CHAPTER XI
Hints on the Preparation of Objects—Preservative Fluids—Mounting Media—Treatment of Special Objects.
The microscopist who relies altogether on the dealer for his permanent preparations may expend a good deal of money, but the satisfaction which he derives from his hobby will be very inferior to that experienced by the worker who endeavours to secure, for exhibition or for reference, specimens of the objects which he finds most interesting and instructive to himself.
It will be our endeavour in the following pages to give a summary of the elementary principles upon which reliance is to be placed, though it must be clearly understood that the technique of the subject, already occupying a vast amount of literature, is extending day by day, so that it is impossible to deal exhaustively even with one single section of it. Reference must be made, for further information, to such publications as the Journal of the Royal Microscopical Society, or that of the Quekett Club, or to the monographs on the various departments. Davies’ work on the general subject will also be found useful by the beginner.
Taking first the question of reagents, we may mention five which leave the cells of a tissue as nearly as possible in the natural condition, but fit for permanent preservation. The first of these, in order of importance and of general applicability, is alcohol, represented for most purposes by methylated spirit, which contains about 84 per cent. of absolute alcohol, though, unfortunately for our purpose, there is a certain quantity of mineral naphtha in it in addition. This last has the effect of making it go milky upon dilution with water, which is a considerable disadvantage, though the milkiness disappears to some extent on standing, and it is rarely worth the while of the ordinary microscopist to go through the formalities necessary to obtain permission to purchase unmineralised spirit, which cannot be had in quantities of less than five gallons (as it is only to be had from the distillers under an Excise permit), and distillers may not supply less.
Four parts of methylated spirit with one of water forms the classical “70 per cent.” alcohol, the most generally useful of all fluids for hardening and preserving purposes. A considerable quantity of this fluid should always be available.
Whatever other fluid may be used to begin with, spirit must almost always be used to finish the process, and fit the tissue for section-cutting and staining.
Of purely preservative, or fixative, fluids, we may mention “formalin,” a 40 per cent. solution of formic aldehyde, which is rapidly coming to the front, as indeed it deserves to do. It is but slightly poisonous, if at all, and leaves in the tissue nothing which requires subsequent removal before proceeding to harden for section-work, whilst it is an admirable preservative of cell-form.
Another admirable but highly poisonous reagent is corrosive sublimate, in saturated solution, with 2 per cent. of acetic acid.
A fourth is osmic acid, used in 1 per cent. solution. This is a highly valuable reagent, but extremely expensive, very poisonous, and giving off fumes which are most irritating to the eyes.
The fifth, a very gentle, but in many respects very satisfactory one, is picric acid in saturated solution. Tissues preserved in this medium must not be washed out with water, as it enters into very feeble combination with protoplasm, and the cells swell and disintegrate as the reagent is dissolved out.
Of mounting media we may mention glycerine, glycerine jelly (made by dissolving starch in glycerine with the aid of heat), and Canada balsam, dissolved in xylol or benzole. The Canada balsam must be dried hard by evaporation over a water-bath, and dissolved as wanted. Under no circumstances should raw balsam be used, as it takes years to set hard, and turns of a deep yellow colour in the process.
Chloroform is frequently used as a solvent, but it has the disadvantage of attacking and extracting a large number of the aniline dyes used for staining structures, an objection from which the mineral solvents are free.
We will now proceed to go through the objects already referred to, and indicate the method of preservation.
For the study of the cell-structures of plants the portion to be examined is to be placed in spirit of about 30 per cent. strength, which is changed after twenty-four hours for 40 per cent., after a further twenty-four hours for 55 per cent., and finally, as regards our present purpose, in 70 per cent. spirit, in which it may remain until required for section-cutting. The effect of this treatment is to extract the bulk of the water from the tissue, with the minimum of shrinkage of the cells, the latter being preserved in their natural relations to surrounding parts.
In some cases, however, it is desirable to examine and preserve delicate structures, or parts, or dissections, in a medium which allows of the retention of the greater part of the natural moisture, and in such a case the tissue is immersed in glycerine diluted very much in the same way as the alcohol in the last process, but with very much longer intervals between the alterations of strength, until it reaches pure glycerine, in which it remains for a considerable time, as the exchange between the tissue and the dense fluid surrounding it goes on very slowly.
A combination of the two methods is also possible, the spirit-hardening being carried out for a portion of the time, and the tissue being thereafter transferred to glycerine, diluted or pure.
The object of using glycerine at all is merely that it has a much lower refractive index than balsam, so that delicate structures may sometimes be better seen in the former medium, but balsam is to be preferred wherever it is possible to use it, i.e. almost always. The writer has not mounted a preparation in glycerine or a medium containing it for many years, nor, with proper staining, does he think it can ever be necessary to do so, except in the case of dissections in which the glycerine can be slowly run in without disturbing the arrangement, as spirit would be pretty sure to do. The harder portions of plants, woody stems, shells of fruit, or the like, require different treatment, and must, as a rule, be allowed to dry thoroughly before being cut.
Starch granules are somewhat troublesome to mount satisfactorily. The writer has tried many methods, and, on the whole, prefers a glycerin-gelatin medium, which keeps for an almost indefinite time, and may be made as follows: Thirty grains of gelatine (Nelson’s “brilliant” or other transparent gelatine is to be preferred) are allowed to soak in water, and the swollen gelatine is drained, and dissolved in the water which it has absorbed, by the aid of a gentle heat. An equal bulk of pure glycerine is then added. In using, a small portion is transferred to a slide with the point of a knife and melted, a small quantity of starch granules added, and stirred into it with a needle. The cover-glass is then laid up on the still-fluid drop, pressed gently down so that the drop is extended to the margin of the cover, and the whole allowed to cool. It is then to be painted round with several layers of Brunswick black, or Hollis’s glue, or zinc-white cement, to prevent evaporation,—Hollis’s glue being perhaps the best medium for the purpose.
