosa ATCC 10145, Staphylococcus aureus ATCC 25923, Streptococcus pyogenes ATCC 19615 are DNA hydrolysis positive.  Staphylococcus epidermidis ATCC 12228 and  Micrococcus luteus ATCC 4698 are DNA hydrolysis negative.