Question

Why is the uninoculated control sector relatively unnecessary in a DNase test?

Answer 1

Answer:

To differentiate Staphylococcus aureus which produces the enzyme deoxyribonuclease from other Staphylococci which do not produce deoxyribonuclease (DNase), this test is presumptively used. Staphylococcus aureus possesses a heat stable enzyme, a thermonuclease. To detect this enzyme, first destroyed organisms by heat and then the free DNase reacts with medium.This test is also given positive by Vibrio, Helicobacter, Serratia, Moraxella and Aeromonas.

The ability of an organism that produce DNase are determined by this test. DNase are extracellular endonuclease that cleave DNA and release free nucleotides and phosphate. To detect these enzymes, DNase agar using no indicators or various indicators (toluidine blue or methyl green) are used to detect the hydrolysis of DNA.

In DNase agar without indicator, the hydrolysis of DNA is observed by a clearing of the agar after addition of HCL (oligonucleotides dissolves in acid but DNA salts are insoluble). The acid precipitates unhydrolyzed DNA making the medium opaque. Therefore, DNase producing colonies hydrolyze DNA and produce a clear zone around the growth.

In case of DNase agar with methyl green, DNA combines with methyl green (act as cation) to produce mint green color. When the DNA is hydrolyzed, the complex is released and the free methyl green is colorless at pH 7.5. So the clear halo is appeared around the areas where DNase producing organism grow.

When toluidine blue O (TBO) is added to the DNase agar, a complex is formed with the DNA, which changes structure when DNA is hydrolyzed, resulting in a bright pink color.

DNse test method:

1. Using a loop which is sterile, several colonies from an 18-24 hours culture is picked.
2. Inoculate the test and control organism in each test area.
3. Incubate the plate at 35-37°C for 24 hours.
4. After incubation observe the color change in DNase with methyl green.
5. In DNase agar without indicators:

• Flood the surface of agar with 1N HCL solution. Tip off the excess acid.
• Allow the reagent to absorb into the plate.
• Observe for clear zone around the colonies within 5 minutes.