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Short answer
The DNase agar test detects whether a bacterium or fungus produces deoxyribonuclease, an enzyme that breaks down DNA. The plate contains DNA as a substrate. Where an organism’s colony digests the DNA, a clear zone forms around it. On the methyl green version of the medium, that zone shows against a green background and needs no added reagent.
What the DNase agar test detects
According to the HiMedia technical data sheet for DNase Test Agar with Methyl Green (M1419), the medium is intended for the detection of deoxyribonuclease activity of bacteria and fungi, and especially for the identification of pathogenic staphylococci from clinical samples. It is an in vitro diagnostic medium for professional use. It supports the growth of both gram-positive and gram-negative organisms, so the result depends on the enzyme, not on a selective effect of the medium.
The test is a differential plate. It does not identify an organism on its own: a DNase result is one piece of evidence that sits alongside colony morphology, Gram reaction and other biochemical tests. Other agar media covered on CASRAI include the bile esculin agar test, MacConkey agar and CNA agar; the general format is explained in what an agar plate is.
How the clear zone forms on methyl green DNase agar
The HiMedia data sheet describes the chemistry. The medium holds DNA as the substrate. Methyl green combines with polymerized DNA to form a stable green complex at pH 7.5. When an organism secretes DNase, the enzyme depolymerizes the DNA. As hydrolysis proceeds, methyl green is released from the complex and fades to colourless, leaving a distinct clear zone around the colonies in an otherwise green medium. The data sheet states that reagent addition is not required.
The published formulation per litre is tryptose 20.000 g, DNA 2.000 g, sodium chloride 5.000 g, methyl green 0.050 g and agar 15.000 g, with a final pH of 7.3 ± 0.2 at 25 °C.
DNase agar versus thermonuclease (toluidine blue) agar
Two sourced formats look similar on a catalogue page but answer different questions. Thermo Fisher’s Remel instructions for use (IFU1895) describe Thermonuclease Agar with Toluidine Blue as a qualitative procedure for the presumptive identification of Staphylococcus aureus. It relies on the fact that the S. aureus nuclease is thermostable while the nucleases of other organisms are thermolabile. Broth cultures are heated before testing, so heat-sensitive nucleases are destroyed and only the thermostable enzyme remains to produce a zone.
| Feature | DNase Test Agar with Methyl Green (HiMedia M1419) | Thermonuclease Agar with Toluidine Blue (Remel IFU1895) |
|---|---|---|
| Stated purpose | Detect DNase activity of bacteria and fungi; identify pathogenic staphylococci | Presumptive identification of S. aureus |
| Enzyme targeted | Any DNase, heat treatment not part of the principle | Thermostable nuclease; heat step removes thermolabile nucleases |
| Dye | Methyl green (green complex fades to colourless) | Toluidine blue (positive shows a pink zone with a darker blue outer ring) |
| Positive result | Clear zone around colonies | Pink zone of clearing at the edge of the test well |
| Incubation stated | 35–37 °C for 18–24 hours | Plate incubated 2–4 hours |
The same organism can give opposite results
The two data sheets list overlapping quality control strains, and the comparison is instructive. Serratia marcescens ATCC 8100 is the positive control on DNase agar with methyl green, showing a clear halo. On the thermonuclease agar it is the negative control. The thermonuclease procedure heats the broth first, and the nuclease of organisms other than S. aureus is described as thermolabile, which fits that result. In contrast, S. aureus ATCC 25923 is positive on both media and S. epidermidis ATCC 12228 is negative on both. A lab that moves between the two media therefore cannot reuse expected results from one data sheet for the other.
Controls and plate-handling checklist for lab staff
Values below are as stated in the two manufacturers’ documents.
- Control strains, methyl green DNase agar (35–37 °C, 18–24 h): S. marcescens ATCC 8100 positive; S. aureus ATCC 25923 positive; Streptococcus pyogenes ATCC 19615 positive; S. epidermidis ATCC 12228 negative. Growth is expected to be luxuriant for all.
- Control strains, thermonuclease agar: S. aureus ATCC 25923 positive; S. epidermidis ATCC 12228 negative; S. marcescens ATCC 8100 negative.
- Culture condition: the HiMedia sheet states that test organisms must be in pure culture and 18–24 hours old.
- Storage: dehydrated powder is kept at 10–30 °C tightly closed; prepared medium is kept at 2–8 °C.
- Medium limits: the Remel insert notes that some staphylococci are inhibited by the thermonuclease medium.
Common misunderstandings
A positive DNase plate does not name the organism
Several species produce DNase. The manufacturers describe their media as aids to identification, and the Remel procedure is explicitly presumptive. Use the result with other tests.
Positive on one DNase medium does not mean positive on another
As the S. marcescens controls show, the medium format changes the expected answer.
A clear zone is not always green-on-colourless
On methyl green agar the zone is colourless against green; on toluidine blue agar a positive is pink. Reading a plate against the wrong colour key leads to misreads.
Any culture will do
The HiMedia instructions require test organisms in pure culture, 18–24 hours old.
FAQ
What is the DNase agar test used for?
It detects deoxyribonuclease activity in bacteria and fungi, and is used especially in identifying pathogenic staphylococci from clinical samples, according to HiMedia’s data sheet.
Why does the plate show a clear zone?
DNase breaks down the DNA that holds methyl green in a green complex. The dye is released and fades, leaving a colourless zone around the colony.
Is a reagent needed to read the methyl green plate?
No. The HiMedia data sheet states that reagent addition is not required.
What is the difference between DNase agar and thermonuclease agar?
Thermonuclease agar uses heated broth cultures so that only the thermostable S. aureus nuclease gives a positive result, and it uses toluidine blue rather than methyl green.
Sources
- HiMedia Laboratories, Technical Data M1419, DNase Test Agar with Methyl Green: himedialabs.com/media/TD/M1419.pdf (checked 2026-10-29).
- Thermo Fisher Scientific / Remel, IFU1895, Thermonuclease Agar with Toluidine Blue: documents.thermofisher.com/TFS-Assets/LSG/manuals/IFU1895.pdf (checked 2026-10-29).
