INDOLE NITRATE MEDIUM

Cat. no. K147 Indole Nitrate Medium with Durham Tube, 15x103mm Tube, 6ml 20 tubes/box

INTENDED USE

Hardy Diagnostics Indole Nitrate Medium is recommended for the detection of indole production and nitrate reduction by microorganisms with a wide range of oxygen tolerances including aerobes, microaerophiles, facultative anaerobes, and obligate anaerobes.

SUMMARY

Indole Nitrate Medium is a combined test medium for indole and/or nitrate determination for a variety of bacteria. The low agar concentration creates varying degrees of anaerobiosis in the media. As a result, the semi-solid media satisfies the oxygen requirements for aerobes, as well as facultative and obligate anaerobes. Hardy Diagnostics Indole Nitrate Medium can be used to assist in the identification of Bacteroides , Prevotella , Fusobacterium , and Clostridium to the species level. However, Indole Nitrate Medium is not recommended for determining indole production by the Enterobacteriaceae because false-negative results may occur. (6)

The indole test is a qualitative procedure for determining the ability of bacteria to produce indole. Casein peptone provides the source of tryptophan in the media. By producing the enzyme tryptophanase, certain microbes can deaminate tryptophan to indole. Indole is detected when it reacts with p-Dimethylamino-benzaldehyde (Kovacs Reagent, Cat. no. Z67) under acidic conditions to produce a red color, indicative of a positive reaction. (4)

The nitrate reduction test is a qualitative procedure for determining the ability of bacteria to reduce nitrate. Organisms which possess the enzyme nitroreductase vary in their ability to reduce nitrate. In the reaction, potassium nitrate is reduced to nitrite, which may then be further reduced to nitrogen gas or ammonia. The end product of nitrate reduction is dependent upon the bacterial species. (5)

If gas is not present in the Durham tube, it is necessary to detect if the organism is able to reduce nitrate to nitrite. The reduction of nitrate to nitrite is determined by the development of a red color complex upon the addition of Anaerobe Nitrate Reagent A (Sulfanilic Acid Solution, Cat. no. Z134) and Anaerobe Nitrate Reagent B (1,6 Cleve's Acid Solution, Cat. no. Z135). The sulfanilic acid reacts with nitrite to form a diazonium salt which then couples with 5-Amino-2-naphthalenesulfonic acid to produce a red dye complex, indicating a positive result for the reduction of nitrate to nitrite.

The absence of a red color reaction after the addition of Anaerobe Nitrate Reagent A and B indicates two possibilities: unreduced nitrate remains in the medium indicating the organism is not capable of nitrate reduction or that the organism has completely reduced nitrate to nitrogen gas or another nitrogen derivative like ammonia, a positive reaction for nitrate reduction. If an organism does not possess the nitroreductase enzyme, nitrate will remain present in the medium. Nitrate can be detected by the addition of Reagent C (Zinc Dust, Cat. no. Z73). Zinc converts nitrate, if present, to nitrite to form a red-dye complex, which confirms the organism is not capable of nitrate reduction.

Alternatively, if the organism has completely reduced nitrate application of zinc dust will not produce a color change, indicative of a positive reaction for the complete reduction of nitrate. If there is gas in the Durham tube and no color change upon the addition of zinc, it can be concluded that nitrate has been fully reduced to nitrogen gas. Yet, if there is no gas in the Durham tube and no color change upon the addition of zinc, it can be assumed that nitrate was fully reduced to ammonia or an another reduced nitrate compound.

FORMULA

Pancreatic Digest of Casein 20.0gm
Disodium Phosphate 2.0gm
Glucose 1.0gm
Potassium Nitrate 1.0gm
Agar 1.0gm

Final pH 7.2 +/- 0.3 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: Samples 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)

Method of Use:

1. Allow the medium to warm to room temperature prior to inoculation. When product is being used with obligate anaerobes, it may be necessary to pre-reduce the media, by boiling for 2 minutes and allowing to cool, prior to use.

2. Select a well-isolated colony of the organism to be tested. Using a sterile loop, inoculate a tube of the prepared medium.

3. Incubate the inoculated media in aerobic atmosphere at 35ºC. for 24-48 hours. If testing for anaerobes ensure the media cap is tightened.

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.

