About this test
What is the purpose of the test?
This test determines whether the microbe produces the enzymes nitrate reductase and nitrite reductase . The two enzymes catalyze two reactions involved in converting starting compound nitrate into end product nitrogen gas.
nitrate reductase nitrite reductase
NO3 ———————————> NO2 ———————————–> N2
nitrate nitrite nitrogen gas
Some bacteria produce both enzymes, some produce nitrate reductase only, and others produce neither.
How is the presence of these enzymes determined?
If a bacterium producing nitrate reductase is grown in a medium containing nitrate , the enzyme converts the nitrate to nitrite . Nitrite reacts with certain chemicals to yield a red-colored product. If the bacterium also produces nitrite reductase , nitrogen gas will be liberated. Bubbles collecting in an inverted Durham tube indicate that nitrogen has been produced.
What medium is used?
The medium used is nitrate broth with Durham tube, a nutrient broth with potassium nitrate added. The inverted Durham tube is placed in the medium as a trap for any nitrogen that might be generated.
How is the test performed?
An inoculum from a pure culture is transferred aseptically to a sterile tube of nitrate broth containing an inverted Durham tube. The inoculated tube is incubated at 35-37 C for 24 hours and the results are determined. A positive test for both enzymes consists of a turbid (cloudy) broth with pronounced gas bubbles trapped in the Durham tube. If results like this are not observed, testing for the individual enzymes can be done through addition of reagents, with a positive test indicated by the broth turning red.
What reagents are added?
Five drops of nitrate reagent A is added, followed by five drops of nitrate reagent B .
Performing this test in the VUMIE Online lab
Inoculation of Medium
1. Select the nitrate broth with Durham tube medium.
2. Complete the process of a tube-to-tube aseptic transfer to inoculate the medium.
Incubation of the Inoculated Medium
3. Place the inoculated tube into the 35-37 C incubator.
4. Press the New Day button to move forward 24 hours.
Determination of Test Results and Addition of Reagents
5. Incubate this test for only 24 hours.
6. Retrieve the incubated culture from the incubator.
7. Observe the culture. If the test was followed as described above, the culture will be cloudy (turbid). If there are bubbles in the Durham tube, this indicates presence of both nitrate reductase and nitrite reductase (nitrates have been converted to nitrites, and then to nitrogen gas – positive for both enzymes).
8. If there are no bubbles in the Durham tube, it will be necessary to add reagents to determine the results. Select the dropper tool and the appropriate reagent needed from the chemical shelf. Remove the cap lid. For this test, select Nitrate reagent A, add it, and then add Nitrate reagent B.
9. If the tube turns red, it is an indication that nitrate reductase is present but nitrite reductase is absent (+ for the first enzyme but — for the second). If the color doesn’t change, and there was no gas in the Durham tube, the bacterium does not have either enzyme.
10. Record test results.
Positive for Nitrate (NO3) Reductase
Negative for Nitrate (NO3) Reductase
Positive for Nitrite (NO2) Reductase (bubbles of N2 gas in Durham tube)
Negative for Nitrite (NO2) Reductase (no bubbles)