The sections after staining may be mounted in Farrant’s solution (p. [59]), or in Canada balsam (p. [61]). If the latter medium is employed, the section should be clarified, after dehydration in alcohol, by means of oil of bergamot, or oil of origanum, instead of oil of cloves, as the latter dissolves out the celloidin and causes the section to break up.
Celloidin is most useful for cutting sections of the coats of the eye, of the internal ear, and of bone marrow. It should always be used for the Weigert-Pal hæmatoxyline method of staining the nervous centres, as it protects the section from being injured by the transference from one fluid to another which is repeatedly required during the process. The stain is completely discharged from the celloidin by the decolourising solution used (p. [90]).
Paraffin.—Paraffin is a very convenient embedding medium for delicate structures, as very thin sections can be obtained and the paraffin need not be removed from the section until the latter is safely on the slide. It is unsuitable for large sections. Staining operations are not easily carried out after cutting in paraffin, and it is better to stain the blocks of tissue in bulk before embedding. The best stains for penetrating are borax carmine (p. [75]), alum carmine (p. [76]), and Kleinenberg’s hæmatoxyline (p. [70]). The tissue must be left in them for four to ten days.
Various kinds of paraffin are employed. It is usual to keep two kinds, one “soft,” melting at 110° F., and another “hard,” melting at 140° F. A mixture of two parts of the hard and one of the soft will be found most generally useful. In winter a large proportion of the soft variety and in hot weather a larger proportion of the hard may be required. A paraffin mass which is always available has been suggested recently by Dr. F. E. Batten, who employs an ordinary white candle, composed of paraffin and wax. If the mass is found to be too hard, it can easily be made of a suitable consistence by adding a little paraffin with a low melting point.
To prepare a piece of tissue for embedding in paraffin, it should be stained, washed in distilled water, and as much moisture as possible removed by blotting paper. The block is then dehydrated, first in methylated spirit for several hours, finally in absolute alcohol. It is taken carefully by means of forceps from the alcohol and placed in xylol for an hour or two according to size. Superfluous xylol is removed from the surface, and the tissue placed in the melted paraffin. This will set round the cold tissue at once, but soon melts again and must be kept at a temperature just above melting point for one to four hours, according to size. The tissue is then transferred to a mould (which can be easily made of paper), about half an inch cube, and melted paraffin poured round it until the mould is full. The mould may be made by folding a piece of paper to form a box about half an inch cube, or a small pill box may be used. Another convenient method is to place two L-shaped pieces of lead in contact with each other so as to enclose a space of suitable size as in the diagram (fig. [3]). The tissue is now hermetically sealed, and can be kept indefinitely if it is not convenient to cut it at the time. To prepare it for cutting, all superfluous paraffin is trimmed away with a warm knife, and the block is fixed on a piece of wood, cut so as to suit the clamp of the microtome, by melting the lower end of the paraffin block with a hot needle or wire and pressing it down on the wood.
Fig. 3.
When sections are cut they may be transferred singly to the slide (which should be lightly smeared beforehand with a saturated solution of celloidin in oil of cloves), or they may be cut so that the back of one section of the paraffin block adheres to the front of the next, and in this way a continuous delicate ribbon of serial sections is obtained. The ribbon is broken up into lengths of about two and a half inches and transferred to the slide, on which several ribbons may be placed side by side, and so a large number of sections kept in the order in which they are cut. A mark should be made on the slide to indicate where the series begins, and each slide should be numbered, so that the exact position of each section in the series can be recognised at once.