12. Alcohol and Ether. Used to dissolve fat-granules and to differentiate between these and albuminous granules.
13. Stains. Fuchsin, methylene blue, methylene green in 1 per cent solutions in physiologic salt-solution and acetic-acid-fuchsin are the best stains used for the examination of fresh-tissues beneath the cover-glass. They are drawn under the cover-glass according to the method given above.
For the rapid staining of sections of fresh tissue cut by freezing a section is floated from the salt-solution onto a slide, which is then carefully lifted from the salt-solution and the excess of the latter removed. Several drops of methylene blue, carbol-thionin or carbol-kresyl-echt-violett are run upon the section with the glass-dropper, and allowed to remain for 15-30 seconds. The stain is then washed off with salt-solution, a cover-slip put on and the section examined in the salt-solution. Thionin has been especially recommended (Wood, Strouse and others) for the rapid staining of frozen sections; but I prefer to use carbol-kresyl-echt-violett (kresyl-echt-violett 1 grm., 5 per cent aqueous solution of phenol 80 cc., 95 per cent alcohol 20 cc.). This gives a very good differentiation in the section examined in water, and the picture is clearer than with thionin; the specific staining-reactions with mucin, amyloid, mast-cells, etc., are also more marked than with the latter stain. Such sections are not as clear as dehydrated and cleared sections and their possibilities of diagnosis are correspondingly limited, even in the hands of an expert with sections of this kind.
To make permanent balsam-mounts of the frozen unfixed sections the latter must be fixed in formol or alcohol, or by heat (hot water). The sections may be placed in 4 per cent formol for several minutes, then into 80 per cent alcohol, then stained, washed, dehydrated in absolute alcohol, cleared in carbol-xylol and finally mounted in balsam. Hæmatoxylin and eosin may be used for the staining. Fresh sections may also be fixed in hot water, stained in hæmatoxylin, dehydrated in alcohol and cleared in xylol. To save time the sections may be fixed on the slide for a few minutes in alcohol, care being taken to prevent the sections from rolling up by dropping the alcohol onto the middle of the section after it has been carefully flattened out on the slide. The section may then be stained with hæmatoxylin, borax-carmine or other stains, dehydrated, cleared and mounted in balsam. After fixing with alcohol on the slide the section may be attached to the slide by blotting it with absorbent paper, then covering section with absolute alcohol, draining this off after a few seconds and then running over the section a thin solution of celloidin, which is allowed to drain off, leaving a very thin film over the section and slide. The latter is then immersed in water for a few seconds, and the celloidin-film on setting holds the section to the slide, provided the celloidin has been of proper consistence. The section can now be stained, washed, dehydrated, cleared in origanum and mounted in balsam. When mounted the thin film of celloidin is invisible (Wright’s method).
I have originated a much better method which is in use in my laboratory, and can be applied to the staining of frozen sections of fresh tissues in large numbers for class use. The sections are floated from the salt-solution on to a warm solution of New Orleans baking molasses diluted ten times, or a dilute sugar-dextrin solution, and thence are floated on to a clean glass plate and arranged in rows. The plate is drained, and then without drying is immersed in absolute alcohol for 15-30 seconds; it is then flooded with a thin celloidin, drained, the celloidin film allowed to set, and the plate then put into warm water, where the celloidin sheet floats off, carrying the sections, which can now be cut out and treated as single celloidin sections, or the whole sheet can be carried through the staining, dehydrating and clearing solutions to be cut up into single sections before mounting.
CHAPTER XIX.
THE PRESERVATION OF MACROSCOPIC PREPARATIONS.
For preserving gross objects for museum specimens alcohol or formol may be employed. The former bleaches the tissues so that ultimately they are almost destitute of color. Formol in a 5 per cent solution gives better color-effects than alcohol, as the blood-containing parts remain darker. The fluid also remains clear and the tissues are firm. When alcohol is used the fluid must be frequently changed, as it becomes turbid and yellowish, and the tissues finally become soft and lose their form. The best methods of preserving the natural color are found in the various modifications of the Kaiserling method. The organs or tissues are placed first in a formol solution until they are just hardened, the formol changing the oxyhæmoglobin into acid hæmatin. They are then transferred to alcohol to bring back the natural color, which is accomplished by the change of the acid hæmatin to an alkali hæmatin, which has a color very closely resembling that of oxyhæmoglobin, so that the natural color is approximately reproduced. The method is carried out as follows:—
| Sol. I.— | Formalin | 200 | cc. |
| Water | 1,000 | cc. | |
| Potassium nitrate | 15 | grms. | |
| Potassium acetate | 30 | grms. |
The tissues are left in this solution, in the dark, for one to several days, being watched carefully to see that they are not over-hardened.
Sol. II.—80 per cent alcohol for 1-6 hours and then 95 per cent until the color is fully restored (2-24 hours). Watch carefully and remove as soon as best color effect is reached, and preserve in—