DNase Test: Introduction, Composition, Principle, Test Requirements, Procedure, Result -Interpretations, and Limitations

Introduction of DNase Test for Bacteria

DNase tests positive and negative bacteria as shown below image. This test is useful for the presumptive identification of Staphylococcus aureus which produces the enzyme deoxyribonuclease (DNase) from other Staphylococci which do not produce DNase. This test is also positive in the following organisms like Aeromonas species, Vibrio cholerae Stenotrophomonas maltophilia, Moraxella catarrhalis, and  Serratia species except for Serratia fonticola.

Composition of Dnase Test Agar with Toluidine Blue

(Hardy Diagnostics formulation)

Ingredients	Gram weight per liter
L-Pancreatic Digest of Casein	15.0gm
Papaic Digest of Soybean Meal	5.0gm
Sodium Chloride	5.0gm
Deoxyribonucleic Acid	2.0gm
Toluidine Blue	0.1gm
Agar	15.0gm
Distilled/Deionized water	1000 ml
Final pH	7.3 +/- 0.2 at 25ºC

Method of Preparation for Dehydrated Culture Media

1. Suspend 42.0 gm of the dehydrated culture media in 1 liter of distilled or deionized water and stir to mix thoroughly.
2. Heat to boiling to dissolve completely.
3. Sterilize in the autoclave at 121ºC. for 15 minutes.
4. Cool to 45-50ºC.
5. Mix well prior to dispensing.
6. Pour into each plate and leave plates on the sterile surface until the agar has solidified.
7. Store the plates in a refrigerator at 2-8°C.

Storage and Shelf Life of Dnase Test Agar

• Store at 2-8ºC  and away from direct light.
• Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), or contamination.
• The product is light and temperature-sensitive; protects from light, excessive heat, moisture, and freezing.

Principle  of DNase Test

Deoxyribonucleases (DNases) are enzymes that hydrolyze deoxyribonucleic acid (DNA) and release free nucleotides and phosphate. The DNases produced by organisms 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 deoxyribonucleic acid (DNA) is observed by a clearing of the agar after the addition of hydrochloric acid (HCl) due to the oligonucleotides dissolving in acid but DNA salts are insoluble.

Media With Indicators (In case of MG indicator)

Medium in presence of the 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 the Case of the 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 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 the detection of the preformed enzymes. 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).

Other Requirements are-

• 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 a sterile inoculating loop from 18-24 hours old culture.
• Inoculate the test and control organisms in each test area.
• Incubate the DNase agar plate at 35-37°C for 24 hours.
• After incubation observes the color change in DNase with methyl green and TBO.
• In DNase Agar Without Indicator, steps 1,2, and 3 are common.
• Flood the surface of the 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 a 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 and Interpretation of DNase Test

DNase agar (without indicator)

Positive: Development of a 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 DNase 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

1. Lynae S. Carcia, Second Edition update, Clinical Microbiology Procedures Handbook
2. Tille, P. M., & Forbes, B. A. (2014). Bailey & Scott’s diagnostic microbiology (Thirteenth edition.). St. Louis, Missouri: Elsevier.
3. B.D. Skerman, A guide to the identification of the genera of bacteria, The Williams & Wilkins Co., Baltimore, MD, (1967)
4. Cowan and Steel’s, manual for the identification of medical bacteria
5. https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/CRITN-DNaseTestAgarTolBlue.htm
6. https://www.fishersci.ie/shop/products/oxoid-dnase-agar/10566323
7. https://himedialabs.com/TD/M482.pdf