Handsectioning and staining

Frequently, although it is important to assess the mycorrhizal status of roots of seedlings used in experiments for the presence of mantle and Hartig net, the sample size precludes embedding materials prior to sectioning. A considerable amount of information can be obtained, however, using simple techniques. Very small roots can be hand-sectioned and stained using modifications of methods published by Frolich (1984) for sectioning and Brundrett et al. (1988) for staining. The procedure is outlined in Fig. 1. Roots to be placed between sheets of parafilm on a dental wax base are kept moist with water. Thin sections cut with a sharp two-sided razor blade are picked up with fine forceps and placed in a small staining basket constructed either by cutting the lid and end off a Beem™ capsule or cutting polyethylene tubing into appropriate lengths and gluing nylon screen (50 /an mesh) to one end with fast-drying epoxy. This basket with sections can be transferred from solution to solution, blotting the basket on absorbent tissue during the transfer. Brundrett et al. (1988) constructed a multiple chamber system by fusing several polyethylene "rings" onto small rings placed on the underside of a larger sheet of nylon screen (see their Fig. 1) for the handling of many samples at the same time.

For determination of the presence of a mantle and Hartig net, sections can be stained for approximately 1 min in an aqueous solution (0.05%) of toluidine blue O, rinsed in water, removed from the basket with a toothpick and mounted in water under a cover-glass on a slide. An alternate and effective method of demonstrating the mantle and Hartig net involves clearing of root hand-sections in 10% KOH for 6-12 h at 90 °C, rinsing, staining in 0.01% chlorazol black E and then mounting them in glycerol under a cover glass for observation (Brundrett et al., 1990). Observation with Nomarski interference contrast optics gives particularly clear images of the Hartig net (Fig. 2). Wilcox and Marsh (1964) used a combination of chlorazol black E and Pianase III-B to demonstrate fungal hyphae in sections of mycorrhizal Pinus roots embedded in paraffin. If it is important to demonstrate features of the root such as Casparian bands in the endodermis and exodermis and lignified tissues, a very effective method has been published by Brundrett et al. (1988). Freehand sections are placed into a basket and stained

Place root between two pieces of parafilm. Add a small drop of water.

Pasteur pipette.

Place parafilm sandwich on a wax block. Slice transverse sections as thinly as possible with a sharp, two-sided razor.

Pasteur pipette.

Wax block

Place sections in staining basket which is in water.

Staining basket Petri dish

Pick up sections with fine forceps.

Wax block

Place sections in staining basket which is in water.

Transfer basket to stain

Transfer basket to stain

Staining basket Petri dish

Glass slide

Glass slide

Stain Blot Rinse Blot

Fig. 1. Method for hand-sectioning and staining roots.

Fig. 2. Transverse hand section of field-collected Fagus grandifolia Ehrh. ectomycorrhiza, cleared in KOH and stained with chlorazol black E. A thick mantle (*), Hartig net hyphae (arrowheads) and extraradical hyphae (double arrowheads) are evident. Photograph courtesy of Martin Damus.

in 0.1% (w/v) berberine hemisulphate (Sigma, CI No. 75160) in distilled water for 1 h, rinsed in several changes of distilled water, blotting each time, stained in 0.5% (w/v) aniline blue WS (Polysciences, CI No. 42755) in distilled water for 30 min and then rinsed as above. Finally, sections are transferred into 0.1% (w/v) FeCl3 in 50% glycerine (prepared by first dissolving FeCl3 in distilled water (0.1%), filtering, and adding 50% glycerine to this solution). Sections are mounted in this solution and observed with ultraviolet illumination on an epifluorescence microscope (Fig. 6). Any number of histochemical procedures, many considered below for embedded tissues, can be used on freehand sections.

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