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 author 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
are made:
-
Root segments of a length of ca. 0.5-1.5 cm are heated at 90oC in 10% KOH
for 1-2 h, depending on the colour and thickness of the roots.
-
Cleared root segments are rinsed in tap water for some minutes.
-
Root segments are heated in 20% HCl for 1 h.
-
Root segments are 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.
