Question

You have successfully isolated pure cultures of a bacterium from soil. What result might you expect if you incolauted it into nitrate broth and why? How would hou determine this result? Please be specific.

Answer 1

Successfully isolated pure cultures of the bacterium from soil. The anaerobic bacteria which grows under the soil is under the anaerobic condition and if these bacterial are under the nitrate broth they would require the nitrate as a nutrient for the growth. A heavy inoculum of test organism is incubated in a broth containing nitrate. These organisms are producing the nitrate reductase enzyme which can easily react on nitrate to get reduced into nitric oxide, nitrous, and nitrogen.

The nitrate reduction test is based on the detection of nitrite and its ability to form a red compound when it reacts with sulfanilic acid to form a complex (nitrite-sulfanilic acid) which then reacts with a α-naphthylamine to give a red precipitate (prontosil), which is a water-soluble azo dye.

However, only when nitrate is present in the medium, red color will be produced. If there’s no red color in the medium after you’ve added sulfanilic acid and α-naphthylamine means only that nitrite is not present in the medium.

There is two explanations for this observation.

1. The nitrate may not have been reduced; the strain is nitrate-negative.
2. The nitrate may have been reduced to nitrite which has then been completely reduced to nitric oxide, nitrous oxide, or nitrogen which will not react with the reagents that react with nitrite; the strain is nitrate-positive.

Thus, when nitrite is not detected, it is necessary to test if the organism has reduced nitrate beyond nitrite. This can be done indirectly by adding small amount of Zinc powder to the culture. Zinc powder catalyses the reduction of nitrate to nitrite. The development of the red colour on addition of Zinc indicates that nitrate was not reduced by the organism which suggests that the test organism is not capable of reducing nitrate. If no color change occurs after the addition of zinc, this indicates that the organism reduced nitrate to one of the other nitrogen compounds and thus is a nitrate reducer.

The experiment was conducted with following steps:

Method

Determination of nitrate reduction to nitrite is a two step process.

1. Inoculating the nitrate broths with bacterial suspension.
2. Incubating the tubes at the optimal temperature 30°C or 37°C for 24 hours.
3. Later on  incubation look for N2 gas first before adding reagents.
4. By Adding 6-8 drops of nitrite reagent A and add the 6-8 drops of nitrite reagent B.
5. The Observation for the reaction (color development) within a minute or less.
6. If no color develops add zinc powder.
7. Again on observing for at least 3 minutes for a red color to develop after addition of zinc.

On conducting the experiment the expected result obtained as follow:

• Positive Test:
  – Development of a cherry red colouration on addition of reagent A and B
  – Absence of a red color development on adding Zn powder
• Negative Test:
  – A development of red color on addition of Zn powder

Nitrate NO3 Nitrate reductase + Sulfanilic acid (Reagent A) Nitrite NO, Colorless (Nitrate-Sulfanilic acid) Nitrite reductase + a-naphthylamine (Reagent B) Nitric Oxide NO Nitric Oxide reductase Red precipitate (Prontosil) Nitrous Oxide N O Nitrous Oxide reductase Nitrogen N2 Microbiology Info.com