DNase test  is useful in the identification of Staphylococcus aureus which produces deoxyribonuclease (DNase) enzymes. This test is particularly useful when plasma is not available to perform a coagulase test or when the results of a coagulase test are difficult to interpret.

Principle

DNase test is used to test the ability of an organism to hydrolyze deoxyribonucleic acid (DNA). In the test, organism is cultured on a medium containing DNA. After overnight incubation, the colonies are tested for DNase production by flooding the plate with a weak hydrochloric acid solution. The acid precipitates unhydrolyzed DNA making the medium opaque. Organisms that hydrolyze DNA produce a clear zone around the growth area.

This test may be used for the identification of Staphylococci but may also be used for the detection of DNase activity in other microorganisms.

Method

• Divide a DNA-ase plate into the required number of strips by marking the underside of the plate.
• Using a sterile loop, inoculate the test and control organisms. Make sure each test area is labelled clearly.
• Incubate the plate aerobically at 35^oC for 13 to 24 hours.
• Flood the surface of the plate with 1 N hydrochloric acid solution. Tip off the excess acid.
• Look for clearing around the colonies within 5 minutes of adding the acid.

Expected Results

Clearing around the colonies . . . . . . . . . . DNase positive strain

No clearing around the colonies . . . . . . . . DNase negative strain

Alternatively,

This test can also be performed on DNase agar that contains methyl green indicator. This method does not require addition of hydrochloric acid for visualization of the result.

Principle

Methyl green is a cation which binds to the negatively-charged DNA. The medium is pale green in color because of DNA-methyl green complex. Organisms producing DNase hydrolyze DNA and breaks down DNA into smaller fragments. When the DNA is broken down, it no longer binds to the methyl green, and a clear halo will appear around the areas where the DNase-producing organism grow.

Method

• Using a sterile loop, inoculate the DNase agar with test and control organisms.
• Incubate the plate aerobically at 35^oC for 13 to 24 hours.

Expected Results

Positive: When DNA is hydrolysed, methyl green is released and combines with highly polymerized DNA at pH 7.5 turning the medium colorless around the test organism

Negative: If there is no hydrolysis of DNA the medium remains green.

Note: Some methicillin resistant S. aureus (MRSA) strains give a negative DNA-ase test. Some coagulase negative staphylococci are weakly positive. Also, S. pyogenes, Moraxella and Serratia species frequently give a positive DNA-ase test.

Quality control strains

Staphylococcus aureus ATCC 25923 – DNase positive

Staphylococcus epidermidis ATCC 12228 – DNase negative

 

Views – 12827