Deoxyribonuclease (DNase) Test – Principle, Procedure, Result

Test Name	Deoxyribonuclease (DNase) Test
Detection	DNases enzyme
Uses	To find out if an organism can break down DNA and use it to get carbon and energy so it can grow. To distinguish Staphylococcus aureus from other Staphylococci.
Result	DNase Test Agar: After adding 1N HCl, a clear zone forms around the growth line, but the rest of the plate is still cloudy. DNase Test Agar w/ Methyl Green: A green agar plate with a growth line and a colourless area around it. DNase Test Agar w/ Toluidine Blue: The rest of the plate is blue, but the area around the growth line is pink.
Require	DNase agar without indicator or DNase agar with methyl green or DNase agar with toluidine blue O
Quality Control	Serratia marcescens – Positive Escherichia coli – Negative

• DNases are enzymes that break down DNA into nucleotides and phosphate that are free to move around.
• DNases are extracellular endonucleases that bacteria make. They cut DNA, which makes a lot of oligonucleotides.
• There are different ways to find these enzymes. Some don’t use an indicator (they need to be mixed with HCl reagent), while others do (toluidine blue O or methyl green).
• DNase agar with methyl green is used as the medium. DNA is added to nutrient agar, which is the medium. The medium made by the indicator methyl green is mint green.
• DNase agar is a differential medium that tests an organism’s ability to make deoxyribonuclease, or DNase, an exoenzyme that breaks down DNA.
• DNase agar has food for the bacteria, DNA, and an indicator called methyl green. Methyl green is a cation that sticks to DNA, which has a negative charge.
• The organisms that make deoxyribonuclease can use it to break DNA into smaller pieces.
• When the DNA is broken down, it no longer binds to the methyl green, and a clear halo will appear around where the DNase-producing organism has grown.
• In 1957, Jeffries and his colleagues came up with a way to show that microorganisms with deoxyribonuclease (DNase) activity have this extracellular enzyme.
• When the plate is flooded with 1N hydrochloric acid, there is a clear zone around the colonies where the DNA has been broken down (HCl).
• Some types of bacteria, like staphylococci, enteric gram-negative bacilli, and pseudomonads, can be found with the help of DNase Test Agar.
• In 1969, changes to DNase Test Agar were described. These changes included an indicator system made of a dye that made it unnecessary to flood the plate with HCl.
• On DNase Test Agar with Methyl Green, organisms that make DNase form colonies that are surrounded by a clear zone. The rest of the medium is green.
• Schreier put toluidine blue in the DNase Test Agar so that Serratia, Klebsiella, and Enterobacter spp. would be easy to tell apart.
• On DNase Test Agar with Toluidine Blue, colonies that make DNase, like Serratia, are surrounded by a pink zone.
• Since HCl does not need to be added to DNase Test Agar with an indicator system, the test isolate is not killed by the acid and is still alive.
• If the plate is negative after one day, it can be put back in the oven, and colonies can be picked right from the surface of the agar for more testing.

Purpose of DNase Test 

• To find out if an organism can break down DNA and use it to get carbon and energy so it can grow.
• To distinguish Staphylococcus aureus from other Staphylococci.

Principle of DNase Test 

• DNases are enzymes that break down DNA into nucleotides and phosphate that are free to move around.
• Bacteria make an enzyme called deoxyribonuclease, which is an extracellular endonuclease that breaks down DNA and makes a lot of oligonucleotides.
• DNase agar with methyl green is used as the medium. DNA is added to nutrient agar, which is the medium. The medium made by the indicator methyl green is mint green.
• The media used to find these enzymes can be made with or without indicators (such as toluidine blue or methyl green) that show when DNA is broken down.
• Casein and soy peptones provide bacteria with the nitrogen, amino acids, and peptides they need to grow.
• Sodium chloride gives you the electrolytes you need to keep the osmotic balance.
• Organisms that make DNase break down DNA into nucleotide parts like mononucleotides and oligonucleotides.
• The DNase Test Agar used changes how DNase activity shows up.
• In the case of DNase Test Agar, when 1N HCl is added to the plate after incubation, it reacts with the polymerized DNA in the medium to make free nucleic acid and a cloudy precipitate.
• Around and below DNase-producing colonies, where DNA has been broken down, the agar is clear, while the rest of the plate is cloudy.
• When DNA is present, methyl green in DNase Test Agar with Methyl Green forms a stable coloured complex.
• If the DNA is broken down, methyl green turns into a colourless compound, and DNase-producing colonies on an otherwise green agar plate are surrounded by clear zones.
• In DNase Test Agar with Toluidine Blue, toluidine blue reacts with DNA to make a blue complex. When DNA is broken down, the structure of toluidine dye changes, making the area around colonies that make DNase pink.

Agar for DNase test 

DNase agar without indicator

Jeffries came up with the original formula. He put DNA into trypticase soy agar. To find polymerized DNA in this medium, acid (HCL) had to be added.

