Nitrate Broth ( Energy Metabolism)

What is tested for in Nitrate Broth?

To test the organism’s ability to reduce nitrate, molecular nitrogen (denitrification) or some other nitrogenous compound

Does Nitrate Test require addition of reagent?

Yes, nitrate reagents A and B then zinc dust

What are components of nitrate broth?

• Pancreatic digest of gelatin

• Potassium nitrate

• durham tube

What is potassium nitrate used for?

As a source of nitrate

What is the purpose of durham tube?

Indicator of gas production

What is the positive result of denitrification?

Air bubble in Durham tune

What is the positive results after incubation and addition of reagents A and B

Red coloration as nitrate react with the reagents

What is the positive results after incubation, addition of reagents A and B then addition of zinc dust?

Red color change as zinc catalyzes the reduction of nitrate to nitrite

If you incubate, add nitrate reagents A and B with zin but the tube remains colorless or no color change, why?

No nitrate present

Brief protocol of Nitrate Reduction test

Day 1:

1. Obtain 4 nitrate broth tubes, label ( E.coli, P.aeruginosa, A.faecalis) and one control

2. Aseptically transfer specimen to labeled tube

3. Incubate for 48 hours at 37°

Day 2:

1. Add 15 drops of nitrate reagent A, 5 drops of nitrate reagent B. Wait for 1 minute

2. Observe change

3. Add 1 small scoop of zinc dust. Vortex for 10 seconds.

4. Record results

5. Remove tapes, place in liquid rank to be autoclaved