TREBLE STAINING. The combination which has given the best effect so far, in treble staining, is picro-carmine, rosanilin, and iodine green. Stain the sections well according to the process already described for picrocarmine, and soak them in acidulated water. Then take a few drops of the solution of hydrochloride of rosanilin, No. 17, dilute with spirit, and immerse the sections for two or three minutes, remove them to methylated spirit and wash off the excess of colouring matter. Then place them in a dilute solution of iodine green. Coming from spirit they will float on top of the watery solution, and this in many cases, when the green stain is not required to be very deep, is quite sufficient. When a deeper stain is required, immerse them altogether, and let them remain a minute or two; but it must be borne in mind that this colour cannot be washed out again if too deep, which the spirituous stain can, so that it is better to have a section apparently over-stained in the rosanilin solution, while it is even under-stained in the iodine green. After washing, the sections are mounted in the usual manner. It will be found, however, that a good deal of the rosanilin will come out in the second immersion in spirit, and it is necessary to change it until no more colour comes away; otherwise the oil of cloves will become coloured, and from it the Canada balsam, in which the specimen is mounted. With the above mentioned three colours, the most beautiful effect may be obtained, but it will take some time and practice to get the process exactly right, and this is a matter which can only be gained by experience. The results will be found to vary with the length of time the section is immersed in each of the two last colours, and also with the strength of the solutions. The sections should be uniformly and deeply stained with picro-carmine. The other two solutions should be saturated in the first instance, and then diluted one-half at least. If they are to be laid aside for some time before mounting they should not be left in spirit, but in oil of cloves. Only a few sections should be stained at one time or some will be found much more deeply stained than others. The best results will also be obtained with material that has been hardened in chromic acid. The staining process is well shown in a section of the base of a cat or dog's tongue, cut through one of the circumvallate papillæ; the section should be sufficiently large to include some of the mucous glands, of which there are a large number in that region. If the staining is well done it will show all the muscle fibres stained with picro-carmine, the connective tissue, protoplasm of cells, &c., stained red; while all the nuclei in the superficial epithelium, serous gland, non-striped muscle tissue in the vessels, and elsewhere, are stained a brilliant green. The most important fact demonstrated by this process is the different chemical reactions shown by the various glands. In the mucous glands, while the epithelium lining the duct is stained in precisely the same manner as the superficial epithelium of the organ, it will be found that the moment the secreting epithelium is reached a new colour presents itself, which differs in toto from either of those employed in the process; thus showing that the secretion has the power of causing these two colours, green and red, to combine, forming different shades from purple to blue, according to which colour predominates. In the serous glands, however, quite another aspect is presented; there is no combination as in the mucous glands, but the protoplasm of the cells is stained more or less deeply with red, while the nuclei have taken on the green; the colour differs, however, from that of the surface epithelium, and appears to have taken on picro-carmine to some extent, which with the rosanilin hydrochloride, gives a dull red colour. In many places will also be seen small masses of adenoid tissue which have stained a bright green throughout. Altogether this makes one of the most brilliant specimens in the whole range of histology, and although the process is rather troublesome, and requires a certain amount of practice to determine the time required for each immersion it amply repays when once properly done. Take only a few sections at a time, and do not hurry over the different processes, and after a few trials the exact time of immersion, will be hit on, and should be recorded. * Iodine green is a very blue green. CHLORIDE OF GOLD AND ANILINES. Some very striking results may be obtained by first staining fresh tissues, especially growing bone, in chloride of gold solution (page 19), and then decalcifying and hardening in spirit. After the material has hardened sufficiently, sections may be made and stained with two colours. It is not quite clear what action the gold chloride has on those parts it does not stain, but that it has some, is evident from the difference of the action of aniline dyes on those specimens prepared in gold, from those hardened in any other manner. A very good material for the purpose is the tail of a young rat or mouse, placed in half per cent. solution of gold chloride for an hour or two, and then decalcified and hardened in the usual way. Very thin transverse sections should be cut, and stained first in rosanilin and then in iodine green. On examining the specimen the gold staining will be seen in the periphery, bringing out the tendon cells, and giving a dark hue to everything for a certain distance from the outside; but within this a great variety of colour will be found, the different tissues being stained in a most gorgeous manner. In the middle the bone trabeculæ will be seen faintly stained, while the calcified cartilage, in their centres, is stained a bright colour, totally different. All these colours may be varied by using different aniline solutions, and a very pretty result may be obtained by |