DNase Test: Introduction, principle, procedure,result interpretations and limitations

DNase test agar

Introduction of DNase test

DNase test is useful for presumptive identification of Staphylococcus aureus which produces the enzyme deoxyribonuclease from other Staphylococci which do not produce DNase. This test is also positive in following organisms like Aeromonas spp., Vibrio cholerae Stenotrophomonas maltophilia, Moraxella catarrhalis and  Serratia spp. except Serratia fonticola.

Principle  of DNase test

Deoxyribonucleases (Dnases) are enzymes that hydrolyze deoxyribonucleic acid (DNA) and release free nucleotides and phosphate. The DNases produced by bacteria are extracellular endonucleases that cleave DNA, yielding a high concentration  of oligonucleotides. There are several media used to detect these enzymes. They are of using without  indicators or with indicators like toluidine blue O (TBO) or methyl green (MG) to detect the hydrolysis of DNA.

Medium without indicator:

The hydrolysis of DNA is observed by a clearing of the agar after addition of HCl  due to the oligonucleotides dissolve in acid but DNA salts are insoluble.

Media with indicators (In case of MG indicator)

Medium in presence of MG indicator, DNA combines with the MG to produce a green color. When the DNA is hydrolyzed, the complex is released and the free MG is colorless at pH 7.5.

In case of TBO indicator-

When TBO is added, a complex is formed with the DNA, which changes structure when DNA is hydrolyzed, resulting in a bright pink color .

Staphylococcus aureus possesses a heat stable enzyme, a thermonuclease. To detect this enzyme, first the organisms are destroyed by heat and then the free DNase reacts with the medium.

Requirements for DNase test

Organisms tested-

  1. Gram-negative rods that are presumptive for Stenotrophomonas maltophilia (positive) and are colistin or polymyxin B resistant to separate from Burkholderia cepacia (negative). MG agar is preferred.
  2. Gram-negative diplococci that are presumptive Moraxella catarrhalis (positive). TBO agar is preferred.
  3.  Gram-positive cocci that are presumptive for Staphylococcus aureus (positive) and are difficult to separate from other closely related species and have a questionable coagulase reaction. Use only TBO for staphylococcus heat-stable testing, since it is more sensitive in detection of preformed enzyme. Some staphylococci do not grow on media containing dyes.
  4. Enterobacteriaceae to identify Serratia spp. (positive) and separate them from Klebsiella and Enterobacter. Serratia fonticola is the only Serratia spp. that is negative for DNase.
  5. Oxidase-positive, indole-positive, gram-negative rods to separate Aeromonas spp. and Vibrio cholerae (positive) from Plesiomonas shigelloides (negative)

DNase agar

TBO agar

MG agar

BHI

Sterile sticks

Needles

Inoculating loops

Pasteur pipettes 

Boiling heat block

Incubators

Control strains

DNase test

Positive controls

  1. Moraxella catarrhalis ATCC 25240—change to pink color (TBO) or colorless (MG
  2. Serratia marcescens ATCC 13880—change to pink color (TBO) or colorless (MG)
  3. Staphylococcus aureus ATCC 25923—change to pink color (TBO) or colorless (MG)

Negative control

  1. Escherichia coli ATCC 25922—no color change

Thermonuclease test

Positive control: Staphylococcus aureus ATCC 25923—change to pink color (TBO)

Negative control: Staphylococcus epidermidis ATCC 12228—no color change

Procedure of DNase test

DNase test method

  • Pick up several colonies using sterile inoculating loop from 18-24 hours old culture.
  • Inoculate the test and control organism in each test area.
  • Incubate the DNase agar plate at 35-37°C for 24 hours.
  • After incubation observe the color change in DNase with methyl green and TBO.
  • In DNase agar without indicator, steps 1,2 and 3 is common.
  • Flood the surface of agar with 1N HCl solution. Tip off the excess acid.
  • Allow the reagent to absorb into the Dnase agar plate.
  • Observe for clear zone around the colonies within 5 minutes.

Thermonuclease method

  1. Inoculate several well-isolated staphylococcal colonies on BHI with a sterile needle.
  2.  Incubate BHI at 35°C for 18 hours.
  3.  Place broth in boiling heat block for 15 minutes.
  4. Cool to room temperature.
  5. Punch a hole in the TBO agar with the large end of a Pasteur glass pipette and remove the agar plug.
  6. Fill the well with 2 drops of cooled broth culture.
  7. Incubate at 35°C for 3 hours and finally observe for color change.

Result interpretation of DNase test

DNase agar (without indicator)

Positive: Development of clear halo around the colony.

Negative: No clear zone in the medium

MG agar

Positive test: the development of a clear halo around the colony or the well in the agar

Negative test: no clear zone in the medium or around the well in the agar and agar remains green.

TBO agar

Positive test: the development of a pink or red halo around the colony or the well in the agar

Negative test: no change in the royal blue color of the medium

Limitations of the DNse test

  1.  For Moraxella and gram-positive cocci with toluidine blue O testing, a low inoculum can result in a false-negative test, since these bacteria may not grow well on the medium.
  2. An inoculum that is too broad may result in complete decolorization of the media, due to the reduction of the dye. If this occurs, the test must be repeated.
  3.  Methyl green medium is better for organisms, such as gram-negative rods, that first grow on the medium and then demonstrate a positive test.

Bibliography

  • Lynae S. Carcia, Second Edition update, Clinical Microbiology Procedures Handbook
  • Tille, P. M., & Forbes, B. A. (2014). Bailey & Scott’s diagnostic microbiology (Thirteenth edition.). St. Louis, Missouri: Elsevier.
  • B.D. Skerman, A guide to the identification of the genera of bacteria, The Williams & Wilkins Co., Baltimore, MD, (1967)
  • Cowan and Steel’s ,manual for the identification of medical bacteria
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