MODIFIED NITRATE ASSIMILATION AGAR

Cat. no. L95 Modified Nitrate Assimilation Agar, 16x125mm Tube, 6ml 20 tubes/box

INTENDED USE

Hardy Diagnostics Modified Nitrate Assimilation Agar is recommended for use in determining nitrate assimilation for the identification of yeasts and molds.

SUMMARY

The ability to utilize nitrate as a sole source of nitrogen is an important taxonomic indicator of some species. Previous research by Hipkin has grouped yeasts on the basis of nitrate utilization, with some species being able to assimilate nitrate ( Brettanomyces, Candida, Hansenula, Pachysolen, and Rhodotorula ) while others are not ( Kluyveromyces, Pichia, Saccharomyces and Schizosaccharomyces ). (8)

Nitrate assimilation is defined simply as the utilization of a nitrogen source by a microorganism in the presence of oxygen. A positive reaction is indicated by the presence of growth or the use of a pH indicator in the medium. The indicator method is a modification of the Wickerham method that was devised by Adams and Cooper. (7) The use of this method is easier to read than conventional techniques, yet it is equally as reliable. (7) Additionally, this method is not affected by carry-over of the inoculum.

Hardy Diagnostics Modified Nitrate Assimilation Agar contains a yeast carbon base composed of essential minerals and vitamins necessary for growth. Potassium nitrate provides the nitrogen source. Bromthymol blue is the pH indicator that changes from green to blue when nitrate is utilized. Agar is the solidifying agent.

FORMULA

Ingredients per liter of deionized water:*

Yeast Carbon Base 0.8gm
Potassium Nitrate 0.7gm
Bromthymol Blue 0.06gm
Nobel Agar 8.0gm

Final pH 6.3 +/- 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 (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: Specimens should be submitted directly to the laboratory without delay and protected from excessive heat and cold. If there is to be a delay in processing, the specimen should be inoculated onto an appropriate transport media and refrigerated until inoculation. Consult listed references for information on specimen collection. (1-5)

1. Allow the medium to come to room temperature prior to use.

2. Using a sterile loop, pick one to three well isolated colonies from a pure culture.

3. Streak the inoculum back and forth over the surface of the slant to evenly distribute the cells.

4. Incubate tubes aerobically, with loosened caps, at 35ºC. (or at the yeast species' optimal temperature) for 24-72 hours.

5. Examine slants for a blue color change within the medium, indicating a positive reaction for nitrate assimilation.

INTERPRETATION OF RESULTS

Positive nitrate assimilation is indicated by a blue color change in the medium.

Negative nitrate assimilation is indicated by no change or a greenish-yellow color in the medium.

LIMITATIONS

Some organisms incapable of utilizing nitrate as a source of nitrogen may not grow at all on this medium.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, swabs, applicator sticks, other culture media, 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
Cryptococcus albidus
ATCC ® 34140**
E 24-72hr 35°C Aerobic Growth; positive assimilation, blue color change
Candida albicans
ATCC ® 10231**
E 24-72hr 35°C Aerobic Growth; negative assimilation, no color change or green-yellow color change

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

USER QUALITY CONTROL

Physical Appearance

Modified Nitrate Assimilation Agar medium should appear slightly opalescent, and dark green 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. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology, J.B. Lippincott Company, Philadelphia, PA.

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. Sutton, D.A., A.W. Fothergill, and M.G. Rinaldi. 1998. Guide to Clinically Significant Fungi. Williams & Wilkins. Baltimore, MD.

8. Walker, G. M. 1998. Yeast Physiology and Biotechnology. John Wiley & Sons, Ltd. New York, NY.


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

032816gr