Rapid DNase Broth

Cat. no. Z24 Rapid DNase Broth, 15x103mm Tube, 0.5ml 20 tubes/box

INTENDED USE

Hardy Diagnostics Rapid DNase Broth is recommended for the detection of deoxyribonuclease (DNase) activity of gram-negative and gram-positive bacteria, particularly Staphylococcus aureus, Moraxella catarrhalis, and Serratia marcescens.

SUMMARY

Jeffries, Holtman, and Guse, in 1957, reported the development of a medium which detected deoxyribonuclease activity of microorganisms.(1) When plates, containing 18-24 hour growth of bacterial isolates, were flooded with 0.1N hydrochloric acid (HCl), organisms possessing deoxyribonuclease (DNase) degraded the DNA into nucleotide fractions resulting in a clear zone around the colonies. DNase with Toluidine Blue O, developed by Schreier in 1969, is a modification of the formula developed by Jeffries, Holtman, and Guse.(1,2) The addition of toluidine blue eliminated the need to add HCl to the plate.

Rapid DNase Broth has been formulated to allow rapid determination of DNase activity, giving results as early as one hour after incubation, thus eliminating the need for overnight incubation of a DNase plated medium. The broth medium is composed of casein and soy peptones, which supply necessary nutrients, and sodium chloride to maintain osmotic equilibrium. DNA is incorporated into the medium to detect organisms which possess the enzyme deoxyribonuclease. Toluidine blue serves as the color indicator.

The complexing of toluidine blue with DNA produces a blue color in the uninoculated medium. Organisms possessing DNase hydrolyze the DNA in the broth, resulting in uncomplexed toluidine blue and a milky white broth appearance. Organisms that are DNase negative do not affect the toluidine blue-DNA complex and the broth remains blue.

Rapid DNase Broth is helpful for Staphylococcus, Moraxella, and Seratia spp., as well as Proteus spp. (approximately half of the Proteus spp. are DNase positive).

FORMULA

Ingredients per liter of deionized water:*

Pancreatic Digest of Casein 15.0gm
Papaic Digest of Soybean Meal 5.0gm
Sodium Chloride 5.0gm
Deoxyribonucleic Acid 2.0gm
Toluidine Blue 0.1gm

Final pH of 7.0 +/- 0.2 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8ºC. away from direct light. Media should not be used if there are any signs of deterioration, discoloration, contamination, or if the expiration date has passed. Product is temperature sensitive; protect from excessive heat and freezing.

PRECAUTIONS

PROCEDURE

Specimen Collection: This medium is not intended for primary isolation of patient specimens. It should be used only with pure cultures of isolated organisms. This product is used in conjunction with other tests for complete identification.

Method of use:

1. Prior to inoculation, allow the medium to equilibrate to room temperature.

2. Obtain organisms from an 18-24 hour pure culture grown on an appropriate plated medium and heavily (equivalent to a 4.0 McFarland turbidity standard or higher) inoculate the broth tube.

3. Incubate the inoculated broth aerobically at 35ºC. for 1-6 hours; incubate with loosened caps. Do not incubate in an atmosphere supplemented with CO2.

4. Following incubation, agitate the tube to mix, then leave the tube undisturbed for one minute.

5. Observe (after one minute) for a milky white broth color, with or without a blue ring at the top.

6. Reincubate negative reactions if desired (see "Limitations" section below).

INTERPRETATION OF RESULTS

A positive DNase reaction is indicated by the appearance of a milky white broth, with or without a blue ring at the top.

A negative DNase reaction is indicated by the medium remaining blue, with or without a dark blue ring at the top.

Strong positive reactions may be observed as early as one hour after incubation. Tubes may be read every hour or batch-read at four to six hours. It is recommended that negatives be incubated up to six hours before calling negative to avoid false-negative readings of weak positives.

LIMITATIONS

Reactions should not be read after more than one minute of the broth resting undisturbed; after one minute, the toluidine blue begins to settle back into solution and could be interpreted as a false-negative reaction.

Due to this reaction being based on a color interpretation, pure isolates should not be taken directly from chromogenic media, as colored colonies may interfere with results.

It is recommended that biochemical and/or serological tests be performed on colonies from pure culture for complete identification.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, slides, staining supplies, other culture media, microscopes, incinerators, incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Staphylococcus aureus
ATCC ® 25923**
E 2-4hr 35°C Aerobic Positive; turns milky white after agitation and resting for one minute
Escherichia coli
ATCC ® 25922**
E 2-4hr 35°C Aerobic Negative; remains blue after agitation and resting for one minute

** Recommended QC strains for User Quality Control according to the CLSI document M22 when applicable.

User Quality Control

PHYSICAL APPEARANCE

Rapid DNase Broth should appear clear, and blue to dark blue in color.

Rapid DNase Broth

Left - Negative DNase Reaction:
Escherichia coli (ATCC® 25922) was incubated in Rapid DNase Broth (Cat. no. Z24) under aerobic conditions for 2 hours at 35ºC. The tube was agitated to mix, then allowed to rest undisturbed for one minute.

Center:
Uninoculated tube of Rapid DNase Broth (Cat. no. Z24).

Right - Positive DNase Reaction:
Staphylococcus aureus (ATCC® 25923) was incubated in Rapid DNase Broth (Cat. no. Z24) under aerobic conditions for 2 hours at 35ºC. The tube was agitated to mix, then allowed to rest undisturbed for one minute.

REFERENCES

1. Jeffries, Holtman and Guse. 1957. J. Bacteriol.; 73:590.

2. Schreier, J.B. 1969. Am. J. Clin. Pathol.; 51:711-716.

3. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

4. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

5. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

6. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.


ATCC is a registered trademark of the American Type Culture Collection.

012617gr