DNA hydrolysis test or Deoxyribonuclease (DNase) test is used to determine the ability of an organism to hydrolyze DNA and utilize it as a source of carbon and energy for growth. An agar medium; DNase agar, a differential medium is used to test the ability of an organism to produce deoxyribonuclease or DNase.
Objectives of DNase Test Agar
DNase test is used for detecting deoxyribonuclease activity of Bacteria and fungi, and particularly for the identification of pathogenic Staphylococci and also used for distinguishing Serratia spp from Enterobacter spp., Staphylococcus aureus from other species, and Moraxella catarrhalis from Neisseria sp.
Principle of DNase Test
DNase test is used to determine the organism that produces deoxyribonuclease an extracellular endonuclease enzyme that breaks down DNA into smaller fragments and releases free nucleotide and phosphate.
In 1956, Weckman and Catlin showed a positive correlation between DNase activity and coagulase activity and suggested that DNase activity could be used to identify potentially pathogenic staphylococci. The original formulation was devised by Jeffries et al.
who incorporated DNA into trypticase soy agar; this medium required the addition of acid to detect polymerized DNA. The addition of dyes to the medium proved beneficial as the acid-addition step could be precluded; the addition of methyl green was reported by Smith, Handcock, and Rhoden, and the toluidine blue O modification was reported by Schreier.
DNase test with Methyl Green Dye
In this case, the DNase agar plate contains Methyl green dye as an indicator. Methyl green intercalates (binds) between the base of the double-stranded DNA with an association constant of approximately (9×10-5 M-1 ) for native DNA and (1×10-5 M-1)for denatured DNA. A maximum number of dye molecules can bind approximately per 5 base pairs of native double-stranded DNA.
Methyl Green binds with the DNA and form a complex, formation of methyl green-DNA complex shows green color at 7.3 or neutral pH. However, at this pH methyl green are remains colorless.
When DNase (deoxyribonuclease) performs enzymatic hydrolysis of phosphodiester bond and breaks the DNA molecules that produce free methyl green molecules. And the free methyl green spontaneously decolorized at the 7.3 or neutral pH is likely due to the result from the tautomerization of dye.
As a result, the zone of clearance on solid media and decrees in color intensity in liquid media are observed based on the ability of DNase activity produced by the test Organism.
DNase test with Toluidine Blue O
Toluidine Blue O (TBO) is a metachromatic dye that changes color when complex to different substances. When TBO complexes with polymerized DNA (uninoculated medium), a royal blue color result.
When DNA is hydrolyzed, TBO complexes with oligonucleotides or mononucleotides and result in a change in the dye structure and absorption spectrum yielding a bright pink color. DNase-positive colonies appear with rose-pink halos on a blue background.
Lior and Patel recommended the use of DNase Agar with TBO when testing Campylobacter species. It should also be mentioned that TBO might be inhibitory to some gram-positive bacteria including some strains of Staphylococcus aureus.
DNase test with No Indicator Dye
In DNase agar without an indicator, the hydrolysis of DNA is observed by a clearing of the agar after the addition of HCL (oligonucleotides dissolve 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.
Media and Requirements for DNase test
- Test organism (18-24 hour old young culture)
- DNase Agar plate
- Tryptone…………………………………. 15 gm/l
- Soya peptone……………………………..5 gm/l
- Deoxyribonucleic acid (DNA)……… 2 gm/l
- Sodium chloride (NaCl)……………… .5 gm/l
- Agar powder……………………………..15 gm/l
- Final pH (at 25oC)…………………… 7.3 ± 0.2
- DNase test agar with Methyl green
- Methyl Green …………………… 0.05 gm/l
- DNase test agar with Toluidine Blue O
- Toluidine Blue O ……………… 0.1 gm/l
- DNAse test without indicator dye
- 1N HCl
Procedure Of DNase Test
- Take a sterile DNase agar medium plate with the respective indicator dye you are using and streak a loop full of culture from a previously incubated test organism plate.
- Then incubate this plate at 37° C for overnight or 24 hours at the aerobic conditions.
- After incubation observe the color change or zone of clearance if you are using indicator dye.
- In the case of DNase without indicator
- Flood the agar plate with the 1N HCl and the tip of access acid from the plate
- Then Allow the reagent to absorb into the plate. Wait for 5 min to react to acid with the substrate.
- Observe the zone of clearance around the colonies.
DNase Test Agar with Methyl Green
DNase Positive (+): Clear zones around bacterial streak and colonies against a green background. Clear zones are best observed against a white background.
DNase Negative (−): No color change, medium remains green
DNase Test Agar with Toluidine Blue O
DNase Positive (+): Bright rose-pink zone around bacterial streak or colonies against a royal blue background.
DNase Negative (−): No color change, medium remains blue
DNase test without indicator
DNase positive (+): DNase positive organisms will be surrounded by clear zones of depolymerized DNA while the medium farther away from the inoculation band will be opaque and whitish due to polymerized DNA.
DNase negative (-): Colonies of DNase negative organisms will not show any clearing around the colonies.
Uses of DNase test
- Used to determine the ability of an organism to hydrolyze deoxyribonucleic acid.
- Used to differentiate Staphylococcus aureus which produces the enzyme deoxyribonuclease from other Staphylococci which do not produce DNase.
- Particularly useful if the plasma is not available to perform a coagulase test or when the result of coagulase tests are difficult to interpret.
- It is also used to distinguish Serratia (positive) from Enterobacter sp. Moraxella catarrhalis (positive) from Neisseria
Limitations of the DNase test
- Many bovine strains of Staphylococcus species are inhibited by methyl green and toluidine blue O, therefore, these mediums should not be used for testing staphylococci of animal origin.
- According to Lior, methyl green is toxic to some strains of Campylobacter necessitating the use of the TBO formulation.
- 1N HCL is bactericidal. Hence if the HCL has been applied the test must be read within 5 minutes.
- Methyl green medium is better for gram-negative rods that first grow on the medium and then demonstrate a positive test.
- Some MRSA strain does not give a positive result and some strain of coagulase-negative Staphylococci such as Staphylococcus capitis gives a weak positive reaction.
- For Moraxella and Gram-positive cocci with TBO testing, a low inoculum can result in a false-negative test, since these organisms may not grow well on the medium.
- Additional tests should be performed on isolated colonies from the pure culture in order to complete identification.
Positive: Staphylococcus aureus (ATCC25923)
Negative: Escherichia coli (ATCC25922)