Skip to content

VLA Protocol

Author

Hilary Major

Updated

22 February 2022 by Cathleen Lapadat

Associated Projects

Global Oaks

Goals

Detailed steps on the organization, clearing process, and image scanning of individual leaves for VLA ( leaf vein length per unit area) work. Method was adapted by Benjamin Blonder to produce leaf skeletons by eroding the lamina tissue and exposing the lignin rich veins to dye.

IMPORTANT: Please wear gloves at all times when handling chemicals

Supplies

  • Pyrex dishes
  • Petri dishes
  • Sodium Hydroxide
  • Ethanol
  • Safranin O
  • Glycerol
  • Duralar
  • Scanner

Steps

Sanitization (For Imported Samples)
  1. Store samples at -80C freezer for 48 hours to kill off any transported fungi or pathogens.
Organization

  1. Arrange the leaves by size.

  2. Match the leaves with the appropriate petri dish or pyrex dish. The larger leaves should be soaked in the pyrex dish.

  3. Write 1-3 word description of leaf characteristics.

Making Leaf Skeletons

  1. Soak leaves in 5% Sodium Hydroxide for 5-30 days

    1. Until they are light beige and uniformly translucent.
    2. No more than 5% of the leaf area varying in color. Largely dependent on the individual sample.
    3. Change out the 5% sodium hydroxide when necessary (when it becomes very dark).
    4. Keep an eye on the leaves. The leaves may disintegrate if kept in the solution for too long.

  2. Pour out sodium hydroxide into hazardous waste container.

  3. Add solution with 50% bleach for 15-90 minutes

    1. Until it is pure white.
    2. 1-2% of leaf area is very light beige.

  4. Pour out bleach into hazardous waste container.

  5. Soak leaves in 50% ethanol for 5-15 minutes.

  6. Pour out ethanol into hazardous waste container.

  7. Add 0.05% Safranin O in 200 proof ethanol for 24-48 hours.

  8. To remove excess dye, rinse leaves in 100% ethanol 3 times for 4-24 hours, or until no more dye leaches from leaf tissue.

Scanning

  1. Mount multiple leaves in glycerol between two sheets of duralar.

  2. Label with the species names.

  3. Scan leaves at 24-bit resolution.

Notes

Please refer to “VLA ImageJ methods” for details steps on processing the images and tracing the veins.

Works Cited

  1. https://benjaminblonder.org/The_secrets_of_leaves/Making_skeletons.html
Last update: October 20, 2022