Preserving and Killing Rotatoria with cilia in situ.—Mr. C. Rousselet’s method of preserving and mounting the Rotatoria[47] has been attended with so much success that the old difficulty attendant upon the preservation of these various beautiful forms of infusorial life has been practically overcome. The process resorted to consists of four stages, namely, narcotising, killing, fixing, and preserving. In dealing with rotifers hitherto, the difficulty has been that of successfully killing them with their rotating organs fully extended. It has been found needful to have recourse in the first instance to a narcotising agent, and one that acts slowly. The most suitable is a weak solution of the hydrochlorate of cocaine, a one per cent. solution, or even weaker. This was first proposed by Mr. Weber for keeping these active little bodies quiet while under observation. Mr. Rousselet carries this agent further; he applied it to narcotise them prior to killing, and this it does most effectually. The rotifers are seen to sink to the bottom of the live-cell, and the cilia gradually to slacken in motion, and the time for killing has arrived. This is effected by Flemming’s chromo-aceto-osmic acid. A rather weak solution must be employed—consisting of 1 per cent. solution of chromic acid, 15 parts; 2 per cent. osmic acid, 4 parts; glacial acetic acid, 1 part—which is at the same time a killing and fixing medium. The word “fixing” must not be taken to imply simply fixing, as it includes rapidly killing and hardening and preventing further change in the tissues of the rotifers by subsequent treatment, as mounting. The animal, therefore, must remain quietly for a few minutes, and then taken out and washed in five or six changes of distilled water, and hence transferred to the preservative fluid. All this must be effected with great care. The best preservative fluid is simply distilled water, rendered antiseptic by a trace of the fixing solution (about eight drops to an ounce of water) giving the slightest tinge of yellow to the solution. This slight tinge of colour is imparted to the rotifers, otherwise they remain transparent and unchanged, while the nervous tissue throughout the body is brought out to perfection.
Some slight difference in treatment is required by certain species, as that of Asplanchna priodonta; after the application of the cocaine solution, which should be added slowly, that is, by letting a few drops trickle down the side of the live-trough; this, being heavier than water, sinks to the bottom, thus narcotising the rotifers, and assisting to kill them with the cilia fully expanded. They should be left quietly for fifteen minutes, then thoroughly washed with distilled water. On further experimenting, Mr. Rousselet found that a weaker solution of osmic acid alone, ¼ per cent., answers quite as well as, if not better than, Flemming’s fluid; even this must be allowed to act for only a very short time—a minute at most; the rotifers then remain white and transparent, excepting the ova, in which a fat-like substance, lecithene, is secreted. If they become too much stained, they may be decolourised by passing them through peroxide of hydrogen. For narcotising the following solution has been found most useful:—Take a 2 per cent. solution of cocaine hydrochlorate, 3 parts; methylated spirit of wood naphtha, 1 part; and distilled water, 6 parts. This must be added as before directed, drop by drop, watching the effect upon the rotifers under the microscope.
All the rotatoria may be killed and preserved in the same way. For mounting, Mr. Rousselet prefers a slightly hollowed-out glass cell, the advantage of which is that the rotifers are kept to the centre, and cannot move to the edge. A little difficulty at first presents itself to exclude air-bubbles, but this, with a little care, can be overcome by placing a drop of a two or three per cent. solution of formalin, just sufficient to fill the cell. Then transfer the rotifers with a dipping pipette to the cell, and lower the cover-glass down very gently, removing any excess of fluid by blotting-paper. The best cement for the cover-glass is gold-size.
Method of Cementing.—After many years’ experience, I have arrived at the conclusion that for cementing down the cover-glass there is nothing better than either gold size or gum dammar varnish. The latter, for some preparations, will be improved by the addition of a small proportion of indiarubber dissolved in naphtha. (See Appendix.)
Should glycerine be preferred, carefully wash away any surplus quantity by gently syringing; then apply a ring of waterproof cement round the cover-glass. An inexpensive one can be made by dissolving ten grains of gum-ammoniac in an ounce of acetic acid, and adding to this solution two drachms of Cox’s gelatine. This liquid flows easily from the brush and is waterproof, rendered more so if subsequently brushed over with a solution of ten grains of bichromate of potash in an ounce of water. An especial recommendation to this cement is its adhesiveness to glass, even should there be a little glycerine left behind on the cover. After the gelatine ring is thoroughly dry any kind of cement may be employed.
A useful cement for fixing minute objects, diatoms, &c., temporarily to thin glass covers, before permanently mounting them in Canada balsam, is made as follows:—Dissolve, without heat, two or three grains of gum arabic in one ounce of distilled water, then add glacial acetic acid, three minims, and the least trace of sugar. Filter carefully through filter paper, and repeat this in the course of three or four weeks. This cement will be unaffected by the balsam.
Mounting Chara.—It is often found difficult to preserve and mount the fruit of chara, but this can be successfully accomplished in glycerine jelly, by taking the following precautions. After cleaning the specimen place it in 92 per cent. of alcohol for several hours, then transfer it to a mixture of equal parts of spirit and glycerine for several hours longer, pour off nearly all the mixture, and add pure glycerine at intervals till the glycerine becomes concentrated. The specimen is then mounted in glycerine jelly in a cell just deep enough to take it without pressure.
There are some objects much more difficult to prepare than others, and which tax the patience of the beginner in a manner which can hardly be imagined by any one who has never made the attempt. The structure of many creatures is so delicate as to require the very greatest care to prevent mutilation, and consequent spoliation, of the specimen. The beginner, therefore, must not be discouraged by a few failures in commencing, but should persevere in his attempts, and constant practice will soon teach him the best way of managing intricate and difficult objects. The room in which he operates should be free from dust, smoke, and intrusion, and everything used should be kept scrupulously clean, since a very small speck of dirt, which may be almost invisible to the naked eye, will assume unpleasant proportions under the microscope, and not only mar, but possibly spoil a fine and delicate preparation.
Few students on commencing to work with the microscope will fully realise the fact that under medium or high powers the natural appearance of almost all objects is changed by the refractive nature of the fluid medium in which they are immersed and which enters more or less into their composition. The remarkable changes effected by the law of diffusion, when alkaloid substances enter into their composition, show the necessity of taking every precaution in the employment of preservative fluids. Glycerine affords an example of the chemical change induced, should the preparation have been passed through an alkaline solution.
Air Bubbles are a constant source of annoyance both in preparing and mounting. These may be removed from the specimen by gently warming the under part of the slide over a spirit lamp, or placing the slide in the warm chamber, when the bubbles will move towards the edge of the cover-glass and ultimately disappear. The air-pump is preferred by many microscopists.