Day 3 - Breathe in, Breathe out. Respiration tests.

Respiration tests do not refer to breathing through lungs, but the ability to reduce molecules for use as energy.  We will perform a nitrate reduction test to determine whether our bacteria can reduce nitrate ions to nitrite ions or nitrogen gas.

Nitrate Reduction Test: 

Materials:

Nitrate broth tube

Pure culture of our bacteria in broth culture

Nitrate reagent A in dropper bottle

Nitrate reagent B in a dropper bottle

Powdered zinc

Small spatula

Disposable gloves

Steps:

1.  Label nitrate broth tube with our group number and test type.

2.  Use aseptic technique to inoculate the nitrate broth tube from our bacteria's broth tube. 

3.  Incubate the tube at 35 degrees for 24-48 hours

4.  Put on gloves and eye protection.  Add 5 drops of reagent A and 5 drops of reagent B to the tube.  Shake gently to mix.  A developing pink or red color in 1-2 minutes would indicate a positive result.

5.  If no color develops, add a small amount of powdered zinc to the tube.  If there is not color change after 10 minutes, it is a positive test result.  A color change to pink or red after the addition of zinc is a negative result.

6.  Discard the tube properly. 

Results:

Discussion:  Our positive result indicates that our bacteria can reduce nitrates to nitrites for energy use.