2. Trichloracetic Acid. Fix tissues in 10% formalin and decalcify in trichloracetic acid 90 cc., formol (40 per cent formaldehyde) 10 cc. Change frequently. Decalcification is rapid, the tissue is but little changed and the staining-power not affected.
3. Concentrated Sulphurous Acid. Fix in 10 per cent formol; decalcify in concentrated sulphurous acid for 24 hours or longer if necessary. Wash thoroughly in alkaline water. This is a very good and rapid method; the staining-power is but little affected.
4. Haug’s Solution. (Pure nitric acid 3-9 cc., absolute alcohol 70 cc., sodium chloride 0.25 grm., water 30 cc.). For tissues fixed in mercuric chloride.
5. Phloroglucin. (Phloroglucin 1 grm., pure nitric acid 10 cc., distilled water 50 cc.). The solution must be carefully dissolved over the flame in a hood. Decalcification is rapid, and the tissue is protected from the acid by the phloroglucin.
6. Ebner’s Fluid. (Hydrochloric acid 5 cc., sodium sulphate 5 grms., alcohol 500 cc., water 1,000 cc.).
7. Schaffer’s Method. Imbed the fixed and hardened tissue in celloidin, harden the celloidin preparation in 85 per cent alcohol, then place celloidin block in a 3-5 per cent water solution of pure nitric acid and agitate in Thoma’s water wheel, for 12 hours, or longer according to the size of the piece. Transfer block to a 5 per cent solution of lithium carbonate or sodium sulphate for 12-24 hours, changing solution several times, wash in running water for 48 hours, dehydrate in graded alcohols up to 85 per cent, and cut.
CHAPTER XXII.
IMBEDDING.
The most perfect methods of fixation and hardening do not permit the cutting of fine sections on a microtome without the freezing of the tissue, or its infiltration and imbedding in some substance which surrounds it with a protective coating, and preserves and holds together its structural elements in their relative positions. For the cutting of very thin sections, or for the preparation of serial sections, it is absolutely necessary to employ the process of imbedding. At the present time paraffin and celloidin are the two substances in general use for this purpose. While each one of these possesses certain advantages over the other, and we find consequently one laboratory worker preferring celloidin and another paraffin for general work, a long and varied experience makes me believe that for a teaching laboratory and for diagnostic work when much material is examined, the paraffin method answers all purposes much better than the celloidin, and that the latter need be employed only in very exceptional cases. Since paraffin sections can be transferred into celloidin by the molasses- or dextrin-fixative method, thus enabling the use of staining-methods that require celloidin sections, very few advantages are left in the favor of celloidin as an imbedding agent. The paraffin method requires a more expensive outfit to start with in the form of a paraffin oven and thermo-regulator, but otherwise the two methods cost about the same. The paraffin method requires more careful attention than the celloidin. As a rule thinner sections can be obtained in paraffin than in celloidin, and for the preparation of serial sections the paraffin method is the only method. Paraffin blocks can be labeled and filed away, and kept indefinitely without any loss of staining-power. With careful attention paid to the different steps of the imbedding process practically everything that can be cut in celloidin can be cut in paraffin. For very large pieces a slow imbedding in celloidin is, however, preferred by most workers. Hard and brittle tissues are as a rule more easily cut in celloidin. For the staining of bacteria in sections paraffin imbedding is necessary. Both methods should be learned and practiced with equal facility; a working knowledge of both is essential in pathologic investigation and diagnosis.
1. CELLOIDIN IMBEDDING. The granular form of Schering’s celloidin is the best preparation to use, although good results can be obtained by using a cheaper well-washed gun-cotton. In purchasing the latter care should be taken to secure a sample that dissolves easily in alcohol and ether, and does not give off yellow fumes when exposed to the light. Schering’s granular celloidin keeps well, and forms on solution a firm, tough, transparent imbedding mass, so that thin sections are obtainable without difficulty. When kept long in stock celloidin becomes hard and dissolves more slowly. For use three solutions are made, thick (10 per cent), thin (2 per cent), and medium (5 per cent). The celloidin granules or shavings are put into a wide-mouthed bottle having a tight stopper, and are covered with absolute alcohol and well shaken, and left for 24 hours. An equal quantity of pure ether is then added, the mixture is well stirred and allowed to stand for another 24 hours, when it is again stirred and evenly mixed, and is then ready for use. When gun-cotton is used it is torn into fine shreds and added to a mixture of equal parts of absolute alcohol and pure ether and shaken until sufficient has been added to give the solution the desired strength.
Slow Celloidin Method.