Pick’s Method:—
| Sol. I.— | Water | 1,000 | cc. |
| Formol | 50 | cc. | |
| Carlsbad salts | 50 | grms. |
Then transfer to 80 and 95 per cent alcohols, as for Kaiserling, and preserve in water 9,000 cc., glycerin 5,400 cc., sodium acetate 2,700 grms.
Westenhoeffer’s method of preserving uric-acid: formol vapor 4-24 hours, then 80-90 per cent alcohol containing mercuric oxide, and preserve in glycerin to which some mercuric oxide covered by cotton or absorbent paper has been added.
Claudius’s Method. The specimen is placed on a grating in a closed vessel containing a concentrated solution of ammonium sulphate, an abundance of the crystals being left on the bottom of the vessel. Carbonic acid or illuminating gas is passed through the ammonium sulphate solution for 48-72 hours, and the specimens are then preserved in the same solution. My experience with this method has not been satisfactory.
Gelatin method of mounting Kaiserling preparations: Soak washed Gold Label gelatin in distilled water for 12-24 hours. Take equal parts of water-logged gelatin and glycerin and dissolve by heating in a double boiler, stirring, for 15-20 minutes. Cool to 40°C. Then clarify with white of egg (well-whipped whites of three eggs to half a gallon of jelly: stir well; steam for ½ hour) and filter through cotton-wool. Add to jelly a few drops of a weak aqueous solution of crystal violet to remove yellow color (Bruère and Kaufmann). To prevent growth of bacteria a small percentage of formol or crystal of phenol may be added. Kaiserling specimens are placed in glass dishes in the melted jelly, and covered with it. When set the dishes containing the mounted specimens may be covered with glass-plates and fastened to these by balsam or cement. I use a deep Petri dish, filling it about two-thirds full with the jelly; over this I pour melted paraffin of a very low melting point so as not to melt the jelly. When the layer of soft paraffin is hard, a thick layer of paraffin of a 52° melting-point is poured over it, and the dish filled even. When the hard paraffin is set, it is varnished with shellac. Liquefaction of the gelatin by bacteria or enzymes constitutes the great drawback to this method.
CHAPTER XX.
THE FIXATION AND HARDENING OF TISSUES.
GENERAL CONSIDERATIONS. For the examination of material that may be injured by freezing, or when very thin sections are required for complicated staining procedures, it becomes necessary to prepare the tissue by fixation and hardening, so that it can be imbedded in some medium permitting the cutting on the microtome of as thin sections as may be desired. Fixation is that process by which the appearances of the tissue are preserved as they were when it was taken for examination; hence in order to obtain pictures resembling as closely as possible those of the living tissue the material should be fixed immediately upon its removal from the living body by operation, or as soon as possible after death when obtained by autopsy. Fixing agents act by coagulating the cell albumins, in this way “setting” or “fixing” the constituents of the cell so that further change is stopped. Fixation, therefore, hardens the cell, and all fixatives are also hardening agents. A practical distinction between fixing and hardening is made, however, resting upon the fact that not all fixatives harden the tissue so completely that the proper consistence for the cutting of thin sections is attained. To achieve this the tissue must be dehydrated. Alcohol and acetone are the only reagents fixing and hardening perfectly at the same time, as they remove the water from the tissue; for all other fixing agents an after-hardening in alcohol is necessary. In the case of such reagents the division of the process into a primary fixation and a second hardening stage has been the cause of the divergence in meaning of the two terms.
The best fixing agents are those that kill the cells at once, but cause a slow coagulation with little or no shrinking. They must penetrate and diffuse through the tissues rapidly so that the deepest cells are quickly reached. Acid media, especially those containing small percentages of acetic acid, are therefore better than alkaline solutions. The tissue-elements, particularly the nuclei, must be preserved as perfectly as possible so that they will not be affected by further procedures of microscopic technique. The chemic properties of physiologic and pathologic substances must likewise be preserved. The preservation of karyokinetic figures is a criterion of good fixation. In pathologic work it is also desirable that the fixing agent should preserve the red blood cells, and permit of the staining of bacteria in sections. Since fixing media are more or less selective in their action, it follows that there is no one fixative that gives equally good results in all cases. Especial fixing reagents must be used for the demonstration of certain substances (fat, etc.), or for the use of certain staining methods. Some stains cannot be used at all after certain fixing agents have been employed. For general use that fixing agent having the widest range of usefulness should be employed; and for this reason fixing media composed of several fixing agents are often employed in preference to the use of a single one.
GENERAL RULES FOR FIXING AND HARDENING.