Ecological Archives E096-056-A3
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Appendix C. Detailed procedures for root staining with trypan blue.
1. Wash out roots with distilled water.
2. Digest in 10% KOH at 90 °C for 30–60 minutes.
3. 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).
5. Acidify roots in 2% HCl until roots appear white.
6. Stain roots in aniline blue solution (0.05% Trypan blue in acidified 70% glycerol) at 90 °C for 3–5 minutes.
7. Destain overnight at room temperature in glycerol and lactic acid (1:1) as appropriate.
8. Mount roots on slides in destaining solution and examine.