Ingredients	Gram/liter
Tryptone	15.0
Soya peptone	5.0
Deoxyribonucleic acid (DNA)	2.0
Sodium chloride	5.0
Bacteriological agar	15.0
1 N HCl

Final pH ( at 25°C) 7.3±0.2

Note: After HCl was added, the agar became clear, which showed that DNA was being broken down (the oligonucleotides dissolve in acid, but DNA salts are insoluble).

1. Suspend 42 grammes in 1000 ml distilled water.
2. Heat the medium and stir it often to dissolve it completely.
3. Use an autoclave for 15 minutes at 12 to 15 lbs of pressure (118°C to 121°C).
4. When it reaches 45°C, pour it into sterile petriplates.

DNase agar with methyl green

Ingredients	Gram/liter
Tryptone	15.0
Soya peptone	5.0
Deoxyribonucleic acid (DNA)	2.0
Sodium chloride	5.0
Bacteriological agar	15.0
methyl green	0.5 gm

Final pH ( at 25°C) 7.3±0.2

According to P. B. SMITH, adding methyl green dye to an agar medium that already contains deoxyribonucleic acid makes it easier to find bacteria that make deoxyribonuclease. When you use the dye, you don’t have to use acid to show that deoxyribonuclease is working. This means that colonies can be subcultured or re-incubated.

DNase agar with methyl green Preparation

1. Suspend 42 grammes in 1000 ml distilled water.
2. Heat the medium and stir it often to dissolve it completely.
3. Use an autoclave for 15 minutes at 12 to 15 lbs of pressure (118°C to 121°C).
4. When it reaches 45°C, pour it into sterile petri plates.
5. Before sterilizing the medium, add 0.5 gm of methyl green, or after incubation, flood the plates with 0.1% methyl green solution.

Note: At pH 7.3, a stable green complex can be made when methyl green dye binds to polymerized DNA. The action of DNase breaks down the structure of DNA, releasing methyl green. Free methyl green loses its colour on its own at a pH of 7.3, creating colourless halos around colonies that are positive for DNase.

DNase agar with toluidine blue O

Ingredients	Gram/liter
Tryptone	15.0
Soya peptone	5.0
Deoxyribonucleic acid (DNA)	2.0
Sodium chloride	5.0
Bacteriological agar	15.0
Toluidine Blue	0.1 g

Final pH ( at 25°C) 7.3±0.2

Schreier says that it was made to make it easy and quick to tell S. marcescens apart from Klebsiella, Enterobacter, and Serratia, which are also in the KES division.

DNase agar with toluidine blue O Preparation

1. Suspend 42 grams in 1000 ml distilled water.
2. Heat the medium and stir it often to dissolve it completely.
3. Use an autoclave for 15 minutes at 12 to 15 lbs of pressure (118°C to 121°C).
4. When it reaches 45°C, pour it into sterile petriplates.
5. Before sterilising the medium, add 0.1 g Toluidine Blue (FD051) or, after incubation, flood the plates with 0.1% Toluidine Blue (FD051) solution.

Note: Toluidine Blue O (TBO) is a metachromatic dye, which means that when it is mixed with other substances, it changes colour. When TBO binds to polymerized DNA (uninoculated medium), the result is a royal blue colour. When DNA is broken down, TBO binds to oligonucleotides or mononucleotides, which changes the structure of the dye and its absorption spectrum, making the colour bright pink. Rose-pink rings show up around colonies that are DNase-positive, which have a blue background. Lior and Patel said that when testing Campylobacter species, DNase Agar with TBO is the best way to do it. Also, TBO might stop some gram-positive bacteria from growing, including some strains of Staphylococcus aureus.

Procedure of DNase Test 

1. The inoculum should come from a pure culture that has grown on Heart Infusion Agar, Sheep Blood Agar, or in BHI Broth for at least one night.
2. Using a direct inoculum, make a straight line from the edge to the middle of the plate (1 to 1 1/2 inches long).
3. On a single plate, you can spread up to eight test organisms. If you want to inoculate a tube, streak the tube from the bottom up, like a fish’s tail.
4. Incubate the plates for 18 to 24 hours at room temperature or 33 to 37°C in the air around them.
5. After the DNase Test Agar has been incubated, flood it with 1N HCl and look for a clear area around the growth line.
6. Both the DNase Test Agar with Methyl Green and the DNase Test Agar with Toluidine Blue do not need 1N HCl added. Look at the “Interpretation of the Test” to find out how well these media support growth.

Detection of DNase activity by flooding with hydrochloric acid

1. Pour 1M hydrochloric acid over the plate to a depth of a few millimetres to separate the DNA that hasn’t been broken down.
2. Let the DNase test agar plate sit for a few minutes so that the reagent can soak in.
3. Pour out any extra hydrochloric acid and look at it against a dark background.
4. Always compare the area around the test strain to the areas that aren’t being tested.
5. In the DNase test agar plate, HCl reacts with DNA salts to precipitate DNA that hasn’t been broken down yet. This makes a white, cloudy area in the agar.

