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Radial Diffusion Assay for Tannins

Author

Rieske-Kinney

Updated

February 2000

Goal

Methods documenting radial diffusion assay for tannins.

Equipment/Supplies

  • Acetic acid, glacial 99.9+%
  • Acetone
  • Agarose, type I
  • Ascorbic acid
  • Boiling plate
  • Bovine serum albumin (BSA)
  • Cork borer
  • Light table (optional)
  • Oven/growth chamber (30C)
  • Parafilm
  • Petri dishes
  • pH meter or paper
  • Pipettes, glass, 10 ml
  • Potassium hydroxide flakes
  • 4% KOH solution = 4 g KOH + 100 ml dH2O
  • Ruler or calipers
  • Stir plate
  • Syringe, Hamilton, 10 ul
  • Tannic acid
  • Test tubes (13 x 100 mm)
  • Thermometer
Detailed equipment/supply list
  • acetic acid, glacial 99.9+% Sigma A-6283 500 ml $16.15
  • acetone Sigma A-4206 1 liter $16.60
  • agarose, type I Sigma A-6013 25 g $38.50
  • ascorbic acid Sigma A-1417 100 g $20.50
  • boiling plate bovine serum albumin (BSA) Sigma P 7656
  • 5 vials $19.25 cork borer
  • light table (optional)
  • oven/growth chamber (30C) parafilm petri dishes
  • pH meter or pH paper
  • pipettes, glass, 10 ml
  • Potassium hydroxide flakes. Aldrich 48,401-6 4%
  • KOH solution = 4 g KOH + 100 ml dH2O
  • ruler or calipers
  • stir plate
  • syringe, Hamilton
  • 10 ul tannic acid Sigma T-0125 250 mg $16.65 test
  • tubes (13 x 100 mm)
  • thermometer

Reagent Preparation

  1. Tannic acid solution

    1. 0.5 g tannic acid dissolved in 10 ml of 45% acetone.

  2. Buffer (50 mM acetic acid)

    1. 2.87 ml acetic acid
      1. Measure 2.87 ml acetic acid with glass pipette, add to 900 ml of dH2O.
      2. Adjust to pH 5.0 with KOH solution, then bring volume up to 1000 ml with dH2O.
      3. Foil, parafilm, and store in refrigerator.

  3. 45% acetone with 10 mM ascorbic acid

    1. 450 ml acetone
    2. 550 ml dH2O
    3. 1.761 g ascorbic acid
      1. Measure appropriate volume of acetone and dH2O. Place in 1000 ml flask. Add ascorbic acid and mix well with stir bar. Cap with foil and parafilm.

Preparation of Agar Plates

  1. Set water bath temperature to 40C.

  2. Weigh out 1.0 g of agarose into a 250 ml beaker, and 100 mg of BSA into a 20 ml scintillation vial.

  3. Add 97 ml of acetic acid buffer to the 1.0 g of agarose.

  4. Bring 97 ml of buffer/agar to full boil on hot plate.

  5. Insert thermometer into buffer/agar and cool to 45C in a 40C water bath.

  6. Mix 100 mg BSA into 2 ml buffer using second stir plate. Stir gently, avoid foaming.

  7. When agar temperature reaches 45C, add BSA solution to agar and mix for ~10 sec on a cool plate.3 Rinse BSA vial with 1 ml of buffer and add the ml to the agar to bring total volume to 100 ml.

  8. Using a glass pipette, dispense 9.5 ml agar into 100 mm petri dishes. Dispense quickly, spiraling from the outside towards the middle. After the 9.5 ml of agar is dispensed, swirl the dishes gently and place on flat surface.

  9. If bubbles form in the agar, remove them with a pasteur pipette.

  10. After agar cools to room temperature, cover and place upside down. Store in plastic bag in refrigerator until ready to analyze unknowns.

  11. Use a cork borer to punch 4 wells in agar just prior to dispensing plant extracts.

Tissue Extraction and Sample Preparation

  1. Tissue should be freeze dried, ground, and stored on dessicant at -15C.

  2. Weigh 100 mg plant tissue and place in 13 x 100 disposable test tube.

  3. Add 1 ml 45% acetone/ascorbic acid solution.

  4. Cork and parafilm.

  5. Hold 1 h and 35 min at room temp.

  6. Centrifuge for 30 min.

Tannin Assay Procedure

  1. Punch 4 mm wells into the bottom of agar slabs. Space 2 cm apart. [Label bottom of petri dish with wax pencil before beginning procedure]

  2. Pipette 16 ul of plant extract into each well. Do two 8 ul aliquots of plant extract and standards into 4 mm wells using a 10 ul micropipette or Hamilton syringe. Agar must have time to absorb first aliquot of material before adding 2nd aliquot. The 1 mg and 0.75 standards should be placed alone in separate petri dishes to avoid overlapping of reaction rings.

  3. After all solutions have been absorbed, seal petri dishes with parafilm and incubate at 30C for 96 h in total darkness. Do not move plates once incubation has begun.

  4. Measure precipitate rings after 96 h.

Standards

  1. Prepare 10 ml of a 50 mg/ml tannic acid solution

    • 500 mg tannic acid
    • 10 ml acetone/ascorbic acid solution
      1. Mix solutions and stir on stir plate.

  2. From this solution prepare the following standards.

Conc. Standard (mg/ml) Vol. of Tannic Acid Vol. of 45% acetone
5 100 900
10 200 800
20 400 600
30 600 400
40 800 200
50 1000 000

  1. Dispense 16 ul of each standard into its correct well. Run duplicates.

  2. Cover, parafilm and incubate plates in 30C oven for 96 h.

  3. After 96 h measure precipitate rings.

Determination of area of reaction rings

  1. Place petri dish on light table. Measure diameter of rings in two places (perpendicularly) using a ruler or caliper. Square the average diameter and record data as diameter squared.

  2. Calculate the amount of plant tissue in volume of extract dried down.

  3. Calculate the amount of plant tissue in 16 ul aliquot used for assay.

  4. Express results as diameter2/ mg plant tissue.

    [Plot calibration curve. Ring diameter on x-axis, [tannic acid] on y-axis.]

Notes

  1. Weigh out agar and BSA ahead of time.

  2. Make agarose in 100 ml batches only. Larger amounts will begin to harden before it is properly dispensed in petri dishes.

  3. Do not add BSA too soon or it will denature from high temperature.

Last update: October 20, 2022