NITRATE BROTH WITH DURHAM TUBE

Cat. no. K42 Nitrate Broth with Durham Tube, 15x103mm Tube, 6ml 20 or 100 tubes/box

INTENDED USE

Hardy Diagnostics Nitrate Broth with Durham Tube is recommended for use in the detection of nitrate reduction by bacteria.

SUMMARY

The ability of bacteria to reduce nitrate is an important biochemical characteristic which aids in the identification of many microorganisms, particularly members of the family Enterobacteriaceae, and members of the Haemophilus, Neisseria, and Branhamella genera.(2,3,5,6)

Organisms which possess the enzyme nitroreductase vary in their ability to reduce nitrate. Some microorganisms reduce nitrate to nitrite while others further reduce the nitrate to form other end products such as ammonia, nitrogen gas, hydroxylamine, etc. The end product of nitrate reduction is dependent upon the bacterial species.(7)

The reduction of nitrate to nitrite is determined by the development of a red color complex upon the addition of sulfanilic acid solution (Nitrate Reagent A, Cat. no. Z71) and N,N-dimethyl-1-naphthylamine (Nitrate Reagent B, Cat. no. Z72). The sulfanilic acid reacts with nitrite to form a diazonium salt which then couples with N,N-dimethyl-1-naphthylamine to produce a red-dye complex. Absence of a red color reaction indicates that the organism has further reduced the nitrites to ammonia or nitrogen gas, or that unreduced nitrate is present, thus indicating the organism does not possess the nitroreductase enzyme.

If an organism does not possess the enzyme, nitrate will remain present in the medium. Application of zinc dust (Nitrate Reagent C, Cat. no. Z73) will convert nitrate to nitrite to form a red-dye complex. This test reaction is considered negative for nitrate reduction. If, however, the organism has reduced nitrate beyond nitrite to nitrogen gas, application of zinc dust will not produce a color change. The test is then considered positive for nitrate reduction.

FORMULA

Ingredients per liter of deionized water:*

Pancreatic Digest of Gelatin 5.0gm
Beef Extract 3.0gm
Potassium Nitrate 1.0gm

Final pH 6.9 +/- 0.2 at 25ºC.

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

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-30ºC. Media should not be used if there are any signs of contamination, deterioration 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: This medium is not intended for primary isolation of patient specimens. It should be used only with cultures of isolated organisms. This product is used in conjunction with other biochemical tests to identify cultures of isolated organisms.

Method of Use:

1. Prior to inoculation, allow the medium to equilibrate to room temperature.

2. Using a loopful of inoculum from an 18-24 hour pure culture, inoculate the broth. A very heavy inoculum is recommended for organisms that do not grow well in the medium (such as some Neisseria spp.). See the "Limitations" section for more information.

3. Incubate the tube with loose cap at 35-37. in an aerobic atmosphere for 24-48 hours.

4. Observe for growth and the presence of gas bubbles in the durham tube. If gas is present and the organism is known to be a non-fermenter, the test is considered positive for nitrate reduction. If growth is apparent but no gas is present, or if gas is present and the organism is known to be a fermenter , proceed with the following steps:

5. Add five drops of Nitrate Reagent A (Cat. no. Z71) and five drops of Nitrate Reagent B (Cat. no. Z72) to the broth. Note: add reagents in the order listed.

6. Gently shake tube to mix reagents.

7. Observe for the development of a deep red color within two minutes following application of the reagents.

8. If a red color does not result after step 6 above, add approximately 6.0mg of Nitrate Reagent C (Cat. no. Z73) to the medium.

9. Observe for the development of a red color within 5-10 minutes following the addition of Nitrate Reagent C. A red color indicates that nitrate was not reduced. Absence of a red color reaction indicates nitrate reduction beyond nitrite.

INTERPRETATION OF RESULTS

A positive nitrate reduction test is indicated by the development of a deep red color after the addition of Nitrate Reagents A and B or no development of color after the addition of Nitrate Reagent C.

A negative nitrate reduction test is indicated by the absence of a deep red color complex after the addition of Nitrate Reagents A and B and formation of a red color complex after the addition of Nitrate Reagent C.

LIMITATIONS

Test isolates must be in pure culture and 18-24 hours old.

A very heavy inoculum is recommended for organisms that do not grow well in the medium (such as some Neisseria spp.). The heavy inoculum will provide sufficient preformed enzymes for the reaction to occur.

Interpretation of color reactions should be made immediately, as color reactions with a positive test may fade rapidly.

If air bubbles are present in the durham tube prior to inoculation, the tube should be inverted until the air is released from the durham tube. Failure to remove air bubbles prior to inoculation may result in reading the result as a false-positive reaction for gas reduction.

A faint pink color may be produced following addition of the nitrate reagents. This is not a positive result. Development of a deep red color is indicative of a positive reaction.

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 very 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.

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

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, slides, staining supplies, Nitrate Reagents A, B and C, other culture media, microscopes, incinerators, incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Reaction
Escherichia coli
ATCC ® 25922
Positive nitrate reduction;
deep red color seen after the addition of Reagents A and B
Acinetobacter baumannii
ATCC ® 19606
Negative nitrate reduction;
no color change seen after the addition of Reagents A and B,
and red color forms after addition of Reagent C

User Quality Control

PHYSICAL APPEARANCE

Nitrate Broth with Durham Tube should appear clear, and light amber in color.

Positive Nitrate Reduction

Escherichia coli (ATCC® 25922) grown in Nitrate Broth with Durham Tube (Cat. no. K42). Incubated aerobically for 24 hours at 35ºC. Five drops of Nitrate Reagent A (Cat. no. Z71) and five drops of Nitrate Reagent B (Cat. no. Z72) were added subsequent to incubation. The red color development was indicative of a positive reaction: the reduction of nitrate to nitrite.

Negative Nitrate Reaction

Acinetobacter baumannii (ATCC® 19606) grown in Nitrate Broth with Durham Tube (Cat. no. K42). Incubated aerobically for 24 hours at 35ºC. Five drops of Nitrate Reagent A (Cat. no. Z71) and five drops of Nitrate Reagent B (Cat. no. Z72) were added subsequent to incubation. No red color development indicated that nitrate was not reduced to nitrite (presumptive negative). To ensure nitrate was not reduced to an end product other than nitrite, all negatives should be confirmed by adding Nitrate Reagent C (Cat. no. Z73). A red color development is indicative of a "true negative" for nitrate reduction.



Reagent C Conformation

Showing confirmation reaction for Acinetobacter baumannii (ATCC® 19606). Since there was not red color development after the addition of Nitrate Reagents A and B, a small amount of Nitrate Reagent C, zinc dust (Cat. no. Z73) was added using the end of a sterile wooden stick. The red color development was indicative as a "true negative" for nitrate reduction.



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

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

5. Ewing, W.H. 1986. Edwards and Ewing's Identification of Enterobacteriaceae, 4th ed. Elsevier Science Publishing Co., Inc., New York.

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

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


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012617gr