Inoculum 56(4) - Mycological Society of America

MYCOLOGICAL NEWS
A Simple Blue Staining Technique for Arbuscular Mycorrhizal
and Other Root-Inhabiting Fungi
Vierheilig et al. (1998) published a method for staining
the fungal symbiont in arbuscular mycorrhizae in which they
used ink and vinegar to reduce the use of dangerous and noxious
chemicals such as Trypan blue. Besides having obvious
health and safety advantages, the method is useful for circumstances
where access to chemicals is restricted or difficult,
as in field studies, work in which young students are involved,
or use by amateur mycologists or gardeners who
might wish to examine organisms in roots.
One disadvantage of the ink and vinegar method is that
acetic acid is somewhat unpleasant to use because of its odor.
Consequently, another safe method of acidifying the roots
with a less odorous compound was sought. Using very dilute
HCl gave good results (see Figure). If, hydrochloric acid is
not available, other weak acids can be used. Trials with Tonic
Water (SUGAR-FREE!) (pH measured at 3.7) worked perfectly,
resulting in an even less hazardous method. However,
in a laboratory setting, HCl is easy to obtain, and at the dilution
suggested, relatively safe. Probably virtually any acidic
liquid will suffice for the acidification process.
In the original recipe, Vierheilig et al. used various inks,
and found them to have different efficacies. One product not
mentioned, but which gives excellent results (see Figure), is
Parker Quink with SOLV-X®, a proprietary solvent that appears
to assist with the staining. This has been found to be
available in several different countries, including Mexico, the
UK, and Belgium. Whichever ink is chosen, ensure it is permanent.
For example, Quink also comes in a Royal Blue
Washable version, which should not be used.
Reagents
For initial clearing of alkali-soluble pigments, 1 M KOH
solution is used. If, for example, in the field, potassium hydroxide
cannot be obtained, a proprietary caustic drain cleaner
(these mainly consist of sodium hydroxide) can be diluted
and used instead, but the result is not as good because the
roots do not soften as much as with KOH.
For roots that retain some pigment after alkali-clearing,
an aqueous solution of 10 % household bleach (resulting in
approximately 0.25 % sodium hypochlorite solution) works
very well. Other bleaching agents, such as hydrogen peroxide
can be used.
The ink can be made up in 1 % HCl (or other acid) at
1:50 v/v, or it can be added with a dropper directly to the
roots after they are placed in the acidifying reagent.
The Protocol
1. Wash the roots with tapwater to remove soil particles
and select those for staining.
68 Inoculum 56(4), August 2005
An arbuscular mycorrhiza between Plantago lanceolata
and an un-named Chinese fungus stained with acidified
Quink. An entry point with a hyphal coil and a colonization
unit with abundant arbuscules is revealed.
The mycorrhiza was provided by courtesy of Yuan Yuan
Ling (China) and Mauritz Vestberg (Finland).
2. On a hotplate or gas burner, bring the roots to the boil in
the alkaline solution, and immediately remove the heat
source (take extreme care and wear suitable safety goggles).
It is also possible to use a microwave oven by
placing the roots in about a 2-cm depth of reagent, starting
the microwave on full power, and when boiling point
is observed, immediately switching off. Alternatively,
very hot (near-boiling) alkali solution can be added, or
the roots can be heated in the alkali solution at about 60-
80 C for 1 hour. If the roots are not heavily pigmented,
they can just be left in cold KOH for 24 hours with quite
a good result.
3. Remove heat and leave to stand for a time up to 24 hours
(minimum, for fine roots with very little pigmentation, 2
hours). It seems to do little harm to leave most roots for
a few days, although some of the finest roots will disintegrate.
It is necessary to experiment with the system for
the particular plant species being stained.
4. Rinse briefly in tapwater to remove excess alkali.
5. If the roots are still dark and opaque because of pigmentation
(as in many woody perennials), they can be
bleached whilst observing them under a dissecting microscope
until the stele just becomes visible. The bleaching
should be for the shortest possible time, as excessive
bleaching reduces the staining efficacy. Transmitted
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