Osmichrome Fixation

With one plate (fig. 1)

FAITHFUL preservation of the general framework of cytoplasm has won for osmium tetroxide pre-eminence as a cytological fixative. In the pre-paration of tissues for study with the light microscope the most common procedures, such as those of Flemming, Altmann, and Champy, have com-bined the action of osmium with that of chromium in a single solution of marked acidity. The discovery of Palade (1952) that the optimum pH for fixation by osmium is 7·2 or even higher, places the favourable range for its activity well above that in which chromate solutions have their maximum effectiveness (Elftman, 1954).

In order to allow both osmium and chromium to act under congenial cir-cumstances we have experimented with their use in sequence. Besides provid-ing each reagent with more favourable conditions, there is an advantage in being able to experiment with the variables separately. We shall refer to the sequence of initial fixation in an essentially neutral osmium tetroxide solution, with post-chromation in a solution of controlled acidity, as ‘osmichrome fixation’.

We shall consider first the osmium reagent, then the chromate reagent, and finally the results achieved by their application.

The osmic reagent employed in osmichrome fixation is essentially the one recommended by Palade (1952) for the fixation of tissue destined for study by the electron microscope. A 1% solution of osmium tetroxide is prepared in M/40 Michaelis veronal buffer and adjusted to pH 7·2 Although the strength of the osmic solution is stated conveniently in terms of the amount of osmium tetroxide used in its preparation, this should not lead to the assumption that the tetroxide persists in unaltered form in the solution. When osmium tetroxide is dissolved it does not remain aloof but joins with water to form H₂OsO₅ (Yost and White, 1928). This may be called perperosmic acid, but the ionization constant is so small (8 × 10⁻¹³) that some biologists consider the name of acid inappropriate. No misunderstanding will result if cytologists continue to refer to solutions of osmium tetroxide, and this usage will be followed in the present paper.

The length of time during which tissues may be subjected to the osmium reagent is governed by two factors. The first is the slowness of penetration. Not only does this limit the size of the block which can be fixed by immersion but it ensures the presence of a fixation gradient, the interior showing some signs of belated access. The second difficulty is the brittleness produced by osmium tetroxide. These difficulties may be minimized by administering the fixative by perfusion. We have found it more practical to prepare our tissue in thin slices, thus decreasing the distance through which diffusion must occur, and to leave them in the i% osmium tetroxide solution for from 5 to 7 hours in a refrigerator at 5° C. Lowering the temperature slows down the chemical action of the osmium reagent more than it does its diffusion.

On the way to the chromating solution the tissue is rinsed gently to prevent the carrying over of an excess of osmium tetroxide. In our experience no good purpose is served by protracted washing at this stage.

The second step in osmichrome fixation is chromation, effected by a solution which is conveniently prepared from potassium dichromate, since potassium is a relatively innocuous cation and the acidity of the solution is close enough to the useful range to allow ready adjustment. We have found 2-5% potassium dichromate a useful concentration.

In a previous paper (Elftman, 1954) attention was called to the fact that the oxidative action of hexavalent chromium is particularly responsive to hydrogen ion concentration but is also accelerated by rise in temperature and is cumulative with time. There is consequently no unique combination of pH, temperature, and time to be prescribed for chromation. For routine use we prefer to restrict the chromation time to 18 hours; this allows tissues collected one morning to be osmicated during the day, chromated overnight, and embedded the following morning. The decision as to which temperature to employ depends to a certain extent on the tissue under study. Less physical distortion occurs when the temperature is not unduly elevated; for most purposes a room temperature of 20° C suffices.

With time and temperature specified, acidity is no longer a free variable. Our best results have been achieved with the chromating solution acting at a pH of 2·5−3·0. In order to stabilize the acidity without introducing deleterious ions we have employed mercuric chloride. The chromating solution which we use most frequently at present is prepared by dissolving potassium dichromate at 2·5% in a stock 5% solution of mercuric chloride. The pH of this solution may be adjusted to 2·5 by the addition of hydrochloric acid to accelerate the early stages of oxidation, or it may be used with its unmodified initial pH of 3·0. In either case the terminal pH after 18 hours of contact with the osmicated tissue is usually about 3·5, a rise considerably greater than that which occurs with fresh tissue during controlled chromation.

