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.
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 6080 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 Continued on following page
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MYCOLOGICAL NEWS
light helps with observations in this process, though it is not essential.
6. Acidify the roots with 1 % HCL or other acid. The acidification can be seen to work, because the roots lighten in color immediately. Move the roots to acidified ink or add ink by dropper directly to the acidified roots. The roots can be examined microscopically after about 30 minutes, though staining generally improves over a longer period. It is best to leave them at least 4 hours, or preferably overnight. They can be kept in acidified glycerol + ink indefinitely. Destaining for a period of a few hours in acidified glycerol is recommended, resulting in improved clarity and contrast, but if one is in a hurry, the stained root fragments can be transferred immediately to a suitable mountant on microscope slides for observation. Polyvinyl alcohol lacto-glyc-
erol (PVLG) (Omar, Boland & Heather 1979) produces a semi-permanent mount (these last many years), or acidified glycerol can be used if there is no need for longer term preservation of specimens. This method reduces even more the use of unpleasant chemicals when compared with the ink and vinegar technique. Nevertheless, all health and safety recommendations and regulations must be followed when using acids, dilute or otherwise, or hot alkaline solutions. References: Omar, M. B., Bolland, L. & Heather, W. A. (1979). Bulletin of the British Mycological Society 13, 3132.; Vierheilig, H., Coughlan, A.P., Wyss, U. & Piché, Y. (1998). Applied Environmental Microbiology 64, 5004-5007. Christopher Walker Royal Botanic Garden, Edinburgh
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Joan Bennett Elected to the National Academy of Sciences Joan W. Bennett, Professor of Cell from 2002-2004, a period of major and Molecular Biology at Tulane Univerchanges in the journal, including an ownsity, was elected to the National Academy ership transition from the New York of Sciences in May. Election to the NaBotanical Garden to the MSA, on-line tional Academy, which serves as a nationpublishing, and most significantly, the al advisory group on matters of science, conversion from an all-paper to an allengineering and medicine, is one of the electronic submission, review and publihighest honors bestowed across the scication system. Dr. Bennett oversaw all of ences. Dr. Bennett will be only the second this turmoil with aplomb, and with all of current Academy member in the Society, those changes now safely in place, it’s along with T.N. Taylor from the Universihard to imagine how it was managed in ty of Kansas. such a short time period. Dr. Bennett is broadly known for her We cannot claim Dr. Bennett as ours Bennett contributions in research, teaching and alone, however. Among other positions in service. Her election to the National the American Society for Microbiology, Academy recognizes her seminal contributions in the areas of she served as its President, and throughout her career has fungal production of secondary metabolites and industrial mi- been instrumental in representing fungi in the broad realm of crobiology. She is an active instructor with responsibilities microbiology, particularly on an international level. She for many courses, and her excellence in teaching and advis- counts among the very small number of fungal biologists ing is recognized through numerous awards. Her record of with membership in the National Academy, and she is certain service, to Tulane and to professional societies, is staggering. to be a strong and effective advocate for mycology. So, it is Dr. Bennett has co-edited five books, and served in an both with pleasure and pride that we congratulate Joan on her editorial capacity on twelve different journals and book se- election to the National Academy. ries. MSA members may be best acquainted with Dr. BenDavid M. Geiser nett from her service as Editor-in-Chief of MYCOLOGIA
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