ANAEROBE NITRATE REAGENT

Cat. no. Z134 Anaerobe Nitrate Reagent A 15ml
Cat. no. Z135 Anaerobe Nitrate Reagent B 15ml
Cat. no. Z73 Nitrate Reagent C 5.0gm

INTENDED USE

Hardy Diagnostics Anaerobe Nitrate Reagents A, B, and C are recommended for use in determining the nitrate reduction reaction of anaerobic bacteria.

SUMMARY

The nitrate reduction test is a qualitative procedure for determining the ability of bacteria to reduce nitrate. In the reaction, nitrate is reduced to nitrite, which may then be further reduced to nitrogen gas or ammonia. Determination of nitrate reduction to nitrite is a two step process. First, the reduction of nitrate to nitrite is determined by the addition of Anaerobe Nitrate Reagents A and B, then if necessary, the reduction of nitrate beyond nitrite is determined by the addition of Nitrate Reagent C (zinc dust). Hardy Diagnostics Anaerobe Nitrate Reagents are more sensitive than regular nitrate reagents when used with microaerophilic, facultatively anaerobic, and obligately anaerobic isolates.

REAGENT FORMULA

Anaerobe Nitrate Reagent A:
Sulfanilic Acid 5.0gm
Acetic Acid, 5N 1000.0ml
Anaerobe Nitrate Reagent B:
5-Amino-2-naphthalenesulfonic Acid (1,6-Cleve's Acid) 3.0gm
Acetic Acid 1000.0ml
Nitrate Reagent C:
Zinc Dust 5.0gm

STORAGE AND SHELF LIFE

Storage: Upon receipt store Anaerobe Nitrate Reagent A and Anaerobe Nitrate Reagent B at 2-30ºC. away from direct light. Nitrate Reagent C can be stored at 15-30ºC. away from direct light. Products should not be used if there are any signs of deterioration, contamination, or if the expiration date has passed.

PRECAUTIONS

PROCEDURE

Specimen Collection: This product is not intended for primary isolation of patient specimens. This product is used in conjunction with other biochemical tests to identify cultures of isolated organism.

Method of Use:

1. Inoculate and incubate Indole Nitrate Medium (Cat. no. K147) according to the technical insert.

2. After incubation, add 5 drops of Anaerobe Nitrate Reagent A and 3 drops of Anaerobe Nitrate Reagent B to the medium. Shake gently to mix the reagents.

3. Examine for the appearance of a deep red color within 1-2 minutes. Positive reactions may fade rapidly (as early as five minutes after addition of reagents A and B).

4. If the result is negative, i.e. no color development, confirm the negative finding by adding Nitrate Reagent C (approximately 6.0mg) to the tube containing the previously added Nitrate Reagents A and B.

5. Examine for the appearance of a red to pink color within 5-10 minutes.

If using nitrate reagents for commercial identification strips, such as API®, consult the manufacturer's literature.

INTERPRETATION OF RESULTS

A positive nitrite reduction is denoted by the appearance of a deep red color change after the addition of Anaerobe Nitrate Reagents A and B. Lack of color development denotes a presumptive negative nitrite reduction test. Development of a pink or red color following the addition of Nitrate Reagent C (zinc dust), confirms the negative nitrite reduction test obtained in the first phase of the test. Lack of color development after the addition of zinc dust indicates that the nitrate was reduced beyond the nitrite reaction to nitrogen gas and constitutes a positive nitrate reduction reaction.

LIMITATIONS

The nitrate reduction test may be used as an aid in the identification of bacteria.

Due to the possible presence of nitrite in culture media only a low nitrite media such as Indole Nitrate Medium (Cat. no. K147) should be used for the nitrate reduction test.

Interpretation of nitrate reduction color reactions should be made immediately, as color reactions with a positive test may fade rapidly (as early as five minutes after addition of reagents A and B).

To avoid false-negative nitrite reduction reactions, reactions must be verified by the addition of Reagent C to the medium.

A negative zinc reduction (no color change) test, in combination with a negative nitrite reaction, is presumptive indication that the nitrate was reduced beyond the nitrite stage. Although a common end product of nitrite reduction is nitrogen gas, other end products may be formed. Additional testing may be required to determine the final end products of the reaction.

Excess zinc dust has been reported to cause false-positive nitrite reduction reactions due to complete reduction of previously unreduced nitrate to ammonia.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, Indole Nitrate Medium (Cat. no. K147), incinerators, incubators, pasteur pipets, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Reaction
Propionibacterium acnes
ATCC® 29399*
Positive nitrate reduction; deep red color seen after Reagents A and B are added
Clostridium sordellii
ATCC® 9714*
Negative nitrate reduction; no color change seen after Reagents A and B are added, and red color forms after addition of Reagent C

User Quality Control

PHYSICAL APPEARANCE

Positive Nitrate Reduction

Showing positive nitrate reduction.
Propionibacterium acnes (ATCC® 29399) grown in Indole Nitrate Medium (Cat. no. K147) aerobically for 24 hours at 35ºC. Picture reflects appearance after addition of Reagent A (Cat. no. Z134) and Reagent B (Cat. no. Z135).

Negative Nitrate Reaction

Showing negative nitrate reduction.
Clostridium sordellii (ATCC® 9714) grown in Indole Nitrate Medium (Cat. no. K147) aerobically for 24 hours at 35ºC. Picture reflects appearance after addition of Reagent A (Cat. no. Z134) and Reagent B (Cat. no. Z135).

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

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

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

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

6. MacFaddin, J.F. Biochemical Tests for Identification of Medical Bacteria,, Lipincott Williams & Wilkins, Philadelphia, PA.

7. Summanen, P., et al. 2002. Wadsworth Anaerobic Bacteriology Manual, 6th ed. Veterans Administration Wadsworth Medical Center, and Departments of Medicine, and Microbiology and Immunology, UCLA School of Medicine, Los Angeles, CA.

API is a registered trademark of bioMeriuex, France.
ATCC is a registered trademark of the American Type Culture Collection.

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