Methyl Green and Pyronin Staining of Frozen-dried Tissue

By J. 0. LAVARACK

(From the Zoology Dept., King's College, Strand, London, W.C. 2)

With 2 plates (figs. 1 and 2)

SUMMARY

Frozen-dried tissue stains readily with methyl green / pyronin. The gross in-tracellular distribution of nucleic acids in frozen-dried pancreatic tissue stainedwith methyl green / pyronin corresponds with that found in chemically fixed tissue.

IT has been suggested by Brachet (1953) that his methyl green / pyroninmethod (1940) is not readily applicable to frozen-dried material. It is the

purpose of the present paper to consider this conclusion of Brachet's.The specificity of methyl green and pyronin has been the subject of much

recent work (Kurnick, 1950; Taft, 1951a and b).

OBSERVATIONS ON THE TECHNIQUE OF METHYL GREEN-PYRONIN STAINING

The following procedures were adopted as standard, and will be sodescribed.

In all cases the material was fresh albino rat tissue.

Frozen-dried tissue

Cooled immediately in liquid propane, the latter cooled by liquid nitrogen.Dried at —400 C. for 2-4 days in a tissue-dryer employing the principle of the

cold surface trap (cooled by solid carbon dioxide / alcohol mixture to about—780) for water vapour under reduced pressure (Bell, 1952).

Infiltrated with 54° paraffin.Sections cut at 7/x and attached to new slides by floating on a drop of aceto-

nitrile on the slide and application of just sufficient heat to flatten.Dried on a hot plate.Paraffin removed and sections rehydrated through alcohols.Stained by (a) the methyl green / pyronin technique suggested by Brachet

(1953), with a solution held with acetate buffer at pH 47 and differentiationin 75 per cent, ethyl alcohol, or (b) the technique suggested by Taft (1951a)with a similarly buffered solution and differentiation in a mixture of tertiarybutyl alcohol and ethyl alcohol.

Xylene; Polystyrene.

Chemically fixed tissue

Immersed immediately in Serra solution (Brachet, 1953) and fixed for 1 to i\hours.

[Quarterly Journal of Microscopical Science, Vol. 96, part 1, pp. 29-33, March 1955.]

30 Lavarack—Methyl Green and Pyronin Staining of

Washed in 95 per cent, alcohol.Absolute alcohol, xylene.Infiltrated with 540 paraffin.Sections cut at 7/A and attached to cleaned slides by floating on warm water

with a smear of albumen-glycerine as adhesive. Dried on a hot plate.Paraffin removed and sections re-hydrated through alcohols. Stained as for

frozen-dried tissue.Xylene; polystyrene.

After trial of the method on a number of chemically fixed tissues, pancreaswas chosen as a test material because of its combination of exocrine cells,intensely staining and with a characteristic distribution of the cytoplasmicpyroninophil material, with groups of weakly staining endocrine cells. Anumber of specimens of frozen-dried and chemically fixed pancreas weresubjected to the standard methods of preparation and staining, being stained inbatches so that frozen-dried and chemically fixed slides traversed the stainingand subsequent treatment together. Under these conditions, with comparisonof the sections by eye under the microscope, the result was unequivocal.Staining, green to blue of the nuclear material, rose-pink to deep red in thecase of nucleoli and cytoplasm, was as intense in frozen-dried as in the control(chemically fixed) pieces of tissue (fig. 1, A, B, C, D). This result was constantlyobtained and was subsequently found to be uninfluenced by the staining methodchosen or by all except one of a number of variations of technique now to bedescribed. It thus appeared that there is nothing in frozen-dried tissue assuch which inhibits its reactions with methyl green and pyronin.

Attempts were now made to define factors which might operate duringmanipulation to alter the staining capacity of frozen-dried tissue. In the courseof these, some incidental conclusions were reached on the technique, and adescription of them is included.

Preparation. During this stage the section of tissue in paraffin is fixed to aslide, its wax is dissolved out, and it is re-hydrated. Until this stage it may beexpected that a considerable proportion of the protein in frozen-dried materialwill remain to be denatured, but that the action of heat and alcohols will bringabout further denaturation. Before this occurs, however, the cells are relativelysusceptible to loss of protein substances soluble in water, including stainablenucleoproteins. Tissue exposed to water at this stage may be expected to losestaining capacity. It is possible that atmospheric water vapour might be afactor, but a more potent one would be an aqueous solution such as the egg-albumen used as a section adhesive.

