DNase test, DNA hydrolysis - Principle, DNAse Media, Procedure, Interpretation

Introduction to DNase test / DNA hydrolysis

DNase test is also known as the DNA hydrolysis test.

As Deoxyribonucleic acid (DNA) is a large polymer of nucleotides, it is too large to be transported intact into the bacterial cell membrane. So, to utilize the external DNA, which is a source of nitrogen, phosphate, and carbon, bacteria secretes an exoenzyme called DNases. These extracellular endonucleases hydrolyze/cleave DNA into nucleotides and phosphate.

The nucleotides are then transported across the bacterial cell wall with the help of transport proteins to make nucleic acids.

Purpose of DNase test, DNA hydrolysis

The production of extracellular DNases has been reported in a subset of prokaryotes including some human pathogens.

This test is used to determine if the test microorganism (bacteria, fungi) is able to hydrolyze DNA and utilize the nucleotides as a source of carbon, phosphate, nitrogen, and energy for growth.

Specifically, the DNA hydrolysis test distinguish

• Moraxella catarrhalis (positive) from Neisseria spp. (negative)

• Staphylococcus aureus (positive) from other Staphylococcus spp

• Serratia (positive) from Enterobacter spp. (negative)

Principle of DNase test, DNA hydrolysis

DNase agar, a differential medium, is used in the DNase test. This agar contains DNA, bacterial nutrients, and methyl green as an indicator. Methyl green works as a cation by binding with the negatively-charged DNA (anion) forming DNA-methyl green complex.

If the test organism grown in the DNase agar breaks down DNA into smaller nucleotides, the DNA in the agar can no longer bond with the indicator Methyl green. As a result, a clear halo will appear around the colonies indicating the organism can produce extracellular endonuclease- DNase.

In cases of the absence of an indicator in the medium, 1N HCl can be added after colony growth. HCL dissolves oligonucleotides but since DNA salts are insoluble, areas of the agar with unhydrolyzed DNA appear opaque. Here too, the clearing of the agar indicates the production of DNase by the test organism.

Media used for DNase test, DNA hydrolysis

DNase medium/media components:

Pancreatic digest of casein (10 g)

yeast extract (10 g)

deoxyribonucleic acid (2 g)

NaCl (5 g)

agar (15 g)

methyl green (0.5 g)

pH 7.5

Procedure for DNase test, DNA hydrolysis

The procedures for the DNase test, and DNA hydrolysis includes:

1. Inoculate (streak) the test organism on the DNase agar

2. Incubate the agar plate at 35-37°C for 18-24 hour

3. Observe for clearing of the agar near colony growth

4. In the case of DNase agar without indicators, flood with 1N HCl solution and tip off the excess acid

5. Observe for the clear zone(s) near colonies within 5 minutes of the flooding

Interpretation

The result of the DNase test, DNA hydrolysis can be interpreted as positive or negative.

Positive

The agar medium becomes colorless around colony/colonies.

Negative

The agar medium retains its green pigment/no clear zone is observed after the addition of HCl.