Nitrate reduction test- Dr C R Meera

 

Nitrates serve as source of nitrogen for many bacteria.  They can also act as final electron acceptor.  Many organisms can be differentiated and identified by their capacity to reduce nitrates to nitrites.  Most of the enterobacteriaceae reduce nitrates.  This character is useful for the identication of the species in Neisseria, Haemophilus and Branhamella.  Some Pseudomonas and nonfermenters reduce nitrate to nitrite and further down to ammonia or to molecular nitrogen.

The enzyme nitrate reductase possessed by organisms reduces nitrates to nitrites. The reduction of nitrates by some aerobic and facultative anaerobic microorganisms occurs in the absence of molecular oxygen, an anaerobic process.  In these organisms anaerobic respiration is an oxidative process whereby the cell uses inorganic substances such as nitrates (NO₃^-) or sulfates (SO₄ ^2-) to supply oxygen that is subsequently utilized as a final hydrogen acceptor during energy formation.  The biochemical transformation may be visualized as follows:

NO₃^-  +  2H⁺  +  2e^{-        Nitrate Reductase}    NO₂^- + H₂O

Nitrate     Hydrogen    Electrons                                                   Nitrite        Water

Some organisms possess the enzymatic capacity to act further on nitrites to reduce them to ammonia (NH₃⁺) or molecular nitrogen (N₂) and this process is called denitrification.  The reaction may be described as follows:

 NO₂^-    ------->         NH₃⁺

Nitrite                                 Ammonia     

 Or

 2NO₃^-   +  12H⁺  +  10e^{-   -------->} N₂   +  6 H₂O

Nitrate           Hydrogen      Electrons                  Molecular nitrogen and water

 Aim

To determine the ability of some microorganisms to reduce nitrates (NO₃^- ) to nitrites (NO₂^-) or beyond the nitrite stage.

Principle

Nitrate reduction can be determined by cultivating organisms in a nitrate broth medium.  The medium is basically a nutrient broth supplemented with 0.1% potassium nitrate (KNO₃) as the nitrate substrate.  In addition, the medium is made into a semisolid by the addition of 0.1% agar.  Following incubation of the cultures, an organism’s ability to reduce nitrates to nitrites is determined by the addition of two reagents: Solution A, which is sulfanilic acid, followed by Solution B, which is α- naphthylamine.  Nitrites in acid environment immediately produce a cherry red coloration due to the formation of a red diazomium dye, p- sulfo benzene-azo-alphanaphthylamine.

Cultures not producing a color change suggest one of two possibilities: (1) nitrates were not reduced by the organism, or (2) the organism possessed such potent nitrate reductase enzymes that nitrates were rapidly reduced beyond nitrites to ammonia or even molecular nitrogen.  To determine whether or not nitrates were reduced past the nitrite stage, a small amount of zinc powder is added to the basically colorless cultures already containing Solutions A and B.  Zinc reduces nitrates to nitrites.  The development of red color therefore verifies that nitrates were not reduced to nitrites by the organism.  If nitrates were not reduced, a negative nitrate reduction reaction has occurred.  If the addition of zinc does not produce a color change, the nitrates in the medium were reduced beyond nitrites to ammonia or nitrogen gas.  This is a positive reaction.

Requirements

24 hr nutrient broth cultures of species Bacillus, Streptococcus, Staphylococcus, Pseudomonas and E.coli.

Nitrate broth, Solution A (sulfanilic acid), Solution B (α- naphthylamine), zinc powder, Bunsen burner, inoculating loop, glass marking pencil etc.

 Procedure

1.  Using sterile technique, inoculate each experimental organism into its appropriately labeled tubes by means of loop inoculation. One uninoculated tube kept as control.

2.  Incubate all inoculated tubes at 37^o C for 24-48 hrs.

3. Add 5 drops of Solution A and then 5 drops of solution B to all nitrate broth cultures and observe for the red color development.

4. Add a minute quantity of zinc to the cultures in which no red color developed.  Observe for the color change to red.

 

Observations

Bacillus, Staphylococcus and E.coli produced cherry red coloration immediately after adding Solutions A and B, indicating that they are nitrate positive.  Pseudomonas and Streptococcus produced no cherry red coloration on addition of Solutions A and B.  Addition of zinc powder to these tubes produced a red color in Streptococcus inoculated ones which indicates a negative nitrate reduction result. No colour formation in the tube inoculated with Pseudomonas indicated the conversion of nitrate beyond nitrites to ammonia or nitrogen gas.

Result

Bacillus, Staphylococcus, E.coli and Pseudomonas are nitrate test positive whereas Streptococcus is nitrate negative.

 

§         Nitrate broth

Peptone                                               5.0 g

Beef extract                                        3.0 g

Potassium nitrate                                 5.0 g

Distilled water                                    1 litre

pH                                                       7.2