CLEARING ROOTS AND STAINING OF MYCORRHIZAE

Generally, the method of clearing of roots and staining of mycorrhizae used is that of Phillips and Hayman (1970). Based on the experience of the authors of this website and suggestions of Professor R. E. Koske, Rhode Island University, USA, two changes were introduced to the original method given by Phillips and Hayman (1970). First, roots earlier cleared in 10% KOH were acidified in 20% HCl instead of 1% HCl as in the original procedure. Second, the concentration of trypan blue was increased from 0.05% to 0.1%.

The following steps were made:
  1. Root segments of a length of ca. 0.5-1.5 cm were heated at 90oC in 10% KOH for 1-2 h, depending on the colour and thickness of the roots.
  2. Cleared root segments were rinsed in tap water for some minutes.
  3. Root segment were heated in 20% HCl for 1 h.
  4. Root segments were heated in 0.1% trypan blue for 1-2 h.

The steps 1, 3, and 4 may be done without heating, remaining roots in KOH, HCl, and trypan blue at room temperature for at least 12 h.

The roots best showing diagnostic properties of mycorrhizae are those of Zea mays.

REFERENCE

Phillips J. M., Hayman D. S. 1970. Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Trans. Brit. Mycol. Soc. 55, 158-161.