Nitrate Broth is a growth media used to carryout  the biochemical test ”nitrate reductase test”.The purpose of nitrate reduction test is to detect nitrate reduction by some bacteria. The ability to reduce nitrate by the enzyme Nitrate reductase found in some bacteria is valuable for differentiating and identifying various types of bacteria especially those of the family of Enterobacteriaceae

Principle of Nitrate reduction Test

• Members of Enterobacteriaceae characteristically reduce nitrate to nitrite which reacts with sulfanilic acid and N,N-dimethyl-1-naphthylamine to produce a red color.

• This reaction is known as Griess reaction. Therefore if an organism grows rapidly and actively reduces nitrate, the test should be performed after an early incubation period since the nitrite may be further reduced to nitrogen.

nitrate reduction test medium composition

The following ingredients are used for the composition Nitrate Broth

Ingredients	Grams / Litre
Peptic digest of animal tissue	5.000
Meat extract	3.000
Potassium nitrate	1.000
Sodium chloride	30.000
Final pH at temperature 25°C	7.0±0.2

How to Prepare Nitrate Broth

1. Start by suspending 39 grams in 1000 ml distilled water
2. Heat if necessary in order to  dissolve the medium completely.
3. Now dispense in tubes then  sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.

Appearance of Medium after preparation

It appears light amber coloured clear to slightly opalescent solution forms in tubes.

Reagents for Nitrate Reduction Test

Reagents	How to Prepare
Sulfanilic Acid	Dissolving 8 grams of sulfanilic acid in 1 litre 5 N acetic acid
Alpha-Naphthylamine reagent	Achieve by dissolve 5 grams of alpha-naphthylamine in 1 litre 5 N acetic acid

Nitrate Reduction Test Procedure

1. Inoculate the broth  with a 18 to 24 hour old pure culture.
2. Then incubate for 24-48 hours at  temperature 20-24 °C aerobically
3. After incubation  of the broth,add about 5 drops of a-naphthylamine and sulfanilic acid to the medium and shake gently in order to mix reagents..
4. If there is no color development in the broth after addition of a-naphthylamine and sulfanilic acid then add a small amount of zinc dust to  to confirm the absence of nitrate in the medium

Result and interpretation

Positive Test	Observe the formation of a pink or red color in the medium within 1-2 minutes following the addition of a-naphthylamine and sulfanilic acid. or no color development within 5-10 minutes after adding zinc dust.
Negative Test	There is no pink or red color development within 1-2 minutes following the addition of a-naphthylamine and sulfanilic acid or red color development within 5-10 minutes after adding zinc dust..

Culture observation and Nitrate reduction reaction

Cultural Response	Nitrate Reduction
Bacillus cereus	Positive reaction, red colour developed within 1-2 minutes
Enterobacter aerogenes	Positive reaction, red colour developed within 1-2 minutes
Escherichia coli	Positive reaction, red colour developed within 1-2 minutes
Salmonella Typhimurium	Positive reaction, red colour developed within 1-2 minutes

Some Precaution to Take into Consideration

• Always make sure to kindly watch for the color change as the color may fade quickly.
• Before final determination of results are made, be sure to add a small amount of zinc dust. Too much zinc dust can a quick reduction of the nitrate resulting in a false negative reaction.
• Also the nitrate reduction test is not a confirmatory test hence complete bacteria  identification should include the morphology, gram reaction, biochemical and serological tests.
• Strongly reducing bacteria may exhibit a brown precipitate.

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 Ewing’s 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