Deoxyribonucleic acid (DNA) is a large polymer of nucleotides that is often too large to enter the cell membrane safely.
To utilize this external DNA, certain cells secrete exoenzymes DNases) from the exterior of the cell wall. The DNA is then hydrolyzed into nucleotides.
These nucleotides can then ride transport proteins in order to be utilized across the cell membrane.
Deoxyribonuclease allows organisms which can interact with it (DNase) to break DNA down into smaller pieces generally.
When the DNA is broken down like this it can often struggle to bind with methyl green, a part of the medium, and results are shown thusly.
This Deoxyribonuclease (DNase) Test can determine the ability of an organism to hydrolyze DNA for carbon energy and growth. The objective of the test is to differentiate organisms based on the production of deoxyribonuclease.
Principle
The hydrolysis of DNa by these microorganisms is identified through the clearing of the agar after HCL is added.
Oligonucleotides dissolve in acid but DNa is insoluble in this liquid.
Unhydrolyzed DNA precipitates acid and changes the medium to opaque, so DNase producing organisms hydrolyze DNA and produce a clear zone.
The test has many uses such as detecting the deoxyribonuclease activity of bacteria and fungi, especially for identifying pathogens.
It’s a general identification test that uses elimination, by which produce DNase and which doesn’t, so that we can eliminate other microorganisms for general identification.
Media
In order to discover deoxyribonuclease the following DNase medium should be used: 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.
This medium will either change color or not depending on the presence of DNase in the medium. Check the results section to see what color you should expect.
Methodology
- With a sterile loop a DNase agar should be inoculated with the organism tested
- This agar plate should be incubated at around 95 – 98.6 Fahrenheit
- Incubation will eventually lead to color change in the DNase with methyl green.
How to read results in a DNase agar without indicators:
- Fill the surface of the agar with 1N HCL solution. Tip off excess acid.
- Allow this reagent to saturate the agar plate
- You want to check for a clear zone around the colonies within the 5 minutes.
What Are The Expected Results Of The Test?
A positive test will remain colorless in the medium and around the test organism, the medium will stay blue for a positive test indicating staph aureus.
A negative test will be shown with a green stripe across the agar with no clearing, this is indicative of staph epidermidis, which means the DNA has not degraded.
Uses Of Test
Firstly, this DNAse test is a great way to simply identify staph aureus and staph epidermidis. A positive test represents the former and a negative test the latter.
If you have identified Staph aureus which produces the deoxyribonuclease from other Staphylococci which do not produce DNase.
Similarly, you can also identify Serratia spp. as it produces a DNase, this separates the latter from non pigmented strains from most other Enterobacteriaceae which is useful for general identification of microorganisms.
In essence, this test is mainly for identification of DNase interacting microorganisms than anything else.
Through identifying which microorganisms can interact with DNase we can eliminate other microorganisms for general identification purposes in the lab.
Limitations To The Test
The agar plate must be inoculated with a suspension of a young broth culture, usually around 4 hours old, or an 18 to 24 hour colony within 1 – 2 ml of saline.
This broad or large inoculum could lead to decolorization of the medium and result in a false test, repeat the test if this occurs.
MRSA strains can often not deliver a positive result and some strains of coagulase negative Staphylococci such as Staphylococcus capitis give weak positive reactions in the medium which can lead to unclear test results.
Moraxella and gram-positive cocci with TBO testing require a low inoculum and can result in a false result since the organisms grow well on the medium regardless.
Once the HCl has been added you must read the test within 5 minutes in order to get the result and this cannot be regained through incubation.
Further Reading
The listed chapters below discuss this test with more accuracy and expertise, check them out to learn more detail about this test:
- Tille, P. M., & Forbes, B. A. (2014). Bailey & Scott’s diagnostic microbiology (Thirteenth edition.). St. Louis, Missouri: Elsevier.
- Cappuccino J.G. and Sherman N. 2008. Microbiology: A Laboratory Manual, 8th ed. Pearson Benjamin Cummings, San Francisco, CA, USA.
Final Thoughts
As you can see, this test is pretty helpful for general identification of microorganisms.
You can generally categorize most microorganisms by their ability to interact with DNase and whether they hydrolyze it or not. It may be rare you need to know this specific data, but it is actually a really helpful identification tool.
When you identify a microorganisms relationship with DNase you can generally eliminate many others, helping you find the microorganism with the qualities and features that you need.
The test does have its limitations and the occasional false positive but in general it can be treated as an effective elimination tool for general identification.
Frequently Asked Questions
What Is DNase Used For?
Molecular biologists use DNase to degrade DNA in applications such as RNA isolation, reverse transcript preparation, DNA-protein interactions and more.
DNase is even used in medical settings to clear mucus in the respiratory tract.
Where Is DNase Found?
Many don’t actually realize that DNase is produced mainly within the body’s digestive systems such as the pancreas and salivary parotid glands.
With this said, there are three types of DNase generally, pancreatic, parotid, and pancreatic-parotid.
Ever since I was a little girl, I have been fascinated by science. Every holiday I would ask for the newest science kit, and spend weeks learning with it.
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