Deoxyribonuclease (DNase)

Test: Principle, Procedure and

results
DECEMBER 23, 2014 BY TANKESHWAR ACHARYA IN BACTERIOLOGY,
BIOCHEMICAL TESTS IN MICROBIOLOGY, LABORATORY DIAGNOSIS OF
BACTERIAL DISEASE

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.
This medium is pale green in color because of DNA-methyl
green (indicator) complex (Note: Methyl green is a cation which
binds to the negatively-charged DNA). It also contains nutrients
for the bacteria.

Figure -1: DNA Hydrolysis test A. Positive; Staphylococcus aureus B.

Positive; Serratia marcescens C. Negative: Staphylococcus epidermidis

If the organism that grows in the medium produces

Deoxyribonuclease, it breaks down DNA into smaller fragments.

When the DNA is broken down, it no longer binds to the methyl

green, and green color fades and the colony is surrounded
by a colorless zone (See fig-1).
Requirements:
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Media: DNase Agar or DNase agar with Methyl green indicator.

Reagent: Hydrochloric acid (1mol/L) only when DNase agar without
indicator is used
Others: Inoculating loop, Bunsen burner

Procedure of DNase (DNA hydrolysis test)

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Dry the Surface of agar plates before use. Each plate may be divided into
sections by drawing lines on the bottom of the plate.
Inoculate the test agar medium: There are two types of inoculation that can
be done.

Spot Inoculation
more

Touch a colony of the organism under test with a loop and inoculate it onto
a small area of the DNase test agar plate, in the middle of one of the
marked sections to form a thick plaque of growth 5-10mm in diameter after
incubation.
Incubate the plate at 37C for 18-24hr.

Band or line streak inoculation

Use a heavy inoculum and draw a line 3-4 cm long from the rim to the
centre of the DNase test agar plate
Incubate the plate at 37C for 18-24hr.
When using DNase agar without indicator,
Flood the plate with 1N Hydrochloric Acid.
Leave the plate to stand for a few minutes to allow the reagent to absorb
into the plate. Decant excess hydrochloric acid and then examine the plate
within 5 minutes against a dark background.

Fig:2: Dnase Test: M. catarrhalis (+ve) and N.gonorrhoeae (-ve). When

DNase is produced by organisms, an acidic end product is formed and the
pH indicator changes from red (alkaline) to yellow (acid).

Expected results:
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Positive: When DNA is hydrolysed, methyl green is released turning the

medium colorless around the test organism.
Negative: If there is no degradation of DNA, the medium remains green.

Test results
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DNase Test positive organisms:

Serratia marcescens
Staphylococcus aureus
Campylobacter jejuni (some strains)
M. Catarrhalis
DNase test negative organisms:
Staphylococcus epidermidis
Neisseria gonorrhoeae

Uses of DNase Test:

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It is used to differentiate S.aureus (DNase +ve) from other Staphylococci

that do not produce such enzyme. The DNase test is particularly useful
when plasma is not available to performed a coagulase test or when the
results of a coagulase test are difficult to interpret.
DNase test distinguishes M. catarrhalis from all other gram-negative
diplococci (e.g. Neisseria gonorrhoeae & Neisseria meningitidis) of human
origin

Limitation of DNase Test

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Some MRSA strains do not give positive DNase test result and some
strains of the coagulase negative staphylococci such as Staphylococcus
capitis may give weak reactions.
Serratia and Moraxella species also produce deoxyribonuclease.

1N HCl is bactericidal for staphylococci. Once the HCl has been applied,
the test must be read within 5 minutes and cannot be continued by reincubation.