Nitrate Broth

Composition**

Final pH (at 25°C) 7.0±0.2

**Formula adjusted, standardized to suit performance parameters

Directions

Suspend 39 grams in 1000 ml 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.

Principle And Interpretation

Nitrate Broth is prepared in accordance with the formula published in 'Pure Culture Study of Bacteria' of the Society of American Bacteriologist (1) and present modified formula is recommended by BIS (2). The ability to reduce nitrate is valuable for differentiating and identifying various types of bacteria especially Enterobacteriaceae family (3). Recently ISO committee (4) has recommended Nitrate Broth for the enumeration of Bacillus cereus - colony count technique at 30°C (4).

Non-fermenters and other miscellaneous gram-negative bacilli vary in their ability to reduce nitrates. Some members of this group are capable of denitrification which is a reduction of nitrate to nitrogen gas. For the glucose fermenting gram-negative bacilli, the production of nitrogen gas from nitrate is an important differential test (4). Reduction of nitrate is generally an anaerobic respiration in which an organism derives its oxygen from nitrate. Members of Enterobacteriaceae characteristically reduce nitrate to nitrite which reacts with sulfanilic acid and N,N-dimethyl-1-naphthylamine to produce the red colour. This reaction is known as Griess reaction. If an organism grows rapidly and reduces nitrate actively, the test should be performed after an early incubation period since the nitrite may be further reduced to nitrogen.

Preparation of Nitrate Test Reagents:

1. Sulfanilic Acid: Dissolve 8 grams of sulfanilic acid in 1 litre 5 N acetic acid.
2. Alpha-Naphthylamine reagent : Dissolve 5 grams of alpha-naphthylamine in 1 litre 5 N acetic acid.

For the test: 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 (5). Nitrate reduction is not a confirmatory test. Complete identification should include the morphology, gram reaction, biochemical and serological tests. Addition of excess zinc may result in false-negative reaction.

Quality Control

Appearance Cream to yellow coloured homogeneous free flowing powder

Colour and Clarity of prepared medium Light amber coloured clear to slightly opalescent solution forms in tubes.

Reaction Reaction of 3.9% w/v aqueous solution at 25°C. pH: 7.0±0.2

Cultural Response

M439S: Cultural characteristics observed after an incubation at 35-37°C for 18-24 hours.

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

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