Rev. 10/30/2019

Sodium-hypochlorite-acid-fuschin method (Byrd et al., 1983).

Clearing Root Tissue

1. Wash roots with water and place them in a 150 ml beaker.  Large root systems may be cut into sections for staining.

2. Add 50 ml of tap water and an appropriate amount of chlorine bleach (5.25% NaOCl) to clear the root tissue.  Soak the roots for 4 minutes in the NaOCl solution and agitate occasionally.  Suggested guidelines for the quantity of bleach to be added are:

    a) 10  ml of 5.25% NaOCl for young roots.

    b) 20  ml of 5.25% NaOCl for roots of moderate age.

    c) 30  ml of 5.25% NaOCl for older or more lignified roots.

Staining Nematodes in Root

3. Rinse the roots with running tap water for about 45 seconds and then immerse them in water for 15 minutes to remove any residual NaOCl which may affect staining with acid fuschin.

4. Drain the water and transfer the roots to a glass beaker with 30-50 ml of tap water.

5. Add 1 ml of stock acid-fuschin stain solution to the water and boil for about 30 seconds on a hotplate or in a microwave oven.  To prepare stock acid-fuschin solution, dissolve 3.5 g acid fuschin in 250 ml acetic acid and 750 ml distilled water.

6. Cool the solution to room temperature, drain the stain solution, and rinse the roots in running tap water.

Destaining the Roots

7. Destain the roots by boiling in 20-30 ml of glycerin acidified with a few drops of 5 N HCl.

8. Distribute the roots in a small amount of glycerin on a Petri dish cover, gently press against the cover with a Petri-dish bottom and observe under a dissecting microscope.  A pair of glass plates may be used instead of Petri dishes. Roots may be stored in acidified glycerin with little loss of contrast between nematodes and roots.

Reference:

Byrd, D.W., Jr., T. Kirkpatrick and K.R. Barker. 1983. An improved technique for clearing and staining plant tissue for detection of nematodes.  Journal of Nematology 14:142-143.