Nitrate Reductase 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.



To perform this test in VirtualUnknown™ Microbiology, complete the following steps:


Inoculation of Medium


1. Select the nitrate broth with Durham tube medium.  

2. Start your Bunsen burner.  

3. Select the inoculating loop tool.  

4. Flame your inoculating loop to sterilize it.

5. Remove the caps from your test tubes.  

6. Flame the mouths of your test tubes.  

7. Use the sterile inoculating tool to pick up an inoculum from the culture tube of the unknown bacterium.

8. Immediately transfer the inoculum into the fresh, sterile medium.  

9. Flame the mouths of your tubes once again.  

10. Replace the caps on the test tubes.  

11. Re-flame the inoculating tool.  


Incubation of the Inoculated Medium


12. Place the inoculated tube into the 35-37 C incubator.  

13. Press the New Day button to move forward 24 hours.  


Determination of Test Results


14. Incubate for the appropriate length of time. For this test, 24 hours is sufficient for determining the result.  

15. Retrieve desired incubated culture from the incubator.  

16. Observe test result. 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). If there are no bubbles in the Durham tube, it will be necessary to add reagents to determine the results.  


Addition of Reagents


17. Select the dropper tool and the appropriate reagent needed from the chemical shelf. For this test, it is necessary to add two reagents in sequence. From the reagent list, first select nitrate reagent A .

18. Remove the cap from the tube.  

19. Place the end of the dropper into the tube and add the reagent to the culture.  

20. Select nitrate reagent B and add this as done before for nitrate reagent A. If the tube turns red, it is an indication that nitrate reductase is present but nitrite reductase is absent. If the color doesn't change, and there was no gas in the Durham tube, the bacterium does not have either enzyme.

21. Record test result.  

22. Dispose of the culture.