3.5.6: Nitrate Reduction Test Activity

In this activity you will test three bacterial samples. Label each nitrate broth tube with the sample it is to be inoculated with.

1. Turn on the Bunsen burner and sterilize the inoculating loop by passing it through the flame of the burner.
2. Obtain a bacterial culture. Remove the cap. Pass the mouth of the tube through the flame of the Bunsen burner.
3. Insert the loop into the tube to acquire a sample of the bacterium.
4. Pass the tube through the flame of the Bunsen burner and recap the test tube, and return the tube to the test tube rack.
5. Pick up the nitrate broth tube to which the sample is to be transferred. Remove the cap and pass the mouth of the tube through the flame of the Bunsen burner.
6. Insert the loop into the tube to introduce the bacterial sample to the media.
7. Pass the mouth of the nitrate broth tube through the flame and recap it, and return it to the test tube rack.
8. Sterilize the inoculating loop by flaming the loop in the flame of the Bunsen burner.

Repeat steps 1 - 8 for the second and third bacterial species and nitrate broth tubes to be inoculated.

9. Incubate the inoculated nitrate broth tubes at 37°C for 24 hours.
10. For each nitrate broth tube, add 5 drops of nitrate reagent A, 5 drops of nitrate reagent B, and mix well. Observe the tubes for a color change.

If the tube remains clear

11. Using a spatula add a small quantity (just covering the tip) of zinc dust to the sample. Mix and observe for color change.

In the table below write the name of the species tested and the results for each of the tests performed.

Experimental Results

Bacterial Species	Color after nitrate reagents A & B	Zinc dust added (Yes or Not required)	Color after addition of zinc	Nitrate Reduction Test Results

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