A Deoxyribonuclease (DNase) test is performed to find out the organism’s ability to hydrolyze DNA and use it as a carbon source, which will in turn use as energy for the organism to grow.
A DNase agar medium, pale green in color which is primarily used as a differential medium. It has the nutrients needed by the bacteria to grow.
The organism in the medium has the ability to produce Deoxyribonuclease if it has broken down the DNA into smaller fragments. An indicator that the DNA has broken down is when it does not bind to methyl green. The color will eventually fade away and you will notice that there is a colorless zone surrounding the colony. (1, 2, 3, and 4)
Image 1: A DNA hydrolysis test; quadrant A and B test positive while quadrant C tests negative.
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Image 2: A positive DNase test is characterized by a clear zone around inoculation line. The test is negative if the agar remains green and clear throughout
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What are the uses of DNase Test?
- It is performed to find out the organism’s ability to hydrolyze deoxyribonucleic acid.
- It aids in differentiating Staphylococcus aureus, which has the ability to produce Deoxyribonuclease enzyme from other Staphylococci that do not have the ability to produce DNase.
- It can be used in performing a coagulase test, especially when plasma is not available. It is also a more reliable test than the coagulase test as there are times when the result of the coagulase test is difficult to interpret.
- It has the ability to distinguish M. catarrhalis from gram-negative diplococcic of human origins such as N. gonorrhoea and N. meningitidis. (3, 4, 5, and 6)
What are the requirements?
- DNase agar (with methyl green indicator)
- Hydrochloric acid but it is only used when the media does not have an indicator.
- Bunsen burner
- Inoculating loop (5)
How does a Deoxyribonuclease (DNase) Test perform?
Note: Make sure that the agar plate’s surface is clean and dry. Divide the agar plate into three parts by simply drawing lines on the plate. (7)
Image 3: The left side of the plate tests negative while the right side tests positive to DNase test.
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Inoculation can be done in two ways. These are the following:
- Spot Inoculation – using a loop, a colony of the organism is touched and inoculate onto a small area of the plate. It is usually placed at the center of one of the sections of the plate. After incubation, you will notice a growth of thick plaque which is about 10 mm in diameter. Incubate the plate for at least 24 hours at a 37 degree Celsius.
- Band/line streak inoculation – Draw a line using an ample amount of inoculum, which is about 4 cm long. Begin at the rim going to the center of the agar plate. Incubate for at least 24 hours at a temperature of 37 degree Celsius.
Note: If the medium you are using does not have an indicator, what you need to do is to put an ample amount of 1N Hydrochloric Acid. Let it stand there for a few minutes so that the reagent can thoroughly absorb into the plate. After a few minutes, check the plate against a dark background. (7, 8, and 9)
A positive result is characterized by a colorless medium around the test organism. It means that the organism being tested has the ability to hydrolyze DNA. Examples of DNase test positive organisms are:
- Staphylococcus aureus
- Serratia marcescens
- M. Catarrhalis
- Some strains of Campylobacter jejuni (1, 10)
The test is negative if the medium remains the same in color (green). It simply means that the organism being tested does not have the ability to hydrolyze DNA. Examples are:
- Staphylococcus epidermidis
- Neisseria gonorrhoeae (3, 5)
- Some strains of MRSA do not test positive to DNase.
- Some Staphylococci that test negative to coagulase test may result in weak reactions to DNase Test.
- If you are using a medium that does not contain an indicator, you need to add 1N HCl and the result should be interpreted within five minutes after the application. Interpreting the result after five minutes may alter the result.
- If you are testing gram-negative rods, it is best to use a methyl green medium.
- If you are testing Gram-positive cocci and Moraxella, you should avoid using a low inoculum as it may result in a false-negative reading. Such organisms do not grow well on the medium. (2, 5, 7, and 9)