TECHNICAL DATA SHEET #310 REV.

DNASE MEDIA
PRODUCT:
Plate and Tube Media:a
DNase Test Agar, item no. P1552
DNase Agar With Toluidine Blue, item no. T6390 (slant), P1560 (plate), T6391 (deep)
DNase Agar With Methyl Green, item no. T6380, P1550

a
see catalog for ordering options

PURPOSE:
DNase agar media are used to determine the production of deoxyribonuclease by organisms, particularly Staphylococcus aureus
and Serratia marcescens.

PRINCIPLES:
Weckman and Catlin9 demonstrated in 1957 that staphylococci produce DNase, which correlated with coagulase production of
the organism and, therefore, could be used as a biochemical marker for their identification. After incubation, the inoculated
plates are flooded with 1 N HCl. Organisms possessing the enzyme degrade the DNA into nucleotide fractions, which are not
affected by the acid, and a clear zone in the media occurs. If no DNase is produced, the plate will appear opaque as the HCl
combines with nucleate salts to yield free nucleic acid, which is cloudy.

In 1969 Schreier6 modified the DNase Test Medium by adding toluidine blue O into the agar. The DNA, and metachromatic dye
complex together, to form a blue color. When DNA is hydrolyzed, the dye becomes complexed with oligonucleotides and
mononucleotides, and the absorption spectrum of the dye shifts, resulting in a metachromatic (or pink) color in the agar. Organisms,
which produce the DNase enzyme, will have a pink zone around the colonies. Toluidine blue may be inhibitory to some gram-positive
organisms, and is therefore best suited for gram-negative bacteria.

In 1950 Kurnick3 developed an improved medium for detecting DNase production with the use of methyl green. Methyl green
combines easily with highly polymerized DNA at pH 7.5 to produce a green color complex in the agar. When the organism
produces DNase, the DNA is hydrolyzed, and methyl green fades. The result is a clearing of the agar around the colony.

FORMULAS:
Approximate, per liter deionized filtered water.

(1) DNase Test Agar:

Pancreatic Digest of Casein ......................... 10.0 g
Yeast Extract ................................................. 10.0
Deoxyribonucleic Acid ................................... 2.0
Sodium Chloride ............................................ 5.0
Agar .............................................................. 15.0
Final pH 7.3 ± 0.2 at 25°C

(2) DNase Agar With Toluidine Blue:

Same as (1) above except it also contains 0.1 g of Toluidine Blue O.

(3) DNase Agar With Methyl Green:

Same as (1) above except it also contains 0.5 g of Methyl Green.

PRECAUTIONS:*
For in vitro diagnostic use. Observe approved biohazard precautions.

Storage: Upon receipt store at 2-8°C away from direct light. Media should not be used if there are signs of contamination,
deterioration (shrinking, cracking, or discoloration), or if the expiration date has passed.

Limitations: DNase Agar With Toluidine Blue can inhibit some strains of gram-positive bacteria, particularly staphylococci.
Some organisms, such as Moraxella catarrhalis, are supported poorly on any of these media. Use of DNase Test Agar without
the dyes added may be the only medium of choice for the more fastidious organisms.

27120 SW 95th Avenue • Wilsonville, OR 97070 • 800.547.0659

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 TECHNICAL DATA SHEET #310 REV. 2
Complete decolorization of the media may take place if the inoculum is too broad, due to the reduction of the dye. Test results
exhibiting this phenomenon must be repeated.

For proper results, DNase Agar must be inoculated with a suspension of a young broth culture not over four hours old, or an 18-
to 24-hour colony in 1-2 ml saline.

For DNase Test Agar, the flooding with HCl renders the plate useless for any further testing.

Failure to incubate with loose caps may produce a false-negative result.

PROCEDURE:*
Specimen Collection: Not applicable since these are not media for primary isolation of organisms from clinical specimens.
These media are used in characterizing pure cultures of isolated organisms. Established isolation techniques and tests for purity
are necessary before inoculating these media. Direct inoculation of specimens will produce erroneous results. Information on
specimen collection may be found in standard reference texts.4

Method of Use, Tubes: Prior to inoculation, the medium should be brought to room temperature. The medium is inoculated with
an 18-to 24-hour colony suspended in 1-2 ml saline, water, or tryptone broth which has been incubated 4-8 hours (light inoculum).
Inoculate the slant with a straight wire and incubate with loose caps for 18 to 24 hours at 35°C.

Method of Use, Plates: To prepare a plated medium from an 18-ml tubed agar deep, heat the tube in a boiling water bath until
the agar is melted, cool to 50°C, pour into a sterile petri dish, and allow to solidify. If using a multipoint inoculation system, lightly
tap the top of one or two well-isolated and morphologically similar colonies and inoculate a broth culture. Deposit a broth spot
5-6 mm in diameter onto the surface of the agar plate using the replicator or any comparable device that could be readily
adaptable. Plates may also be inoculated by placing a broth spot on the plated agar surface using a loopful of the organism to
be tested. Incubate aerobically at 35°C for 18 to 24 hours.

Interpretation: DNase Test Agar: After good growth is obtained and the plates have been flooded with 5-10 ml of 1 N HCl, a
positive result is indicated by a clear zone around the inoculum, signifying depolymerization of DNA. A negative reaction is
growth with opacity surrounding the inoculum.

DNase Agar With Toluidine Blue: After incubation, a positive reaction is indicated by a pink to red to violet color through the slant
or around the inoculum. A negative reaction is growth with no change in color (blue).

DNase With Methyl Green: After incubation, a positive reaction is indicated by a colorless zone through the slant or around the
inoculum. A negative reaction is growth with no color change (green).

Materials Required but Not Provided: Standard microbiological supplies and equipment such as loops, needles, incubator,
incinerator, and inoculation system are not provided.

QUALITY CONTROL:*
Microorganisms Used (ATCC #): Expected Results:
Staphylococcus aureus (25913) (+); wk(+)/ng on DNase with Toluidine Blue.
Escherichia coli (25922) (-)
Serratia marcescens (8100) (+)
Key: See “Interpretation”

User Quality Control: Check for signs of contamination and deterioration.

BIBLIOGRAPHY:
1. Finegold, S. M., and E. J. Baron, Bailey and Scott’s Diagnostic Microbiology, 7th ed., C. V. Mosby, St. Louis, 1986.
2. Jeffries, V. D., et al., J. Bacteriol., 73:590, 1957.
3. Kurnick, N. B., Arch. Biochem., 29:41-53, 1950.
4. Lennette, E. H., et al., Manual of Clinical Microbiology, 4th ed., American Society for Microbiology, Washington, D. C., 1985.
5. MacFaddin, J. F., Biochemical Tests for Identification of Medical Bacteria, 2nd ed., Williams and Wilkins, Baltimore, 1980.
6. Schreier, J. B., Am. J. Clin. Pathol., 51:711-716, 1969.
7. Smith, P. B., et al., Appl. Microbiol., 18:991-993, 1969.
8. Washington, J. A., Laboratory Procedures in Clinical Microbiology, Springer Verlag, New York, 1981.
9. Weckman, G. B., and B. W. Catlin, J. Bacteriol., 73:749, 1957.

*For more detailed information, consult appropriate references and/or details in the preface of the PML Technical Manual.

PML MICROBIOLOGICALS, INC.

Data #310
Copyright, 1989 by PML Microbiologicals, Inc.
Revision Date: January 2001

27120 SW 95th Avenue • Wilsonville, OR 97070 • 800.547.0659

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