The preparation of osmichrome-fixed tissue for embedding follows the usual routine. Of the numerous clearing agents which are available, methyl benzoate (oil of Niobe) deserves commendation. For the small blocks of tissue currently of interest in this laboratory immersion in methyl benzoate for an hour and a half at 37° C is sufficient. Its relatively rapid action and constancy of composition are points of superiority over cedar oil. The subsequent infiltration with paraffin should not be prolonged unnecessarily. Sections may be cut readily at 2 μ.

Characteristic results obtained with osmichrome fixation may be studied in the photomicrographs, fig. 1, A−D. We have been able to utilize the usual range of staining techniques with a minimum of the difficulty so frequently associated with osmic fixation.

The periodic acid / Schiff procedure has resulted in the staining of granules of the Golgi apparatus in the uterus (fig. 1, A). This should not be taken as evidence of the presence of carbohydrate, since oxidation of unsaturated bonds of phospholipids provides a precursor for this reaction and osmium itself can affect the reagent.

Altmann’s anilin / acid fuchsin not only stains the mitochondria of the intestine (fig. 1, D) but also provides a clear presentation of the centrioles of the testis (fig. 1, B). The acroblasts are faintly visible in this section; they may be stained more deeply by the periodic acid / Schiff method.

The use of Sudan black is illustrated by the villus of fig. 1, c. The Golgi lipid is deeply stained and the apical mitochondria have taken up more of the dye than have the basal ones. Variation of the staining reaction with distance in from the tip of the villus follows a regular sequence and may be indicative of changes in the cells as they progress towards the apex. A comparison of Sudan black staining of osmichrome tissue with that treated by controlled chromation reveals a difference between basal and apical mitochondria in their reaction to osmium tetroxide.

In order to interpret the results of osmichrome fixation, some consideration must be given to the method of action of the reagents. The chemistry of fixation by osmium has been reviewed succinctly by Baker (1945) and in detail by Berg (1927). More recent experiments have been described by Porter and Kailman (1953) and by Bahr (1954). The physical results of the action of osmium on proteins may be described as gelation without coagulation.

The action of osmium tetroxide on lipids has caused considerable perplexity since unsaturated triglycerides will blacken rapidly while phospholipids require prolonged treatment before final reduction of the osmium is accomplished. An explanation suggested by the work of Criegee (1936, 1942) envisages a two-stage process. The first stage results in an ester which, though not black to transmitted light, will be opaque to electrons and therefore be useful in electron microscopy. The second stage, reduction of the osmium to the dioxide or the metal, is not always a simple matter. Empirical methods of accomplishing this second reaction may be found in the numerous osmic procedures for the Golgi apparatus.

With fixation of proteins accomplished by osmium tetroxide, the particular function which remains to be performed by the chromating reagent is concerned with the phospholipids. They must be rendered insoluble in the reagents to be applied later and left in a chemical state which will allow analytical procedures to be performed upon them. In osmichrome fixation the action of the hexavalent chromium is influenced to a certain extent by the first-stage reaction of osmium tetroxide with the phospholipids.

Osmichrome fixation adds one more to the long list of procedures in which osmium tetroxide and dichromate have worked in partnership. It is hoped that the separation of variables made possible by applying the reagents in sequence will facilitate further experimentation with problems of fixation.

This investigation was aided by a grant from the National Science Foundation. It is a pleasure to acknowledge the expert technical assistance of Mary Miksic and Gloria Wayne.

FIG. 1 (plate). Tissues of Long-Evans rats after osmichrome fixation. Sections 5 μ thick. Photomicrographs taken on Kodak Commercial Ortho film with a Wratten 60 filter.

A, uterine gland, stained with the periodic acid / Schiff procedure, showing a positive reaction of the Golgi material.

B, testis, stained with Altmann’s anilin I acid fuchsin and methyl blue. The acid fuchsin is concentrated in the centrioles. The acroblasts are more faintly visible.

c, tip of villus of small intestine, stained with Sudan black in ethylene glycol. The clear >val spaces are nuclei (no nuclear stain was used). The Golgi apparatus and the mitochondria ere evident.

D, epithelium of small intestine, stained with Altmann’s anilin / acid fuchsin and methyl due. Threadlike apical mitochondria and more rounded basal ones may be seen. In the lower portion of the section the Golgi apparatus has also taken up the acid-fuchsin.