Nitrate Reduction/Nitrate Reductase Test

Some bacterial species are able to reduce nitrate (NO3)  to nitrite (NO2)  using the enzyme nitrate reductase in an anaerobic process by using molecules other then oxygen, nitrate, as a terminal electron acceptor.  To determine if a bacteria is able to reduce nitrate they are grown in a nitrate broth and a series of chemical are added to detect nitrate reduction.

(A)                                                 (B)                                                     (C)     


Nitrate reduction test for the reduction of nitrate in nitrate media, to determine if the bacteria contains nitrate and/or nitrite reductase. Organism were incubated for 48 hr's at 37 degree's in nitrate media then reagents added.  The addition of these reagents test for the presence of nitrites and the media will turn red if nitrites are present.

Nitrate broths after the addition of 10 drops reagents A (sulfanilic acid in acetic acid) and B (nn-dimethyl-1-naphthylamine in acetic acid).
 A) Alcaligenes faecalis showed no color change indicating that zinc must be added to complete test.

B) Escherichia coli turned red after the addition of reagents A and B indicating that nitrate had been reduced to nitrite, this organism has nitrate reductase. Zinc does not need to be added, test is complete.

C) Pseudomonas aeruginosa showed no color change indicating that zinc must be added to complete test.

Nitrate Broth----->addition of reagent A and B----> broth turns red then NO3 reduced to NO2 by nitrate reductase.
or

 Nitrate Broth----->addition of reagent A and B----> broth remain clear---->add Zinc



(A)                                                    (B)                                            (C)          

  A) Alcaligenes faecalis turned red, indicating that nitrate is still present and has not been reduced, nitrate reductase is not present.  Nitrate reacts with zinc and if still present in the media then the Zinc reacts with the Nitrate causing it to be reduced, the media to turns red.  The Zinc/Nitrate reaction turned the media red not the bacteria!

     B) Escherichia coli turned red after the addition of reagents A and B, indicating that nitrate has been reduced to nitrite so zinc was not added.

C) Pseudomonas aeruginosa showed no color change indicating that nitrate has been completely reduced beyond nitrite to ammonia or molecular nitrogen....ie nitrate/nitrite is not present in the media and does not react with the chemical added to detect nitrate/nitrite.

 Nitrate Broth----->addition of reagent A and B----> broth remain clear---->add Zinc

Zinc-----> broth turns red---->Zinc reacted to Nitrate in the broth-----> no reductase present.

or

Zinc----->broth remains clear-----> no nitrate present in media----->reductase present and nitrate reduced beyond nitrite to ammonia or molecular nitrogen.


All Images copyright T. Sturm 

 

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