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 tissue | 5.000 |
| Meat extract | 3.000 |
| Potassium nitrate | 1.000 |
| Sodium chloride | 30.000 |
| 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.
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
| Appearance | Cream to yellow coloured homogeneous free flowing powder |
| Color 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 |
| pH | 6.80-7.20 |
| Cultural Response | Cultural characteristics observed after an incubation at 35-37°C for 18-24 hours |
Bacteria Culture Response on Nitrate Broth
| Organisms | Nitrate Reduction |
| Bacillus cereus ATCC 10876 | Positive reaction, red colour developed within 1-2 minutes |
| Enterobacter aerogenes ATCC 13048 | Positive reaction, red colour developed within 1-2 minutes |
| Escherichia coli ATCC 25922 | Positive reaction, red colour developed within 1-2 minutes |
| Salmonella Typhimurium ATCC 14028 | Positive 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.
