Day 1) Setting up the experiment and initial sample collection¶

• Transfer 1 mL of a 1,000 ppm ammonia stock to a 500 mL volumetric flask. Bring up to the mark using distilled water. This is your 2 ppm ammonia stock;
• Divide the 2 ppm solution into two beakers and label one as “Gravel” and another as “Gravel + Bacteria”.
• Take a scoop (25–50 g) of aquarium gravel from the store bought bag. Place in the beaker labelled “Gravel”.
• To obtain nitrification bacteria, remove an equivalent amount of gravel from an established aquarium as your source of nitrification bacteria. Place in the beaker labelled “Gravel + Bacteria”.

• Transfer 1.5 mL of sample to a clean microfuge tube for ammonia measurements.
• Transfer 1.0 mL of sample to a clean microfuge tube for nitrate measurements.
• Label these two samples as “T=0”. Store them at –20°C.
• Repeat sampling every 24 hours for the following 4 days of the experiment, and label them “T=1”, …, “T=4”, respectively. You will collect a total of 10 samples (5 for ammonia, and 5 for nitrate measurements).

Day 2) Prepare solutions¶

Prepare the following solutions for making the ammonia and nitrate measurements.

1) Hypochlorite solution:

• Place 1 ml of bleach into a 100 mL volumetric flask and fill with 70 mL of DI water;
• Add 0.5 grams of NaOH and mix until dissolved;
• Fill flask to the 100 mL mark.

2) Salicylate/Catalyst solution:

• Place 10 g of sodium salicylate into a 100 mL volumetric flask and fill with 70 mL of DI water until dissolved.
• Add 0.04 grams of sodium nitroferricyanide and mix until dissolved.
• Add 0.5 grams NaOH to adjust the pH to the ~12.0 range.
• Fill flask to the 100 mL mark. Transfer solution into a dark, airtight glass bottle for maximum longevity. Due to limited storage life, prepare fresh solutions weekly.

3) 25 mM EDTA

• Dissolve 9.3g EDTA in 1L of distilled water.

4) Phosphate buffer (25 mM KH2PO4, 0.025 mM EDTA, pH 7.5)

• Dissolve 3.75 g of potassium phosphate (KH2PO4) and 1.4 g potassium hydroxide (KOH) in 800 mL of distilled water in a 1L volumetric flask
• Add 1 mL of 25 mM EDTA and fill to the mark

5) 2 units/mL nitrate reductase

• Add 1 mL of NECi proprietary enzyme diluent to 2 units of freeze-dried enzyme and reconstitute following the instructions supplied with the enzyme.

6) 1 mg/mL NADH

• Dissolve 0.1 g of NADH (FW=709.4) in 100 mL distilled water. Aliquot and store unused NADH in the freezer.

7) 1% sulfanilamide solution

• Weigh out 0.15g of sulfanilamide into a small amber bottle. Add 15 mL of 3M HCl.

8) 0.02% NED

• Weigh out 0.02 g of NED into an amber bottle. Add 100 mL of distilled water.

Day 3) Prepare ammonia standard curve¶

• Transfer 20 mL of the 10 ppm ammonia standard solution to a 100 mL volumetric flask. Fill flask to the 100 mL mark with distilled water and invert several times to mix. Label flask as 2.0 ppm ammonia.
• Label nine large test tubes #1-9. Pipette the indicated volumes of 2.0 ppm ammonia and distilled water into the test tubes as shown in the Table below.

• Transfer 1 mL of each sample to be tested into a new test tube. Note: we recommend doing the measurements in triplicate.
• Add 250 µL of hypochlorite solution and mix
• Add 250 µL of salicylate/catalyst solution and mix
• Let tubes stand for 5-10 mins to develop color.
• Launch the Educational Colorimeter Plotting program, and select the Red color channel.
• Transfer the contents of Tube #1 (0.0 µM ammonia) into a cuvette, and use it for calibration.
• Before removing the calibration sample from the enclosure, click “Measure” (Absorbance value should be ~0.00).
• In the cell next to the measurement, enter the concentration value in µM (which in this case is 0.0).
• Transfer the contents back into the test tube. (It is good practice to rinse the cuvette with distilled water between samples)
• Transfer the next solution (Tube #2) into the cuvette, place it inside the enclosure, and click “Measure”.
• In the cell next to the measurement, enter the concentration value in µM.
• Repeat steps 11–13 for the remaining samples.
• After completing all measurements, and entering all concentration values, click “Plot” to graph your data. The points should roughly follow a linear trend (see sample graphs below).
• Using the file menu, go to “File>Export”, choose a filename for storing your sample curve (eg, student1_ammonia_sc), and click “OK”.

Image of cuvettes with ammonia standard curve in triplicate

Sample ammonia standard curve

Day 4) Prepare nitrate standard curve¶

• Remove the NADH and nitrate reductase prepared in Step 2 from the freezer. Thaw the NADH. In a test tube, prepare a “10 x master mix” of enzyme, NADH and phosphate buffer as shown in the Table below.

• Label nine tubes #M1, M2, …, M9. To each tube transfer 1 mL of the master mix.
• To prepare the standard curve samples, label nine test tubes #1–9. Pipette the indicated volumes of 10 ppm nitrate standard and distilled water into these test tubes as shown in the Table below.

• Add 50 µL of each standard curve sample to the corresponding tube containing the master mix (eg, from tube #1 to tube #M1, etc.). Mix thoroughly and incubate for 20–30 minutes.
• Add 500 µL of 1% sulfanimide to each tube and mix
• Add 500 µL of 0.02% NED to each tube and mix.
• Launch the Educational Colorimeter Plotting program, and select the Green color channel.
• Transfer the contents of Tube #M1 (0 ppm nitrate) into a cuvette, and use it for calibration.
• Before removing the calibration sample from the enclosure, click “Measure” (Absorbance value should be ~0.00).
• In the cell next to the measurement, enter the concentration value in µM (which in this case is 0.0).
• Transfer the contents back into the test tube. (It is good practice to rinse the cuvette with distilled water between samples)
• Transfer the next solution (Tube #M2) into the cuvette, place it inside the enclosure, and click “Measure”.
• In the cell next to the measurement, enter the concentration value in µM.
• Repeat steps 11–13 for the remaining samples.
• After completing all measurements, and entering all concentration values, click “Plot” to graph your data.
• Using the options menu, go to “Options>Export”, choose a filename for storing your sample curve (eg, student1_nitrate_sc), and click “OK”.

Image of cuvettes with nitrate standard curve in duplicate

Sample nitrate standard curve

Day 5) Measure nitrate and ammonia in samples¶

Take your last sample from the experiment (“T=4”). Remove from the freezer the samples collected on previous days (“T=0”, …, “T=3”) and thaw.

• Process all 10 samples (5 for nitrate, and 5 for ammonia measurements) the same way as you did for generating the standard curves on Days 3 and 4. Don’t forget to also process a distilled water sample for calibration.
• Once you have the samples ready for measurement, open the Educational Colorimeter Concentration program.
• Starting with ammonia, select the standard curve generated on Day 3 from the drop down list (eg, “student1_ammonia_sc”). Note that the Red color channel will be automatically selected.
• Calibrate the colorimeter with the 0 µM ammonia control, and measure all of your ammonia samples. Label the corresponding measurements as “Day 0”, …, “Day 4”.
• Once you have finished click the “Plot” button. Save the displayed graph.
• Repeat steps above for nitrate. Select the standard curve generated on Day 4 from the drop down list (eg, “student1_nitrate_sc”). Note the Green color channel will be automatically selected.