Frozen-dried paraffin sections (a) stuck to slides by the standard methodwere compared with sections (b) pressed on to a slide, the surface of whichwas smeared with albumen-glycerine adhesive and with sections (c) floatedon a drop of distilled water on the slide and subjected to heat sufficient toflatten. The slides, after attachment of the sections, were all dried on a hotplate and subjected to the standard rehydration, staining, dehydration, and

Frozen-dried Tissue 31

mounting procedures. If the sections were on a thick layer of albumen-glycerinesuch as might require over an hour to dry on the hot plate, the tissue spreadand disintegrated; with thinner films of adhesive and with water, drying outquickly, sections were obtained which could be stained. Compared with thecontrols (a) these sections all showed spreading and various grades of reductionin staining capacity. In the case of slides floated on water, staining was minimal,although differentiation between pyronin and methyl green staining com-ponents of the cell was still recognizable (fig. 2, c, D).

Other possible mechanisms for this effect are that the tissue itself or the egg-albumen solution may contain nucleases, whose action would be favoured byhydration.

Staining. Satisfactory staining was obtained with both dyes, whichever ofthe standard staining procedures was used. It is known that results dependupon the quality of the dyes. The methyl green of this series was a RevectorMicroscopic Stain produced by Hopkins and Williams, and this stainedsatisfactorily. Two pyronins were tried, of which one stained feebly, particu-larly when used according to the procedure of Brachet. The other, producedby Geigy, gave very satisfactory results. This finding was confirmed by test-ing comparable sections in staining solutions differing only in the brand ofpyronin used. Intensity of pyronin staining differed considerably.

Dehydration and mounting. The well-known lability of pyronin in tissueduring differentiation with alcohol was observed here in the use of the originalpyronin. In this instance the method of staining and dehydration with tertiarybutyl alcohol / ethyl alcohol mixture as recommended by Taft was found togive the more stable staining and the greater final intensity. With the methylgreen of Hopkins and Williams, the pyronin of Geigy, and the staining solu-tions of Brachet and Taft, staining was stable and, in fact, the stained tissuecould be subjected to differentiation for 10 minutes in 70 per cent., 95 per cent.,or absolute ethyl alcohol without excessive loss of pyronin.

Since differences in reaction to alcohol during differentiation might formthe basis of differences of staining, pairs of slides, one in each pair fixed inSerra's fluid, the other frozen-dried, were subjected to differentiation for 10minutes in varied strengths of alcohol from 70 per cent, to absolute. Nodifferences were observed.

INTRACELLULAR DISTRIBUTION OF STAINABLE SUBSTANCES

The distribution of stainable substances in pancreatic exocrine cells of therat, frozen-dried, is shown in fig. 1, A, B. The regularly outlined nuclei staindiffusely green, and through this background more intensely green-stainingmaterial is dispersed in irregular masses and particularly concentrated in azone immediately surrounding the nucleolus. There is also a thin zone ofdeeper green-staining in the region of the nuclear membrane. The nucleoli,of which there is one or more in each nucleus, stain a clear rose-pink. Thecytoplasm of these cells contains pyronin-staining material with a distinctly

32 Lavarack—Methyl Green and Pyronin Staining of

zonal distribution. In a region that is variable in width but comprises aboutone-half to two-thirds of the depth of the cell from the internal cell marginbounding the gland lumen, the pyronin is distributed in an approximatelyreticular pattern, appearing to occupy space between large granules of non-staining substance. In an external region extending to the outer border of thecell, the pyronin-stained material is confluent and deep red, and this regionusually completely contains the nucleus or the latter may project partially intothe internal region.

The distribution of these substances in pancreatic exocrine cells fixed inSerra's solution is shown in fig. i, c, D. It conforms grossly to that withinfrozen-dried cells though the latter appear better preserved. Compared withthe frozen-dried cells there appears to be some shift of cytoplasmic substance(compare Bell, 1952); but, since the distribution may depend on the functionalstate of the gland cell, no conclusions on its relation to chemical fixation will bedrawn from this material.

The cytoplasm of the frozen-dried pancreatic endocrine cells stains palelythroughout without regional variation, the nucleus also palely, the nucleolusrelatively deeply (fig. 2, A, B).

