Cross-section of Woad leaf at 10 magnification taken through a microscope:
The dark cell outlines of this woad leaf cross- section are indigo, which was precipitated in the leaf cells using ammonia gas.
The indigo appears as a dark circle around the cell and may look olive green or navy in your browser.
Notice the cell outline in dark blue/green. This is the indigo precipitated within the cell structure of the leaf.
The above leaf fragment of Isatis tinctoria, has been growing under frost conditions.
Using a method described by Molisch in 1899 and referred to in Hurry's book (p. 42-46), I wanted to demonstrate the presence of indigo in Isatis tinctoria leaves that had been exposed to frost.
The leaves for my experiment were harvested October 28th, 2007, after two months of exposure to below 0°C, night temperatures, and two weeks of daytime frost. The plants were not dormant. Woad remains leafy and evergreen under snow.
To precipitate the indigo within the leaf, I exposed the leaf to ammonia gas, in a closed glass jar, for 7 days. This turned the leaf blue-green.
I then had to remove the chlorophyll from the leaf by soaking the ammonia treated leaf for 24 hours in 99.9% isopropyl alcohol.
The treated leaf was very brittle. I soaked the leaf in warm water for 2 minutes and this corrected the brittleness, allowing me to prepare a cross section microscope slide and examine it under a microscope.
I didn't have access to Canada Balsam, as suggested by James B. Hurry, (The Woad Plant and Its Dye, 1930, p. 42) so I prepared my microscope slides of Isatis tinctoria using clear nail polish. These specimens have not been stained.
This is a British landrace variety.
The indigo is seen in the dark cells. The brighter yellow-green areas are without indigo
Cross -Section of a woad leaf at 10 magnification
The darker areas are the indigo within the chlorophyll cells, after the chlorophyll has been removed with alcohol.
10X Magnification of the edge of a woad leaf showing the absence of indigo in the leaf hairs.
There is some indigo showing on the inner edges of the leaf, but the leaf hairs are transparent and colourless. It was hard to take a clear picture of the leaf edge due to the back lighting on the slide.
French selection from "Bleu de Lectoure" seed.
The indigo in this leaf tip sample has very deep indigo in the cells but also something the other leaf samples did not reveal...
Indirubin, an isomer of indigo.
The indirubin shows up in the cross section as pink, mauve or purple. In the cells with indirubin, indigo blue is also present. Indirubin is a result of the conditions in which the indigo precursors are transformed within the leaf.
This sample was taken from the tip of the leaf, after frost.
There is a significant decrease in indigo precursors within the leaves of Isatis tinctoria during the growing season, with the highest yields occurring in the late July or Early August in my area, after a period of warm, sunny weather. Subsequent harvests have a lower indigo yield per weight of leaves.
2nd year plants, flowers and seeds also contain indigo precursors, but at a lower concentration.
After frost exposure, during the growing season, Isatis tinctoria may still be used for dyeing but the indigo yield is less.
Second year plants may also be used for dyeing, before the scape emerges. The indigo precursor content is less than with 1st year leaves.