Nitrate Reduction Test
A visual guide to the nitrate reduction test, illustrating the three distinct metabolic endpoints (reduction to nitrite, reduction to nitrogen gas, or no reduction) using a Durham tube and zinc dust.
Nitrate Reduction Broths A-D
Differentiate reduction of nitrate to nitrite, complete reduction to gaseous nitrogen (N2), and negative reactions after adding reagents A & B and zinc dust.
Red color after adding sulfanilic acid (Reagent A) and alpha-naphthylamine (Reagent B); indicates reduction of nitrate (NO3-) to nitrite (NO2-). Durham tube has no gas.
Red color after adding Reagents A and B, with a gas bubble in the Durham tube. Indicates nitrate reduction to nitrite plus gas production from fermentation.
No color after Reagents A & B, and remains colorless after adding zinc dust. Gas bubble in Durham tube indicates complete reduction to nitrogen gas (denitrification).
Baseline medium. Amber broth, no gas bubble in Durham tube, serving as a negative control.
Red color after adding sulfanilic acid (Reagent A) and alpha-naphthylamine (Reagent B); indicates reduction of nitrate (NO3-) to nitrite (NO2-). Durham tube has no g...
Red color after adding Reagents A and B, with a gas bubble in the Durham tube. Indicates nitrate reduction to nitrite plus gas production from fermentation.
No color after Reagents A & B, and remains colorless after adding zinc dust. Gas bubble in Durham tube indicates complete reduction to nitrogen gas (denitrification)...
Baseline medium. Amber broth, no gas bubble in Durham tube, serving as a negative control.
Deciphering the reaction steps
| Visual clue | Meaning | Student shorthand |
|---|---|---|
| Red color after Reagents A & B | Nitrate reduced to nitrite (NO2-); reacts with sulfanilic acid & alpha-naphthylamine to form red azo dye | Positive (NO3+ to NO2-) |
| No color after Reagents A & B | Either nitrate is unreduced, OR reduced beyond nitrite to nongaseous/gaseous products | Inconclusive; must add zinc |
| Red color after Zinc dust | Zinc chemically reduced remaining nitrate to nitrite, which then reacted with Reagents A & B | Negative (NO3- not reduced) |
| No color after Zinc dust | No nitrate remained for zinc to reduce; nitrate was biologically reduced to N2, NH3, or other products | Positive (NO3+ to N2 or NH3) |
| Gas bubble in Durham tube | Gaseous nitrogen (N2) or fermentation gas produced | Gas production indicator |
Nitrate Reduction Interpretive Matrix
| Pattern | What to call it | How to think about it |
|---|---|---|
| Red after Reagents A & B | Nitrate reduced to Nitrite (NO3+) | Organism produces nitrate reductase. Common in Escherichia coli (positive control). |
| Colorless after A & B, Colorless after Zinc, Gas present | Nitrate reduced to Nitrogen Gas (NO3+, gas+) | Denitrification complete. Common in Pseudomonas aeruginosa (positive control). |
| Colorless after A & B, Colorless after Zinc, No gas | Nitrate reduced to Ammonia/other nongaseous products (NO3+) | Nitrate reduced beyond nitrite to nongaseous end products. |
| Colorless after A & B, Red after Zinc | Negative for nitrate reduction | Nitrate remained intact in broth; zinc reduced it to nitrite. Common in Acinetobacter baumannii. |
| Uninoculated Control | No reaction | Baseline comparison for color changes and Durham tube integrity. |
- Nitrate reduction determines the ability of an organism to reduce nitrate (NO3-) to nitrite (NO2-) or other nitrogenous compounds.
- The test requires a sequential two-step reagent addition: Reagents A & B first, followed by zinc dust if no color develops.
- Enterobacteriaceae typically reduce nitrate to nitrite. Non-fermenters like Pseudomonas aeruginosa often perform complete denitrification to nitrogen gas.
Best mental model: Reagents A & B detect nitrite (red = positive). Zinc dust detects nitrate (red = negative). If neither is detected, nitrate was reduced completely beyond nitrite (colorless = positive).