Nitrate Reduction Results


Purpose:  Trypticase-nitrate broth is used to detect an organisms ability to reduce nitrate to nitrite or a further reduced nitrogenous compound such as nitrous oxide or nitrogen gas.

Principle:  Nitrate may be reduce to multiple comounds by two processes.  Anaerobic respiration and denitrification.  In anaerobic respiration the bacterium uses nitrate as it's terminal electron acceptor, reducing nitrate to a variety of compounds, while denitrification reduces nitrate solely to molecular nitrogen.  Sulfanilic acid and dimethyl 1-naphthylamine are added to detect nitrite, which will complex with these molecules forming a red color.  If no red color is observed there are two possibilities;  the nitrate has not been reduced, or it has been reduced further than nitrite.  To differentiate between these two possibilities, zinc powder is added, which will complex with nitrate forming a red color.  Thus if the tube turns red after zinc, nitrate has not been reduced and the result is negative.  If no red color is observed then the nitrate has been reduced further than nitrite and the result is positive.


 
 
The tubes above have been treated with dimethyl 1-naptholamine and sulfanilic acid.  The tube on the left illustrated no change, thus it was inconclusive.  The center tube also illustrated no change as expected.  The tube on the right indicates that the organism present reduced nitrate to nitrite.  In order to further determine positive/negative nitrate reduction, zinc powder must be added to detect the presence of nitrate.
 
A tube will turn a deep red color on the addition of zinc if nitrate is present.  The inconclusive tube did not turn red indicating that the organism present reduced nitrate to something besides nitrite (positive).  The uninoculated control turned red indicating, as expected, that nitrate is still present in the tube.

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