5. 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:

To Test for Indole Production:

1. Aliquot approximately 1.5ml of Indole Nitrate Medium into an additional test tube. Add 4-5 drops of the Kovacs Indole Reagent (Cat. no. Z67) and shake gently. Observe for a pink to red color, indicative of a positive result.

To Test for Nitrate Reduction:

1. Use the original tube of media to test for nitrate reduction.

2. A Durham tube is included for detecting those organisms capable of reducing nitrate to nitrogen gas. A positive result can be presumptively noted if gas is present in the Durham tube. However, it is necessary to proceed through steps 3-5 to confirm that gas production is from the complete reduction of nitrate to nitrogen gas.

3. Add five drops of Anaerobe Nitrate Reagent A (Cat. no. Z134) and three drops of Anaerobe Nitrate Reagent B (Cat. no. Z135) to the broth. Note: Add reagents in the order listed. Gently shake tube to mix reagents. Observe for the development of a deep red color within two minutes following application of the reagents. Color reactions with a positive test may fade rapidly (as early as five minutes).

4. If a red color does not result after step 3, add approximately 6.0mg of Reagent C (Cat. no. Z73) to the medium. Observe for the development of a red color within 5-10 minutes following the addition of zinc dust. A red color indicates that nitrate was not reduced.

5. After the addition of zinc, the absence of a red color reaction indicates the complete reduction of nitrate. Absence of a red color upon the addition of zinc and gas in the Durham tube confirms that the gas produced is due to the reduction of nitrate to nitrogen gas. If there is no gas in the Durham tube and no color change upon the addition of zinc, it indicates that nitrate was completely reduced to ammonia.

INTERPRETATION OF RESULTS

Interpretation of Indole Production:

A positive Kovacs tube test reaction is denoted by the appearance of a pink to red color in the top alcohol layer. Negative reactions remain colorless or light yellow.

Interpretation of Nitrate Reduction:

A positive nitrate reduction test is detected by either of two results: the development of a deep red color after the addition of Anaerobe Nitrate Reagents A and B or no development of color after the addition of Reagent C. Gas in the Durham tube presumptively indicates nitrate was reduced to nitrogen gas, especially if the organism is a known non-fermenter. The reduction of nitrate to nitrogen gas can be confirmed by the presence of gas in the Durham tube and no color reaction after the addition of Reagent C.

A negative nitrate reduction test is confirmed by a two step process: the absence of a deep red color complex after the addition of Anaerobe Nitrate Reagents A and B and the formation of a red color complex after the addition of Reagent C.

LIMITATIONS

Indole Nitrate Medium is not recommended for the determination of indole production by coliforms and other enterics as these organisms strongly reduce nitrate to nitrite, hindering the indole reaction.

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

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

As nitrogen reduction may take up to 48 hours for some organisms, it is recommended that multiple tubes of Indole Nitrate Media be inoculated if results are needed prior to 48 hours. If prior tests are negative, a final confirmatory test should be conducted at 48 hours of incubation.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as test tubes, loops, other culture media, swabs, applicator sticks, incinerators, anaerobic gas generators, anaerobic jars, and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Propionibacterium acnes
ATCC ® 29399**
A 24-48hr 35°C Aerobic Growth; nitrate positive, indole negative
Clostridium sordellii
ATCC ® 9714**
A 24-48hr 35°C Aerobic Growth; nitrate negative, indole positive

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

User Quality Control

PHYSICAL APPEARANCE

Indole Nitrate Medium should appear clear, and light amber in color.

Positive Nitrate Test

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

Negative Nitrate Test

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



Negative Indole Test

Showing negative indole test.
Propionibacterium acnes (ATCC ® 29399) grown in Indole Nitrate Medium (Cat. no. K147). Incubated aerobically for 24 hours at 35ºC. 1.5ml aliquot of medium after addition of Kovac's Indole Reagent (Cat. no. Z67).

Positive Indole Test

Showing positive indole test.
Clostridium sordellii (ATCC ® 9714) grown in Indole Nitrate Medium (Cat. no. K147). Incubated aerobically for 24 hours at 35ºC. 1.5ml aliquot of medium after addition of Kovac's Indole Reagent (Cat. no. Z67).



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. Wadsworth, et al. 2002. Anaerobic Bacteriology Manual, 6th ed. Star Publishing Company, Belmont, CA.


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

012617gr