STAIN TECHNOLOGY VOLUME 5% OCTOBER, 1947 NUMBER 4

IMPROVEMENTS IN THE PARAFFIN METHOD

ADRIANCE S. FOSTER AND ERNEST M. GIFFORD, JR.,

Department of Botany, University of Califmnia, Berkeley, California

ABsTRAcT.-Dilute hydrofluoric acid alone and in conjunction with glycerin and ethyl alcohol was employed successfully to soften various types of refractory plant materials embedded in par&. Serial sections were cut at 6-8 p and no appreciable deleterious effects on cell walls, cell contents, or staining procedures occurred. “T&s” and “phlobaphene compounds” can be removed from tissues softened by this method by treating the sections for 1 2 4 8 hours with an aqueous solution of chromic acid, potassium bichro- mate and glacial acetic acid prepared according to the formula given by Johansen (1940).

I. SOFTENING OF REFRACTORY PLANT L%TERIAL

ENBEDDED IN PARAFFIN Certain plant materials, as well as animal tissues, tend to be ex-

tremely difficult to section after being embedded in paraffin. Meth- ods utilizing mixtures of glycerin-alcohol and glycerin-anilin-oil, have already been recommended for softening embedded animal tissues (Baker, 1941; Lendrum, 1944). In a modified form these methods can also be adopted for plant tissues. Exposing embedded plant material to warm water, as suggested by Johansen (1940), Sass (1940), and Ball (1941), has also been advocated but this technic may result in the serious maceration of tissues, especially if the treatment is prolonged.

It has been assumed rather generally that difficulty in sectioning such material as woody stems, coriaceous leaves, roots, etc., was produced by the lignin and silica in the walls of fibers, sdereids, and tracheary elements. Kerr (1934), however, has shown that failure to microtome woody materials successfully is due largely to the condi- tion of the cellulose in the walls rather than to their mineral and lignin content. According to Kerr, the softening action of hydro-

ST4IX TECASOLOGT, YOL. 99. S O . +. OCTOBER 1947

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150 STBIS TECHSOLOGY

fiuoric acid on woody tissues “is associated with a partial degrada- tion of cellulose and the formation of hydrocellulose”. This results in a change “in the physical properties of the fibers”.

In the light of the above methods, the writers have tested the softening action of hydrofluoric acid, either alone or in conjunction with glycerin-alcohol, on a aide range of plant tissues after embed- ding in paraffin. Specimen vials lined with paraffin were used and prior to treatment a small area of the embedded tissue was exposed by trimming with a razor blade. All materials used in these experi- ments were embedded in “Tissuemat”. The composition of the various solutions and the exposure times are presented in the follow- ing table.

TABLE 1. COMPOSITION OF FLUIDS ASD EXPOSURE TIMES. I

a.

Schedde I 20 ml. glvcerin 1 tieek 80 ml. oi70% ethyl alcohol follon-ed by: l0yo hydmfluoric acid 3 t o 7 days

Composition

3.

Exposure-period Room Temp. W C . &

10 ml. glvcerin 10 ml. hidrofluoric acid 80 ml. 95% ethyl alcohol

3 days to 2 weeks

1. I 10-15% hydrofiuoricacid 1 3to7days

The exposure times listed in the above table proved satisfactory for the authors’ materials but doubtless will vary depending upon the “hardness” of the tissues in each case. The softening action of the reagents may be accelerated by placing the specimen vials in a 3’2” C. electric oven. Tests with a sharp razor blade should be made at intervals until the desired texture is obtained.

Any of the above schedules appreciably softened the materials investigated but schedules 2 and 3 usually yielded smoother ribbons, apparently because of the “lubricating” action of the glycerin. Ma- terial allowed to remain in aqueous hydrofluoric acid for long periods may show signs of maceration. This may be alleviated by the use of ethyl alcohol. It has been employed successfully in microtoming such material as the coriaceous leaves of Mouriria Huberi Cogn. (Foster, 1947) and the scaly winter buds of species of Drimys, Pseudowintera, Illicium and Trochodendron.

The writers have followed the tannic acid, iron chloride, safranin schedule of Foster (1934). In addition, the common fast-green-safranin

Schedule 3 is recommended for general use.

This procedure has been followed by successful staining.

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IMPROVEMEXTS I S THE PARAFFIX METHOD 131

schedule is not altered. No visible alterations or deleterious effects of these treatments have been observed in the plant tissues so treated.

11. THE REVOVAL OF TANNINS AND PHLQBAPHENE COM- POUSDS FROM PARAFFIN SECTIOXS

Often associated with processed material which is difficult to section is the presence of tannins and phlobaphene compounds. This is particularly true of many tissues in bud scales, leaves, stems and roots. Johansen (1940) has described a method, originated by Dr. Palmer Stockwell, of treating paraffin sections prior to staining. The formula is as follows:

Chromic acid.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . f g. Potassium biochromate. . . . . . . . . . . . . . . . . . . . . . . . 1 g. Glacial acetic acid. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 ml. Distilled HzO.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 ml.

Depending on the material in question, it has been found that 12 to 48 hours is required to remove adequately the tannin compounds. Slides-should then be washed in running water for 1 to 2 hours before proceeding to the usual staining schedules.

This procedure is of great value in histogenetic studies since there is a tendency for the embryonic layers of certain leaves, bud scales, and young stems to accumulate “tannins” to the point of obscuring nuclear divisions and plastid structure. Following this treatment, staining is noticeably improved. If the maximum time of treatment is required, there is a tendency for the sections to drop off unless the adhesive described by Sass (1940 p. 50-51, Formula 111) is used.

The potency of the bleaching fluid decreases with age so that a fresh solution should be prepared after marked oxidation has occurred.

LITERATURE CITED

BAKER, JOHN R. 1941. A fluid for softening tissues embedded in paraffin wax. J.

BALL, ERNEST. 1941. Microtechnique for the shoot apex. Amer. J. Bot., 28,

FOSTER, ADRraNeE S. The use of tannic acid and iron chloride for staining

FOSTER, A%RI.meE S. Structure and ontogeny of the terminal selereids in the

JOH.~NSEN, DONALD -4. 1940. Plant Microtechnique. McGras-Hill, xe’eff York. KERR, T~obias . 1934. Action of hydrofluoric acid in softening wood. Tropical

LENDRLW, AWN C. On the cutting of tough and hard tissues emhedded in

SASS, JOHN E. 1940. Elements of Botanical Xicrotechnique. XcGraw-Hi& Sen-

. .

Roy. Micr. Soe., 61,75-8.

B M 3 .

cell +Is in meristematic tissue.

leaf of Mouriria Huberi Cogn.

1934.

1947. Stain Techn., 9, 9 1 4 .

Amer. J. Bot., 34. In press.

Woods. SO. 40,37-19.

paraffin. Stain Techn., 19, 143-4.

York.

1944.

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