CRITERION™ DNASE TEST AGAR WITH TOLUIDINE BLUE

Cat. no. C5670 CRITERION™ DNase Test Agar with Toluidine Blue 84gm
Cat. no. C5671 CRITERION™ DNase Test Agar with Toluidine Blue 500gm
Cat. no. C5672 CRITERION™ DNase Test Agar with Toluidine Blue 2kg
Cat. no. C5673 CRITERION™ DNase Test Agar with Toluidine Blue 10kg
Cat. no. C5674 CRITERION™ DNase Test Agar with Toluidine Blue 50kg

INTENDED USE

Hardy Diagnostics CRITERION™ DNase Test Agar with Toluidine Blue is recommended for the detection of DNase in gram-negative bacteria, especially Serratia spp.

This dehydrated culture medium is a raw material intended to be used in the making of prepared media products, which will require further processing, additional ingredients, or supplements.

SUMMARY

DNase with Toluidine Blue O, developed by Schreier in 1969, is a modification of the formula developed by Jeffries, Holtman, and Guse(7,8) The basal medium is composed of casein and soy peptones which supply necessary nutrients. Sodium chloride is added to maintain osmotic equilibrium. DNA is incorporated into the medium to detect organisms which possess the enzyme deoxyribonuclease, while toluidine blue O serves as the color indicator. The addition of toluidine blue eliminates the need to add HCl to the plate.

The complexing of toluidine blue O with DNA produces a blue color in the uninoculated medium. Organisms which depolymerize DNA result in the formation of a dye, oligonucleotide, and mononucleotide complex. Metachromatic properties of the indicator thereby produce a visible bright rose-pink color in the surrounding area of the organisms possessing the DNase enzyme.

Because toluidine blue may be inhibitory to some gram-positive organisms, it is recommended for the detection of DNase in gram-negative microorganisms.

FORMULA

Gram weight per liter: 42.0gm/L
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
Agar 15.0gm

Final pH 7.3 +/- 0.2 at 25ºC.

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

STORAGE AND SHELF LIFE

Store the sealed bottle(s) containing dehydrated culture medium at 2-30ºC. Dehydrated culture medium is very hygroscopic. Keep lid tightly sealed. Protect dehydrated culture media from moisture and light. The dehydrated culture media should be discarded if it is not free-flowing or if the color has changed from its original light bluish-beige.

Store the prepared culture media at 2-8ºC.

PRECAUTIONS

METHOD OF PREPARATION FOR DEHYDRATED CULTURE MEDIA

1. Suspend 42.0gm of the dehydrated culture media in 1 liter of distilled or deionized water. Stir to mix thoroughly.

2. Heat to boiling to dissolve completely.

3. Sterilize in the autoclave at 121ºC. for 15 minutes.

4. Cool to 45-50ºC.

PROCEDURE AND INTERPRETATION OF RESULTS

For information on procedures and interpretation of results, consult listed references or refer to the prepared media Instructions for Use (IFU) for Cat. No. G24.

LIMITATIONS

DNase with Toluidine Blue may be inhibitory to some strains of gram-positive bacteria, particularly staphylococci. When testing for Branhamella catarrhalis, a very heavy inoculum must be used.

An inoculum that is too broad may result in complete decolorization of the media, due to the reduction of the dye. If this occurs, the test results must be repeated.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as autoclaves, incinerators, and incubators, etc., are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Serratia marcescens
ATCC ® 8100
E 24-48hr 35°C Aerobic Growth; red zone around colonies
Escherichia coli
ATCC ® 25922
E 24-48hr 35°C Aerobic Growth; no color change

User Quality Control

PHYSICAL APPEARANCE

CRITERION™ DNase Test Agar with Toluidine Blue powder should appear homogeneous, free-flowing, and light bluish-beige in color. The prepared media should appear clear, slightly opalescent, and dark blue in color.

REFERENCES

1. 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.

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

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

4. MacFaddin, J.F. 1985. Media for Isolation, Cultivation, Identification, Maintenance of Bacteria, Vol. I. Williams & Wilkins, Baltimore, MD.

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

6. Quality Assurance for Commercially Prepared Microbiological Culture Media, M22. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, PA.

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

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


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

052316gr