Nutrient Analyses on Microplate Reader
Reagents
Vanadium solution (VCl 3 )
In 100 ml volumetric flask
- 0.80 g vanadium (III) chloride in 50 ml of 1 M HCl
CAUTION: Work quickly because Cl 3 V powder will react with air!
Flush with N 2 , store in the dark at 4°C, or freeze, should be blue.
2.0 % (w/v) Sulfanilamide solution (SULF)
In 100 ml volumetric flask
- 2 g Sulfanilamide
- Bring to volume with 5% (v/v) HCl
Flush with N 2 ; may be stored in the dark at 4°C for several months, and discard if colored.
0.1% (w/v) NEDD solution
In 100 ml volumetric flask
- 100 mg N-(1-naphthyl)-ethylenediamine dihydrochloride
- Bring to volume with DI water.
Flush with N2; may be stored in the dark at 4°C for several months, and discard if colored.
Premix solutions just prior to analysis to a ratio of 2:1:1 (20 ml VCl 3 : 10 ml SULF : 10 ml NEDD)
Standard Dilutions
Concentration (ppm )
Standard (ml)
1 M KCl
(ml)
0
0
10
1
0.25
9.75
2
0.5
9.5
4
1.0
9.0
8
2.0
8.0
16
4.0
6.0
24
6.0
4.0
40
10
0.0
Standard (40 ppm NO 3 )
In 100 ml volumetric flask
- 4 ml 1,000 ppm nitrate (NO 3 ) standard
- Bring to volume in 1 M KCl.
Procedure
Soil Solution Extraction
- 10 g Oven-dried equivalent field fresh, sieved soil
- 20 ml 1M KCl
Shake for 1 hour and centrifuge (10 min @ 3000 rpm).
Ambient (initial):
- Add 100 µl of reagent to 100 µl of sample, or standard in clear 96-wellplate.
Incubated (or farm soils):
- Add 50 µl KCl and 100 µl of reagent to 50 µl of sample, or standard in clear 96-wellplate.
Incubate at 37°C and measure when color develops. Measure absorbance at 540 nm. Samples should turn pink; if dark magenta (OVRFLW), redo by adding only 10 µl of sample with 90 µl of KCl and 100 µl of reagent.
Adapted from Miranda et al., 2001, Nitric Oxide-Biology and Chemistry
Created: 03/06/07 Revised: 10/10/11