Practical pathology

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.

The tissue is blocked by removing it from the thick celloidin on a section-lifter and placing it on a block of vulcanized fiber or wood with enough of the thick celloidin about it to form a good matrix. The preparation is then allowed to evaporate in the air until the surface of the celloidin becomes firm (does not stick to the finger). The block is then placed in 80 per cent alcohol or pure chloroform until hard enough for cutting (1-24 hours). Cork should not be used for blocking, nor should wood unless the tannin has been removed by long treatment with alcohol-ether. The celloidin will adhere more firmly to the fiber block if the latter is dry, and if there is a sufficient layer of celloidin between the tissue and the block. The imbedded tissues may be preserved in 80 per cent alcohol for a long time, but gradually lose their staining power. They may also be kept dry by coating them with melted paraffin. The blocks when preserved in alcohol may be marked with an indelible pencil.

For imbedding large pieces of tissue in celloidin a glass dish may be filled with thick celloidin and the infiltrated tissue placed in it with cutting surface down. The celloidin is then allowed to evaporate slowly under a glass cover, and fresh celloidin may be added as shrinkage occurs. When well-hardened the celloidin is cut out of the dish and the block trimmed to the proper proportions, and attached directly to the object-holder of the microtome or to a block of wood by a few drops of thick celloidin, allowing it to dry for a minute or so and then immersing in 80 per cent alcohol. The block may be cut on the freezing microtome by soaking the hardened celloidin in water to remove the alcohol (when block sinks), then coating it with saturated gum arabic solution, and freezing.

Rapid Celloidin Method.

I have used the following method in my laboratory for a number of years as a regular procedure in practical diagnostic work, and the results have been uniformly good.

The quick celloidin methods recommended in the literature (Kaufmann, Stepanow, Scholz and others) require more time, and do not give better results. Material received from operative clinics late in the afternoon can be sectioned and stained usually by ten o’clock the next morning.

2. PARAFFIN IMBEDDING. A paraffin of a melting-point sufficiently high enough to withstand summer heat is advisable; a 52°C. paraffin answers for this latitude. The use of softer paraffins is not necessary. The paraffin-oven should be regulated at a constant temperature of 54-55°C. Over-heating of the tissue while in the oven must be carefully safeguarded, so that in the management of a paraffin-oven the care of the thermo-regulator is the most important thing.

Slow Paraffin Method.

The use of xylol-paraffin is not necessary. For blocking staining dishes, watch-glasses, glass salt-cellars, paper-boxes, metal frames., etc., may be used. A thin smear of tincture of green soap or glycerin is rubbed over the inside of the imbedding box, and it is nearly filled with fresh melted paraffin. With warm forceps the tissue is taken out of the bottle of second paraffin in the oven, and arranged in the melted paraffin in the imbedding dish in the proper position for cutting. Care must be taken that the melted paraffin is not hot enough to “burn” the tissue, else its staining-power may be affected. The surface of the paraffin is then cooled by blowing upon it, and as soon as a film appears upon the surface, the dish is carefully immersed in cold water, so that the paraffin may set quickly. When cool the paraffin-block should slip out of the dish and float to the surface. It is then trimmed to the desired shape, leaving a good matrix of paraffin around the tissue. The paraffin-block is then fastened to a wooden block or to the object-holder of the microtome by means of melted paraffin (a hot knife is drawn along the under-surface of the block and the latter immediately pressed upon the wooden block or object-holder). Chloroform, cedar oil, benzene, carbon bisulphide, etc., may be used instead of xylol, and each one possesses certain advantages for certain purposes. Benzene is advisable for osmic-acid preparations.

Rapid Paraffin Method.

The above method can be greatly shortened for uterine curettings, thin bits of tissue, etc., if the various steps are closely watched, and if the entire process is carried on in the oven. The whole process may be carried out in 1-3 hours, a very great advantage over the quick celloidin method. A simpler and cheaper method is that recommended by Heller, Henke and Brunk, as follows:—

Acetone Method.

By this method the entire process of fixing, hardening, imbedding, cutting and staining can be carried out in half an hour, and by it the freezing-microtome can be dispensed with in a large part of quick diagnostic work.

Pyridin Method.

This method requires a longer time than the acetone method, and is not so good.

Combinations of celloidin and paraffin may be employed by imbedding first in celloidin, transferring block to origanum oil, then xylol and finally paraffin. Formol-agar has been recommended by Bolton and Harris for simultaneous fixation and imbedding. It offers no especial advantages. Wright uses formol-gelatin for imbedding tissues for sectioning on the freezing microtome. The bits of tissue are placed in warm 20 per cent pure gelatin; this is allowed to set, and the block placed in 10 per cent formol for 24 hours; it is then frozen and cut. Soap-gelatin, glycerin-gelatin, gum-glycerin, etc., are now rarely used for imbedding.

Note:—When a number of pieces of tissue are blocked at the same time, either in celloidin or paraffin, they may be tagged with paper labels fastened to the tissue with a drop of gum tragacanth or gum arabic. As the gum is not soluble in any of the infiltrating and imbedding media the labels remain attached until the block is ready for cutting.