Colorimetric Assay for the Quantification of Non-Structural Carbohydrates in Plant Tissues

First, weigh out replicates from each tissue sample into glass 15-milliliter tubes. Label the tubes, and record the exact mass of each sample.

Next, add 1 milliliter of 0.1 molar sulfuric acid to each tube, and close the caps tightly. Then, place the tubes in a boiling water bath for 1 hour.

Transfer the tubes to a tepid water bath to cool. Then, pour the contents of the tubes into labeled 1.5-milliliter microcentrifuge tubes. After this, centrifuge the tubes at 15,000 g for 10 minutes. Use a micropipette to transfer the supernatant to new labeled 1.5-milliliter tubes.

Using a pipette, transfer 15 microliters of each unknown sample to its own test tube. Then, add 385 microliters of distilled water to each tube.

In a fume hood, add 400 microliters of 5% phenol to each standard and unknown sample test tube. Immediately thereafter, add 2 milliliters of sulfuric acid to each tube.

Make sure to add the sulfuric acid to the surface of the solution.

After incubating the tubes for 10 minutes, vortex the tubes. Then, incubate the tube for an additional 30 minutes.

Transfer 800 microliters from each sample tube to three polystyrene 1.5-milliliter semi-micro cuvettes. Then, set the spectrophotometer to read at 490 nanometers, and calibrate it with a blank cuvette.

Finally, run each cuvette through the spectrophotometer, and record the absorbance.