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DNA Hydrolysis Test or Deoxyribonuclease

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Osama Bakheet

This document describes the DNA hydrolysis (DNase) test, which is used to determine if an organism can hydrolyze DNA to use as an energy source. It involves inoculating bacteria onto DNase agar containing methyl green dye and DNA. If the bacteria produce DNase enzyme, it will degrade the DNA and release methyl green, leaving a colorless zone around the colony. Staphylococcus aureus is DNase positive and used as a control, while Staphylococcus epidermidis is negative. The test procedure and expected results are provided.

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DNA Hydrolysis Test or Deoxyribonuclease

Uploaded by

Osama Bakheet
419 views2 pages
This document describes the DNA hydrolysis (DNase) test, which is used to determine if an organism can hydrolyze DNA to use as an energy source. It involves inoculating bacteria onto DNase agar containing methyl green dye and DNA. If the bacteria produce DNase enzyme, it will degrade the DNA and release methyl green, leaving a colorless zone around the colony. Staphylococcus aureus is DNase positive and used as a control, while Staphylococcus epidermidis is negative. The test procedure and expected results are provided.

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DNA Hydrolysis Test or Deoxyribonuclease

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This document describes the DNA hydrolysis (DNase) test, which is used to determine if an organism can hydrolyze DNA to use as an energy source. It involves inoculating bacteria onto DNase agar containing methyl green dye and DNA. If the bacteria produce DNase enzyme, it will degrade the DNA and release methyl green, leaving a colorless zone around the colony. Staphylococcus aureus is DNase positive and used as a control, while Staphylococcus epidermidis is negative. The test procedure and expected results are provided.

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© All Rights Reserved
Download as DOCX, PDF, TXT or read online from Scribd
Download as docx, pdf, or txt
419 views2 pages

DNA Hydrolysis Test or Deoxyribonuclease

Uploaded by

Osama Bakheet
This document describes the DNA hydrolysis (DNase) test, which is used to determine if an organism can hydrolyze DNA to use as an energy source. It involves inoculating bacteria onto DNase agar containing methyl green dye and DNA. If the bacteria produce DNase enzyme, it will degrade the DNA and release methyl green, leaving a colorless zone around the colony. Staphylococcus aureus is DNase positive and used as a control, while Staphylococcus epidermidis is negative. The test procedure and expected results are provided.

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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.
It is used to differentiate S.aureus (that produce DNase enzyme)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.
An agar medium; DNase agar, a differential medium is used to test the ability of an organism to
produce an exoenzyme, called deoxyribonuclease or DNase, that hydrolyzes DNA.

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: 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.

Requirements:
1. Media: DNase Agar or DNase agar with Methyl green indicator.
2. Reagent: Hydrochloric acid (1mol/L) only when DNase agar without indicator is used
3. Others: Inoculating loop, Bunsen burner
Procedure of DNase (DNA hydrolysis test)
1. Dry the Surface of agar plates before use. Each plate may be divided into sections by
drawing lines on the bottom of the plate.
2. Inoculate the test agar medium: There are two types of inoculation that can be done.

Spot Inoculation

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.

3. 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.

Expected results:
1. Positive: When DNA is hydrolysed, methyl green is released turning the medium
colorless around the test organism.
2. Negative: If there is no degradation of DNA, the medium remains green.
Quality Control:
1. Staphylococcus aureus ATCC 25923DNase positive
2. Staphylococcus epidermidis ATCC 12228 DNase negative
3. Serratia marcescens ATCC 13880 DNase positive
4.

Klebsiella pneumoniae ATCC 33495 DNase negative

Test results
1. DNase Test Positive organisms
A.

Serratia marcescens

B.

Staphylococcus aureus

C.

Campylobacter jejuni (some strains)

2. Negative Control for DNase test: Staphylococcus epidermidis


Limitation of DNase Test
1. Some MRSA strains do not give positive DNA se test result and some strains of the
coagulase negative staphylococci such as Staphylococcus capitis may give weak
reactions.
2. Serratia and Moraxella species also produce deoxyribonuclease.
3. 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 re-incubation.

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