DISCUSSION

When the routine technique described here is used, frozen-dried tissue issuitable for the application of methyl green / pyronin staining reactions.Further, the result is reliable and the staining is stable and not disturbed byvariations of differentiation technique. The frozen-dried and chemically fixedtissues seem to behave in the same way during staining and to the eye haveapproximately the same intensity of colour. Taft also reported satisfactoryresults (1951a). To understand why some workers have failed to stain frozen-dried tissue, it is necessary to seek factors which may contribute to differencesbetween its behaviour and that of chemically fixed tissue. One such is theaction of water. If sections are floated on water as a preparation for alcoholicdehydration, a striking difference in staining behaviour results (compare figs1, A, B and 2, c, D). Contact with water at this stage has no effect on thesubsequent reaction in the case of chemically fixed tissue but may almostabolish it in the case of frozen-dried tissue. This effect might be due tothe diffusibility of the frozen-dried substances or possibly to their suscepti-bility to nucleases which may be present. Another possible action which re-quires consideration, in view of the suggestion of Brachet (1953) that lipidesretained in frozen-dried material may influence the course of the staining

FIG. 1 (plate), A and B, exocrine cells of rat pancreas, frozen-dried and stained withmethyl green / pyronin. A shows the distribution of pyronin-staining (internal and externalcytoplasm, nucleoli). B shows distribution of methyl green (nuclear periphery and round thenucleoli).

C and D, exocrine cells of rat pancreas, fixed with Serra and stained with methyl green /pyronin. c shows the distribution of pyronin staining; D that of methyl green. Compare withA and B.

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Frozen-dried Tissue 33

reaction, is one of solution of these lipides by acetonitrile used in attaching thesections. Though it may occur, removal of lipides in this way is apparently nota prerequisite for the staining, since equally good results are obtained if thesections are simply attached to the slide by heat.

There is general agreement that the nucleoproteins are included in thebasiphil substances of the cell and this view is based on the Feulgen aldehydereaction, the action of nucleases, and absorption measurements. Localizationof these nucleoproteins in cells requires careful consideration of the conditionsunder which they were observed. The Feulgen-positive and the basiphilsubstances of pancreatic cells have been plotted in chemically fixed and infrozen-dried material. Chemically fixed material has also been used forobservations on basiphil substances combined with selective destruction bynucleases (Brachet, 1940). Absorption measurements have been made onfrozen-dried material, in this case unstained and simply transferred throughparaffin to glycerine (Caspersson, 1950). That deoxyribose nucleic acid isdistributed in the nucleus and ribose nucleic acid in the nucleolus andcytoplasm and that there are cytoplasmic zones of ribose nucleic acid are con-clusions derived from observation on cells, whether frozen-dried or chemicallyfixed, stained or unstained. So far, therefore, although the evidence is notexhaustive, it is consistent with a supposition that this is the distribution ofnucleic acids during life.

It is a pleasure to thank Prof. J. F. Danielli and Dr. L. G. E. Bell for muchinstruction and help in many ways during my stay in the Department ofZoology, King's College, Prof. J. Brachet for his kindness and decisiveassistance during a short visit to his department, and Mr. A. T. Green for themicrophotographs. The work was done during the tenure of a C. J. MartinFellowship of the Department of Health, Australia. The assistance of theDepartment is gratefully acknowledged.

REFERENCESBELL, L. G. E., 1952. Int. Rev. Cytol., 1, 35.BRACHET, J., 1940. C. R. Soc. biol., 133, 88.

'953- Quart. J. micr. Sci., 94, 1.CASPERSSON, T., 1950. Cell growth and cell function. New York (W. W. Norton & Co.).KURNICK, N. B., 1950. J. Gen. Physiol., 33, 243.TAFT, E. B., 1951a. Stain Techn., 26, 205.

19516. Exp. cell. Res., 2, 312.

FIG. 2 (plate), A and B, islet of endocrine cells of rat pancreas, frozen-dried and stainedwith methyl green / pyronin. A shows the distribution of pyronin staining, B that of methylgreen.

c and D, exocrine cells of rat pancreas. The photomicrographs show the effect of exposureto water after freezing-drying, with subsequent staining by pyronin (c) and methyl green (D).