Nitrate Broth & Nitrate Reducing Test : Principle, Composition and Results Interpretation

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The ability to reduce nitrate is useful for differentiating and identifying different types of bacteria, particularly those in the Enterobacteriaceae family. The ability of non-fermenters and other gram-negative bacilli to reduce nitrates varies. Some members of this group can denitrify, which is the conversion of nitrate to nitrogen gas. The production of nitrogen gas from nitrate is an important differential test for glucose fermenting gram-negative bacilli. Nitrate reduction is a type of anaerobic respiration in which an organism gets its oxygen from nitrate.

Nitrate Broth is recommended for detecting nitrate reduction by bacteria and is prepared in accordance with the formula published in the Society of American Bacteriologists‘ ‘Pure Culture Study of Bacteria,’ and the present modified formula is recommended by BIS.

Principle of Nitrate Broth

Members of the Enterobacteriaceae typically reduce nitrate to nitrite, which then reacts with sulfanilic acid and N, N-dimethyl-1-naphthylamine to produce the red color. This is known as the Griess reaction. If an organism grows quickly and actively reduces nitrate, the test should be performed after a short incubation period, because the nitrite may be further reduced to nitrogen.

Composition of Nitrate Broth

Peptic digest of animal tissue5.000
Meat extract3.000
Potassium nitrate1.000
Sodium chloride30.000
Final pH ( at 25°C)7.0±0.2

Preparation procedure of Nitrate Broth

  • Dissolve 39 grams in 1000 mL of distilled water.
  • Heat if necessary to dissolve the medium completely.
  • Dispense in tubes and sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.

Preparation of Nitrate Test Reagents

  • To make sulfanilic acid reagent, dissolve 8 grams sulfanilic acid in 1 liter 5 N acetic acid.
  • To make Alpha-Naphthylamine Reagent, dissolve 5 g of alpha-naphthylamine in 1 litre of 5 N acetic acid.

Nitrate Reducing Test Procedure

  • Put few drops of each reagent into the tube containing culture to be tested.
  • A distinct red or pink colour indicates nitrate reduction.
  • The results should be recorded within 5 – 10 seconds as the colour fades on standing.
  • A control (uninoculated) tube should also be tested.
  • If there is no pink colour formation, add pinch of zinc dust to confirm the absence of nitrate in the medium.
Nitrate Reduction Test Results
Nitrate Reduction Test Results

The reduction of nitrates is not a confirmatory test. Morphology, gram reaction, biochemical, and serological tests should all be included in a complete identification. Excess zinc may result in a false-negative reaction.

Quality Control of Nitrate Broth

AppearanceCream to yellow coloured homogeneous free flowing powder
Color and Clarity of prepared mediumLight amber coloured clear to slightly opalescent solution forms in tubes.
ReactionReaction of 3.9% w/v aqueous solution at 25°C. pH : 7.0±0.2
pH6.80-7.20
Cultural ResponseCultural characteristics observed after an incubation at 35-37°C for 18-24 hours
Uninoculated Nitrate Broth
Uninoculated Nitrate Broth

Bacteria Culture Response on Nitrate Broth

OrganismsNitrate Reduction
Bacillus cereus ATCC 10876Positive reaction, red colour developed within 1-2 minutes
Enterobacter aerogenes ATCC 13048Positive reaction, red colour developed within 1-2 minutes
Escherichia coli ATCC 25922Positive reaction, red colour developed within 1-2 minutes
Salmonella Typhimurium ATCC 14028Positive reaction, red colour developed within 1-2 minutes

Storage and Shelf Life

Store below 30°C in tightly closed container and the prepared medium at 2-8°C.Use before expiry date on the label.

Reference

  • Society of American Bacteriologist, Pure Culture Study of Bacteria, 1994, 12: Leaflet 11:8.
  • Bureau of Indian Standards IS: 5887 (Part IV) 1976.
  • Ewing, 1986, Edwards and Ewings Identification of Enterobacteriaceae, 4th ed., Elsevier Science Pub. Co., Inc., N.Y.
  • International Organization for Standardization (ISO), 1993, Draft ISO/DIS 7932.
  • MacFaddin, 1980, Biochemical Tests for the Identification of Medical Bacteria, 2nd ed., Williams and Wilkins, Baltimore.

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About the Author: Labweeks

KEUMENI DEFFE Arthur luciano is a medical laboratory technologist, community health advocate and currently a master student in tropical medicine and infectious disease.

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