The Preparation & Mounting of Microscopic Objects

by microscopist Thomas Davies

Of the various modes of cleaning and mounting the Diatomaceæ, I believe that the following may be safely recommended as affording results of the very best quality. My friend, Mr. T. G. Rylands, gave it to me as that which he prefers, and I can safely say that his numerous slides are at least equal to any I have ever seen. I will give it just as I received it from him, though there may be some little repetition of what has been said elsewhere, as he does not appropriate any part of it as his own. He says:—In this branch of mounting, general rules alone can be laid down, because the gatherings may contain iron, lime, fine silt, or vegetable matter under conditions for special treatment, and consequently the first step should be to experiment on various kinds.

In gathering diatoms much labour is saved by judgment and care; hence it is desirable to get acquainted with them in their growing condition, so that when recognised upon the sands or other spots they may be carefully removed by the aid of the spoon or small tin scoop before described. When growing upon algæ or other plants, the plants and diatoms together may be carried home, in which case they must be simply drained and not washed or pressed, in order that the diatoms be not lost. As it is always desirable to examine the gathering on the ground, a “Gairdner’s hand microscope” with powers from 80 to 200 diameters will be found very useful. The best gatherings are those which represent one species abundantly. Those which are mixed may be rejected, unless they are seen to contain something valuable or important, as the object should be not so much to supply microscopical curiosities as to collect material which is available for the study of nature.

The gathering when carried home should always be carefully examined before anything is done with it; not only on account of the additional information thus acquired, but also because it often happens that a specimen should be mounted in fluid (see [Chapter IV.]) in the condition in which it is gathered, as well as cleaned and mounted in balsam ([Chapter III.]) and dry.

Where the gathering is taken from sand, the whole may be shaken up in water as a preliminary operation, when much of the sand will be separated by its own weight. The lime test, however, should be applied, viz.—a small portion of hydrochloric acid, and if there be effervescence it must be dissolved out by this means. From Algæ and other weeds diatoms may be detached by agitating the whole together in a weak solution of nitric acid—about one of pure acid to twenty or thirty of water, as it must be sufficiently weak to free the diatoms without destroying the matter to which they adhere. The diatoms may then be separated by sifting through coarse muslin, which will retain the Algæ, &c. The process of cleaning will vary according to circumstances. Some gatherings require to be boiled only a few minutes in nitric acid; but the more general plan where they are mixed with organic or other foreign matter, is to boil them in pure sulphuric acid until they cease to grow darker in colour (usually from a half to one minute), and then to add, drop by drop to avoid explosions, a cold saturated solution of chlorate of potash until the colour is discharged, or, in case the colour does not disappear, the quantity of the solution used is at least equal to that of the acid. This operation is best performed in a wide-mouthed ordinary beaker glass,[B] a test-tube being too narrow. The mixture whilst boiling should be poured into thirty times its bulk of cold water, and the whole allowed to subside. The fluid must then be carefully decanted and the vessel re-supplied once or twice with pure water, so as to get rid of all the acid. The gathering may then be transferred to a small boiling glass or test-tube, and—the water being carefully decanted—boiled in the smallest available quantity of nitric acid, and washed as before. This last process has been found necessary from the frequent appearance of minute crystals, which cannot otherwise be readily disposed of without the loss of a considerable proportion of diatoms.

[B] These glasses are round, about six inches high, and usually contain about eight ounces. They are rather wider at the bottom, tapering gradually to the top, and may be generally procured at the chemists, &c.

I may here mention that the washing glasses used by Mr. Rylands are stoppered conical bottles varying in capacity from two ounces to one quart; the conical form being employed to prevent the adherence of anything to the side; they are “stoppered” to render them available in the shaking process about to be described.

The gathering, freed from acid, is now put into two inches depth of water, shaken vigorously for a minute or two, and allowed to subside for half an hour, after which the turbid fluid must be carefully decanted. This operation must be repeated until all the matter is removed which will not settle in half an hour. The fluid removed should be examined by a drop being put upon a slide, as in some cases very light diatoms have been found to come off almost pure in one or more of these earlier washings. The quantity of water and time of subsidence given may be taken generally, but may require to be modified according to circumstances and the judgment of the operator. By the repetition and variation of this process—the shaking being the most important part—the gathering, if a pure one, will be sufficiently clean. If, however, it contains a variety of species and forms, it may require to be divided into different densities.

In some cases, however, it is best to divide the gathering as a preliminary operation, which may be done by agitating it in a quantity of water and decanting what does not readily subside. The heavier and the lighter portions are then to be treated as two separate boilings. But when the cleansing has been carried to the above stage and this division is required, the plan must be somewhat as follows:—The gathering must be shaken in a test-tube with six inches of water, and then allowed to subside until one inch at the top remains pure. About three inches are then to be carefully withdrawn by a pipette, when the tube may be filled up and the operation repeated. The three lower inches also may then be decanted and examined. The gathering is thus divided into three portions, viz.—that which was withdrawn by the pipette, that which remained floating in the lower three inches of water in the tube, and that which had settled at the bottom. An examination of these will inform the operator how to obtain that particular density of gathering which he desires, and how far it is worth while to refine this process of elutriation; for in cases of necessity any one, or all three, of these densities may be operated upon in the same way to separate a particular diatom.

As occasional aids, it may be remarked, that in some cases liquor ammoniæ may be used in place of water, as it often separates fine dirt, which is not otherwise easily got rid of. Some fossil deposits require to be treated with a boiling solution of carbonate of soda to disintegrate them; but this operation requires great care, lest the alkali should destroy the diatoms. Vegetable silicates also sometimes require to be removed by a solution of carbonate of soda; but as the frustules of the diatoms themselves are but vegetable silica, even more care is required in this case. It may be well to mention, that some diatoms are so imperfectly siliceous that they will not bear boiling in acid at all. Some of these may be allowed to stand in cold nitric acid some time, whilst others of a smaller and more delicate character should, when possible, be treated with distilled water alone.

We will now consider the mode of mounting the prepared diatoms, which, if used dry (as described in this chapter), should be carefully washed two or three times with the purest distilled water. In this branch, as in every other, each collector gives preference to that method in which he is an adept. Thus the diatoms may be placed on the under side of the cover, to be as near to the object-glass as possible, or upon the slide itself; and each plan has its advocates. Whichsoever of these is used, nothing seems more simple to the novice than a tolerably equal dispersion of the objects upon the slide or cover; but this is by no means so readily accomplished, consequently I give Mr. Rylands’ method, as his slides are perfect in this respect also. He always places the diatoms upon the thin glass cover. It is not sufficient, as is frequently thought, to take a drop of liquid containing the cleansed material and spread it upon the cover or slide, as without some additional precaution that uniform and regular distribution of the specimens is not obtained which is desirable. In order to effect this, let a drop of the cleansed gathering be diluted sufficiently for the purpose—how much must be determined in each case by experiment—and let the covers to be mounted be cleaned and laid upon the brass plate. (See [Chapter I.]) By means of a glass tube, about one-twelfth of an inch in diameter, stopped by the wetted finger at the upper end, take up as much of the diluted material as will form a moderately convex drop extending over the whole cover. When all the covers required are thus prepared, apply a lamp below the brass plate, and raise the temperature to a point just short of boiling. By this means the covers will be dried in a few minutes, and the specimens equally distributed over the whole area. The spread of the fluid upon the covers is facilitated by breathing upon them; and, to insure uniformity, care must be taken to avoid shaking them whilst drying. The best plan is to mount at least half a dozen at once.