Clearing and staining

Based on the clearing and staining procedure described in Bruzzese and Hasan (1983).

Clearing-staining solution (Makes approx. 1 L)

• 300 mL ethanol (95%)
• 150 mL chloroform
• 125 mL lactic acid 125 mL
• 150 g phenol
• 450 g chloral hydrate
• 0.6 g aniline blue

Stockwell's bleach

This solution had been published in the 1940's and 1970's. Morawetz (2013) is the clearest recipe available online.

• 10 mL 10% chromic acid (or 1 g powdered with dH₂O)
• 10 mL glacial acetic acid
• 1 g potassium dichromate
• 80 mL dH₂O

Clearing and staining

1. Cut the leaves into 1-3 cm² pieces.
2. Immerse the leaves in the clearing staining solution (2 mL·cm²) in stoppered glass vial.
3. Leave for 48 hrs at RT (approx. 25°C).
4. Remove the leaf pieces and place in chloral hydrate (2.5 g·mL^-1 in water).
5. Leave for 12-24 hrs at RT.
6. Remove the piece and rinse with distilled water.

If after chloral hydrate treatment the leaves are not completely cleared, soak in Stockwell's bleach and repeat the clearing and staining procedure. Mount the stained leaf pieces in/on a permanent mounting medium.

References

Bruzzese, E., and S. Hasan. 1983. “A Whole Leaf Clearing and Staining Technique for Host Specificity Studies of Rust Fungi.” Plant Pathology 32 (3): 335–38. doi: 10.1111/j.1365-3059.1983.tb02841.x. http://doi.wiley.com/10.1111/j.1365-3059.1983.tb02841.x

Morawetz, Jeffery J. 2013. “A Clearing Protocol for Whole Tissues: An Example Using Haustoria of Orobanchaceae.” Applications in Plant Sciences 1 (1): apps.1200361. doi:10.3732/apps.1200361