Detection of DNase activity by flooding with toluidine blue O (TBO) solution

1. Flood the plate with a few millimetres of TBO to make a complex with either DNA that has been broken down or DNA that has not been broken down.
2. Wait 3–5 minutes before touching the DNase test agar plate.
3. Pour out extra TBO and check right away
4. Always compare the area around the test strain to the areas that aren’t being tested.
5. Read for up to 30 minutes in 5 minute chunks.
6. TBO binds to DNA that has been broken up by water to make a complex that makes bright pink zones around colonies on a royal blue background. Organisms that don’t have DNase don’t change the colour of the background.

Inoculation Types

There are two ways to get an immunisation. Spot inoculations or band (line) streak inoculations are two of them.

Spot inoculation

1. Touch a colony of the organism being tested with a loop and put it on a small area of the DNase test agar plate in the middle of one of the marked sections. After incubation, a thick plaque of growth 5–10 mm in diameter will form. It also helps to poke holes in the agar and plate it out.
2. The plate should be kept at 37°C for 18–24 hours.

Band or line streak inoculation3

1. Use a heavy inoculum and draw a line from the edge of the DNase test agar plate to the middle that is 3–4 cm long.
2. The plate should be kept at 37°C for 18–24 hours.

Result Interpretation Of Deoxyribonuclease (DNase) Test

DNase Test Agar

• Positive Test – After adding 1N HCl, a clear zone forms around the growth line, but the rest of the plate is still cloudy.
• Negative Test – When 1N HCl is added, there is no clear area around the growth line, and the whole plate is cloudy.

DNase Test Agar w/ Methyl Green:

• Positive Test – A green agar plate with a growth line and a colourless area around it.
• Negative Test – There is no area around the growth line, and the whole plate is green.

DNase Test Agar w/ Toluidine Blue:

• Positive Test – The rest of the plate is blue, but the area around the growth line is pink.
• Negative Test – There is no pink around the growth line, and the whole plate is blue.

Organism	Result
Pseudomonas aeruginosa	Negative
Serratia marcescens	Positive
Campylobacter jejuni	Positive
Corynebacterium diphtheriae	Positive
Streptococcus pyogenes	positive
Staphylococcus aureus	Positive
Moraxella catarrhalis	Positive
Klebsiella pneumoniae	Negative
Escherichia coli	Negative
Staphylococcus epidermidis	Negative

Quality Control

Using the following quality control organisms, all lot numbers of DNase Test Agar with and without Additives have been tested and found to be good. Control organisms should be tested using quality control methods that have already been set up in the lab. If there are problems with quality control, patient results shouldn’t be sent out.

Organism	Result
Serratia marcescens	Positive
Staphylococcus aureus	Positive
Escherichia coli	Negative
Staphylococcus epidermidis	Negative

Limitations of DNase Test 

• On DNase Test Agar with Toluidine Blue, some strains of staphylococci and other gram-positive bacteria may not be able to grow.
• Some S. aureus stains don’t grow well on DNase Test Agar, but growth isn’t necessary to find DNase activity.
• DNase Test Agar with and without Additives is meant to be used as an extra test to identify different organisms. For sure identification of the test isolate, you may need to do more biochemical tests.
• Some types of MRSA do not react to the enzyme DNase.
• Some Staphylococci that don’t react to the coagulase test might only react weakly to the DNase test.
• When testing for gram-negative rods, the best medium to use is methyl green.
• If you are using a medium that doesn’t have an indicator, you need to add 1N HCl, and you should know what the result means within five minutes. If you look at the result after five minutes, it might be different.
• If you are testing for Gram-positive cocci or Moraxella, don’t use a low inoculum because it could give you a false-negative result. The medium is not good for growing these kinds of organisms.
• Lior says that some strains of Campylobacter can’t handle methyl green, which is why the TBO formulation is used.
• Methyl green and toludine blue O stop the growth of many bovine strains of Staphylococcus species. Because of this, these mediums should not be used to test staphylococci from animals.

Uses of DNase Test 

• The Deoxyribonuclease (DNase) test shows if an organism can break down DNA and use it as a source of carbon and energy to grow.
• To identify potential pathogenic staphylococci. And to tell the difference between Staphylococcus aureus subsp. aureus (+) and S. epidermidis (-)
• To help tell Klebsiella-Enterobacter (-) apart from Klebsiella, Enterobacter, and Serratia spp. (+), except for S. proteamaculans subsp. protemacuans (V+) and S. fonticola (-).
• To tell the difference between Aeromonas spp. (-) and Plesiomonas shigelloides (-),
• To tell Campylobacter jejuni (V), C. coli (V+), C. coli (V+), C. lari (+), and Helicobacter pylori (+) apart from other Campylobacter spp. (-)
• To tell Moraxella catarrhalis (+) apart from other Moraxella spp. (-) that are often found.

References

• Deoxyribonuclease (DNAse) Test. (2021, August 1). https://bio.libretexts.org/@go/page/3665
• https://microbiologie-clinique.com/dnase-test-agar-principle-interpretation.html#:~:text=Deoxyribonuclease%20(DNase)%20test%20is%20used,release%20free%20nucleotides%20and%20phosphate.
• https://himedialabs.com/TD/M482.pdf
• https://www.austincc.edu/microbugz/dnase_test.php
• https://vumicro.com/docs/deoxyribonuclease-dnase-test/
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