帝王会所

Nutrient Analyses on Microplate Reader

By Jared L. DeForest

Reagents

Vanadium solution (VCl₃)

In 100 ml volumetric flask

• 0.80 g vanadium (III) chloride in 50 ml of 1 M HCl

CAUTION: Work quickly because Cl₃V powder will react with air!

Flush with N₂, store in the dark at 4掳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 N₂; may be stored in the dark at 4掳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掳C for several months, and discard if colored.

Premix solutions just prior to analysis to a ratio of 2:1:1 (20 ml VCl₃ : 10 ml SULF : 10 ml NEDD)

Standard Dilutions

Standard (40 ppm NO₃)

In 100 ml volumetric flask

• 4 ml 1,000 ppm nitrate (NO₃) 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 碌l of reagent to 100 碌l of sample, or standard in clear 96-wellplate.

Incubated (or farm soils):

• Add 50 碌l KCl and 100 碌l of reagent to 50 碌l of sample, or standard in clear 96-wellplate.

Incubate at 37掳C and measure when color develops. Measure absorbance at 540 nm. Samples should turn pink; if dark magenta (OVRFLW), redo by adding only 10 碌l of sample with 90 碌l of KCl and 100 碌l of reagent.

Adapted from Miranda et al., 2001, Nitric Oxide-Biology and Chemistry

Created: 03/06/07 Revised: 10/10/11