HARDYDISK™ NITRATE

Cat. no. Z7071 HardyDisk™ Nitrate 50 disks/cartridge

INTENDED USE

Hardy Diagnostics HardyDisks™ Nitrate are for the differentiation of anaerobes based on the ability to reduce nitrate.

SUMMARY

Organisms that possess the enzyme nitrate reductase are able to utilize nitrogen as a terminal acceptor in their electron transport chain. In this reaction, nitrate is reduced to nitrite, which may then be further reduced to nitrogen gas or ammonias. The HardyDisk™ Nitrate detects the nitrite that these organisms produce by the colored end product that is formed after addition of nitrate reagents.

Nitrate, when combined with sulfanilic acid and N,N-dimethyl-alpha-naphthylamine forms a red compound, p-sulfobenzene-azo-napthylamine, which is indicative of a positive reaction. If there is no color production (a negative reaction) zinc dust is added to confirm the negative reaction. Zinc will reduce residual nitrate to nitrite producing a red color (a confirmed negative reaction). If both the nitrate and zinc reactions are negative, the nitrate has been broken down beyond nitrite (a positive reaction for nitrate reduction).

FORMULA

HardyDisks™ Nitrate are impregnated with potassium nitrate and sodium molybdate. The disks are imprinted with the letters NRT.

STORAGE AND SHELF LIFE

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

PRECAUTIONS

PROCEDURE

1. Streak a reduced blood agar plate to obtain confluent growth.

2. Aseptically place a Nitrate HardyDisk™ on the plate and press it to the agar with a sterile pair of forceps.

3. Incubate the plate, inverted, for 24-48 hours at 35ºC.

4. After the plates have been incubated, add one drop of Sulfanilic Acid (Nitrate Reagent A, Cat. no. Z71) and one drop of N,N-dimethyl-alpha-naphthylamine (Nitrate Reagent B, Cat. no. Z72).

5. Observe for a color change within 3-5 minutes.

6. If there is no color development, add a small amount of Nitrate Reagent C, Cat. no. Z73 (zinc dust), and examine for a red color change within 10 minutes.

INTERPRETATION OF RESULTS

A red color development after addition of Nitrate Reagents A and B is a positive test for the reduction of nitrate. If no color develops after addition of the reagents, the test is presumptively negative. Development of a red color after the addition of the Nitrate Reagent C is a confirmed negative reaction. When nitrate is reduced beyond nitrite, no color change occurs after the addition of the Nitrate Reagent C. This is considered a positive test for nitrate reduction.

LIMITATIONS

Nitrate HardyDisks™ are intended to aid in the identification of anaerobic bacteria. Additional biochemical and serological tests may be required for complete identification. See listed references for more information.(1-4)

Rapidly growing organisms may discolor Nitrate HardyDisks™, making interpretation of the color reaction difficult. In this case another method for determining nitrate reductase activity, such as the tube nitrate test, is recommended.(5)

Organisms that do not grow sufficiently may fail to produce enough nitrate reductase to visualize a positive reaction, giving a false-negative test result.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, other culture media, swabs, applicator sticks, incinerators, incubators, Nitrate Reagent A (Cat. no. Z71) and Nitrate Reagent B (Cat. no. Z72), and Nitrate Reagent C (Cat. no. Z73), etc., as well as other serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Propionibacterium acnes
ATCC ® 11827
B 24-48hr 35°C Anaerobic Positive; Red color development after the addition of Nitrate Reagents A and B
Clostridium difficile
ATCC ® 9689
B 24-48hr 35°C Anaerobic Negative; Red color development only after the addition of Reagent C (zinc dust)

User Quality Control

PHYSICAL APPEARANCE

HardyDisks™ Nitrate should appear as 1/4 inch filter paper disks imprinted with the letters NRT, and should appear white in color.

Positive Reduction, Negative

Showing a positive nitrate reduction reaction.
Propionibacterium acnes (ATCC® 11827) colonies growing on Brucella Agar with H and K (Cat. no. A30) with a Nitrate HardyDisk™ (Cat. no. Z7071) aseptically placed on the plate prior to anaerobic incubation (24 hours at 35 deg. C.). One drop of Nitrate Reagent A (Cat. no. Z71) and one drop of Nitrate Reagent B (Cat. no. Z72) were dropped onto the disk. The red color development was indicative of a positive reaction.

Nitrate Reduction, Negative

Showing a negative nitrate reduction reaction.
Clostridium difficile (ATCC® 9689) colonies growing on Brucella Agar with H and K (Cat. no. A30) with a Nitrate HardyDisk™ (Cat. no. Z7071) aseptically placed on the plate prior to anaerobic incubation (24 hours at 35 deg. C.). One drop of Nitrate Reagent A (Cat. no. Z71) and one drop of Nitrate Reagent B (Cat. no. Z72) were dropped onto the disk. The absence of a red color development was indicative of a negative reaction.

REFERENCES

1. Jorgensen., et al. Manual of Clinical Microbiology, 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. Wideman, P.A., et al. 1977. Simple disk technique for detection of nitrate reduction by anaerobic bacteria. J. Clin. Micro.; 5:315-319.


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080516gr