Root Staining Procedures
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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.
Transcript of Root Staining Procedures
Root Staining Procedures
Staining Roots for the Detection of Mycorrhizae and Fungal PathogensThis 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 solution0.05% methyl blue or aniline blue in EITHER acidified 70% glycerol OR lactic acid.
Acidified glycerolGlycerol 700 mL; water 300 mL containing 2 mL concentrated HCl.
ReferenceGrace, C. and Stribley, D.P. (1991). A safer procedure for routine staining of vesicular-arbuscular mycorrhizal
fungi. Mycological Research 95:1160-1162.
