Technical specifications

Chemicals:
10% KOH
Household vinegar (which is usually 4 or 5% acetic acid)
Ink: Blue, red and black (click to see effective inks)

Procedure:
  1. Roots were cleared by boiling in 10% (wt/vol) KOH (boiling times differed according to the type of plant [Click to see suggested time and tips]
  2. The roots were rinsed several times with tap water.
  3. Cleared roots were boiled for 3 min in a 5% ink-vinegar solution (e.g., 5 ml ink in 95 ml vinegar) with ordinary household vinegar (5% acetic acid).
  4. 4. After boiling roots were destained by rinsing in tap water (acidified with a few drops of vinegar) or alternatively by rinsing in pure vinegar (destaining times differed according to the type of ink [Click to see the effect of different inks]. Caution: not every type of ink works.
  5. After destaining roots were kept in the refrigerator (4C).
  6. Observation is done under a stereo microscopes (see below tips #3) or a conventional light microscope.


Microscopic observation:
  1. If clearing and staining is properly done this technique allows the observation of stained intraradical and extraradical AM fungal structures. Colonized roots are easily distinguished from noncolonized roots. Individual hyphae in heavily and partially colonized sections of roots are clearly visible. Arbuscules, vesicles, intraradical and extraradical hyphae, and penetration units are all stained by the ink dye.
  2. For photographic and assessment purposes, the best contrast was achieved with Shaeffer black ink. After destaining, roots were pale brownish red, whereas fungal structures remained black.
  3. Destaining of the blue and the black inks was most successful when tap water (with a few drops of vinegar) rather than vinegar was used.

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