NOCARDIA HYDROLYSIS KIT

Cat. no. Z604 Nocardia Hydrolysis Kit 20 tubes/kit
Contains five tubes each of the following formulas:
Cat. no. Q60A Casein Agar, 20x125mm Tube, 18ml Deep
Cat. no. Q60B Xanthine, 20x125mm Tube, 18ml Deep
Cat. no. Q60C Hypoxanthine, 20x125mm Tube, 18ml Deep
Cat. no. Q60D Tyrosine, 20x125mm Tube, 18ml Deep

INTENDED USE

Hardy Diagnostics Nocardia Hydrolysis Kit is designed to aid in the differentiation of aerobic Actinomycetes, Nocardia, Actinomadura, and Streptomyces species by determining the presence of hydrolysis enzymes within these species.

SUMMARY

These agars are useful in differentiating Nocardia , Actinomadura , and Streptomyces species on the basis of their patterns of hydrolysis. Certain species of aerobic Actinomycetes produce hydrolytic enzymes that degrade proteins, purines and amino acids. If hydrolysis occurs, a clearing of the media takes place.

FORMULA

Ingredients per liter of deionized water:*

Casein Agar (Q60A):
Skim Milk 75.0gm
Agar 15.0gm
Xanthine Agar (Q60B):
Pancreatic Digest of Gelatin 5.0gm
Xanthine 4.0gm
Beef Extract 3.0gm
Agar 15.0gm
Hypoxanthine Agar (Q60C):
Hypoxanthine 5.0gm
Pancreatic Digest of Gelatin 5.0gm
Beef Extract 3.0gm
Agar 15.0gm
Tyrosine Agar (Q60D):
Tyrosine 5.0gm
Pancreatic Digest of Gelatin 5.0gm
Beef Extract 3.0gm
Agar 15.0gm

Final pH 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. in the dark. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

PRECAUTIONS

PROCEDURE

Specimen Collection: Not applicable since these media are not primary isolation. These media are used in characterizing pure cultures. 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. (3,4,6)

Place the tubes in a boiling waterbath to melt agar.

After agar has melted, allow to cool to just short of solidification, and pour into chilled (2-8ºC.) 15x100mm petri dishes.

Inoculate by streaking or spot inoculating (about the size of a quarter) each plate with a pure suspension of previously isolated organisms. Incubate at 25ºC. (or 35ºC. if necessary) for up to 3 weeks. Examine each section for growth and clearing or hydrolysis at 7, 14, and 21 days.

Expected Results:
Organism Casein Tyrosine Xanthine Hypoxanthine
Nocardia otitidiscaviarum
( caviae )
- - + +
Nocardia asteroides - - - -
Nocardia brasiliensis + + - +

INTERPRETATION OF RESULTS

Positive Test - Clearing is observed around and/or beneath colony growth (hydrolysis).

Negative Test - No clearing is observed around and/or beneath the inoculum.

LIMITATIONS

It is essential that the tyrosine and xanthine be kept in suspension while the media solidifies. After boiling the agar deeps, allow the molten agar to cool just short of solidification. Refrigerate the petri dishes and remove just prior to pouring. Mix the agar deeps thoroughly, and distribute evenly into the cold plate.

It is essential that a control organism be placed on each plate to determine the efficacy of the media.

Media should be freshly prepared for optimum reactions.

All crystals should dissolve completely before pouring into the plate.

Sealing the plates may be necessary to reduce dehydration and possible aerial dissemination of hyphal structures (MycoSeal™ Cat. no. SS9225).

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, other culture media, swabs, applicator sticks, MycoSeal™ (Cat. no. SS9225), incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms
Inoculation Method*
Incubation
Results
Time
Temperature
Atmosphere
Nocardia brasiliensis
ATCC ® 19296
G 2 weeks 15-30°C Aerobic Growth on all and positive for casein, hypoxanthine and tyrosine hydrolysis
Nocardia otitidiscaviarum
( caviae )
ATCC ® 14629

G 2 weeks 15-30°C Aerobic Growth on all and positive for xanthine and hypoxanthine hydrolysis
Nocardia asteroides
ATCC ® 3308
G 2 weeks 15-30°C Aerobic Growth on all and negative for casein, xanthine, hypoxanthine and tyrosine hydrolysis

User Quality Control

PHYSICAL APPEARANCE

N. otitidiscaviarum growing on Hypoxanthine Agar

Nocardia otitidiscaviarum (ATCC ® 14629) growing on Hypoxanthine Agar (Cat. no. Q60C) from the Nocardia Hydrolysis Kit (Cat. no. Z604). Showing positive growth, positive hydrolysis. Incubated aerobically for 2 weeks at 30ºC.

REFERENCES

1. Campbell, M.C. and J.L. Stewart. 1980. The Medical Mycology Handbook, John Wiley and Sons, New York, NY.

2. Haley, L.D. and C.S. Calloway. 1978. Laboratory Methods in Medical Mycology, 4th ed. U.S. Department of Health, Education, and Welfare. Publication No. (CDC) 78-8361, Washington, D.C.

3. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology, J.B. Lippincott Company, Philadelphia, PA.

4. Lennette, E.H., et al. 1985. Manual of Clinical Microbiology, 4th ed. American Society for Microbiology, Washington, D.C.

5. Balows, A., K.L. Hermann, H. Isenberg, H.J. Shadomy, and W.J. Hausler, Jr. 1991. Manual of Clinical Microbiology, 5th ed. ASM, Washington, D.C.

6. Baron, E.J. and S.M. Finegold. 1990. Bailey and Scott's Diagnostic Microbiology, 8th ed. The C.V. Mosby Company, St. Louis. MO.

7. McGinnis, M.R., R.F. D'Amato and G.A. Land. 1987. Pictorial Handbook of Medically Important Fungi and Aerobic Actinomycetes, Praeger Scientific, New York, NY.

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

9. Cumitech 3A; Quality Control and Quality Assurance Practices in Clinical Microbiology, ASM, 1990.


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

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