Botanic Gardens Trust, Sydney, Australia

Department of Environment, Climate Change and Water NSW

Root Staining Procedures

Staining Roots for the Detection of Mycorrhizae and Fungal Pathogens

This technique is especially useful for observing vesicular-arbuscular mycorrhizae in roots. It will also demonstrate the presence of other pathogenic fungi. It is most conveniently done with the roots placed in a test tube or small flask, to which each solution is added in turn. One of the steps uses hot potassium hydroxide solution, which is very corrosive and may cause serious injury if it is spilled on skin or gets into the eyes. Gloves and protective eye-wear should be used.

  1. Wash out roots with water. Store samples in 50% ethanol if required.
  2. Digest in 10% KOH at 90° (in steamer, or in test tubes in a beaker of boiling water) for 30-60 minutes. CAUTION - hot alkali is very corrosive. Use 30 minutes for leeks, onions, wheat, etc; 45 minutes for most herbaceous dicots, maize; 60 minutes for roots of woody species.
  3. If the roots are discoloured, bleach for 10-30 minutes at room temperature in alkaline hydrogen peroxide (3 mL 20% NH4OH in 30 mL 3% H2O2).
  4. Rinse roots well in tap water (3 changes, or running water for 2 minutes.) Acidify roots in 2% HCl until roots appear white.
  5. Stain roots in aniline blue solution (see below) at 90° for 8-10 minutes.
  6. Destain overnight at room temperature, or 5-10 minutes at 90°, in 70% glycerol or lactic acid as appropriate.
  7. Mount roots on slides in destaining solution and examine.

Staining solution

0.05% methyl blue or aniline blue in EITHER acidified 70% glycerol OR lactic acid.

Acidified glycerol

Glycerol 700 mL; water 300 mL containing 2 mL concentrated HCl.

Reference

Grace, C. and Stribley, D.P. (1991). A safer procedure for routine staining of vesicular-arbuscular mycorrhizal fungi. Mycological Research 95:1160-1162.

Staining Roots for the Detection of Nematodes

This procedure will work with fresh roots, or roots fixed in any of the common fixatives (formalin, alcohol, FAA etc.). It is important that the roots be either stained or fixed as soon as possible after collection since nematodes may migrate from the roots. Fungi are not usually stained by this method.

  1. Wash soil from roots with tap water.
  2. Place roots in 250 mL conical flask with approximately 70 mL of 1.5% sodium hypochlorite (or half strength household bleach) and bleach for 5 minutes with occasional stirring.
  3. Rinse roots with water, then soak for 15 minutes in 1% acetic acid. This acid rinse step is critical for consistent staining, especially of fixed roots.
  4. Drain off acid solution, then place roots in 30 mL distilled water to which 1 mL stain (see below) has been added. Heat over a low flame until boiling. Boil gently for 30 seconds, then allow to cool for 30 minutes at room temperature.
  5. Remove excess stain by rinsing with water. Place roots in 20 mL acidified glycerol, and heat to boiling. Remove from heat IMMEDIATELY boiling commences, and cool quickly by standing flask in shallow water.
  6. Pour roots in glycerol into a Petri dish. Gently tease apart and mount on a microscope slide. Nematodes stain red; root tissue should remain unstained.

 
Stock staining solution

0.35 g acid fuchsin
25 mL acetic acid
75 mL water

Acidified glycerol

2 mL HCl, dissolved in 300 mL water
Then add to 700 mL glycerol

Reference

Byrd, D.W.T., Kirkpatrick, T. and Barker, K.R. (1983) An improved technique for clearing and staining plant tissues for detection of nematodes. Journal of Nematology 15:142-143. 

  

InfectedRootTissueStained.jpg 
Staining roots for detection of nematodes

 
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