Petals, or other parts of which it is desired to obtain a surface view, must be mounted in cells, which may be made by the use of button-moulds of suitable size, cemented to the glass slide with marine glue. The slide must be free from grease, as the tissue must be fixed in position by the use of gum, and allowed to dry thoroughly before closing the cell, or the cover-glass will be bedewed with moisture when the cell is closed. The best plan is, after air-drying for a couple of days, to place the preparation on a metal plate over a beaker of boiling water for an hour or more, and then to close the cell immediately with Brunswick black, maintaining the heat at first to ensure rapid drying, and then slowly withdrawing it. When cool, another coat should be given, and rather thick covers should be used, as these preparations are never required to be examined with high powers.
To mount pollen-grains, they should be sprinkled upon the surface of a slide which has been previously moistened with thin gum, and allowed to dry until it has become just “tacky”; the drying is then completed by gentle heat and a drop of balsam placed upon the grains, with a cover-glass over all. Bubbles will probably form, but with Canada balsam this is not of the slightest importance, as they always come out of their own accord, and balsam mounts should never be closed with cement of any kind until thoroughly dry.
Air-bubbles in other media may be eliminated by the use of the air-pump shown in Fig. 16, which may be obtained from Baker at a very reasonable rate, and which is useful not only for that purpose, but for accelerating the drying of moist tissues. To do this, there is placed upon the plate of the pump a porcelain dish containing strong sulphuric acid, and upon this is placed a little triangle of platinum wire, which serves to support the preparation. The air is now exhausted; the tissue parts with moisture to supply its place, and this moisture is in turn greedily absorbed by the sulphuric acid, so that drying is rapid and continuous, as well as very thorough, whilst the process has the great advantage of dispensing entirely with the use of heat.
Fig. 16.
Portions of many of the delicate algæ may be mounted in glycerine, having previously been soaked in it as already described; whilst the unicellular forms, such as desmids and diatoms, may be preserved in almost exactly the natural condition by simply mounting them in a saturated solution of picric acid.
Probably formalin, in a solution of 10 per cent. strength, would answer the purpose equally well, but the writer has not tried it. It is hardly necessary to say that, with such extremely fluid media, great care is required in closing the cell. A thin layer of Hollis’s glue should be first painted on, to secure the cover in position, and when this is thoroughly dry, several successive layers must be added in the same way.
It may be said here, that it is advisable in all cases to use circular cover-glasses, as far as possible, as they lend themselves with great facility to a mechanically accurate closure. This slide is placed upon a turn-table, carefully adjusted until the cover is seen to be central when rotated, and a brush, preferably a small camel-hair pencil, charged with the desired fluid, but not in large excess, is held against the junction of the slide and cover, whilst the table is rapidly spun. A little experience will teach better than any description what amount of fluid there should be in the brush, and how thick the cement should be. If too thick, it will drag off the cover; if too thin, it will flow over the latter and over the slide.
The preparation of diatom-skeletons as permanent objects is easy. Consisting, as they do, of pure silex, or flint,—i.e., practically glass,—they resist long boiling in acids, so that there is little difficulty in isolating them from any organic matter with which they are mingled. It is generally recommended to treat them with strong nitric acid. This is a mistake. The acid acts much more powerfully and less violently when diluted with an equal bulk of water, and it is in an acid so diluted that portions of water-plants, or other diatomaceous material, should be boiled in a glass beaker until all the organic matter is dissolved. The beaker should be covered with a glass plate, to prevent dissipation of the acid fumes. When the process is complete, usually in about half an hour, the contents of the beaker are thoroughly stirred with a glass rod, poured rapidly off into a larger bulk of cold water, and allowed to settle for another half-hour. The process is then repeated with a smaller bulk of water, several times, to allow the removal of the last traces of acid, and finally with distilled water. The separation of the diatoms into grades is effected by settlement. The final result is poured into a tall glass vessel, and allowed to settle for, at first, a minute, the supernatant fluid again poured off, and allowed to settle for two minutes, and so on, the period being gradually increased, and each sediment preserved apart. The first will probably only be sand, but the proportion of diatoms will increase with each separation, though there will always be a certain proportion of sand of such a size as to settle at the same rate as the diatoms. Marine plants especially will furnish a rich harvest by treatment as described.
Solid diatomaceous deposits, such as kiesel-guhr, mountain-meal, and especially the famous Oamaru deposit from New Zealand, demand different treatment, and perhaps the best way is to disintegrate the mass, either by boiling with Sunlight soap (though the alkali attacks the flint to some extent) or to mix the mass with a super-saturated solution of acetate of soda (made by saturating water with the crystals whilst boiling), and by successive coolings, heatings, and stirrings to cause the process of crystallisation to break up the mass, which it will do very thoroughly. The diatoms are then separated by sedimentation, as above described.
A small portion of the deposit may now be spread thinly on a glass slide, allowed to dry thoroughly, be treated with balsam, and covered.
If it be desired to select individual diatoms, this must be done under the microscope, by means of a bristle fixed in a handle either with glue or sealing-wax. The diatom selected will adhere to the bristle if the latter be slightly greasy, and should then be transferred to a slightly adhesive slide, coated either with thin solution of white shellac, or with thin gum nearly dry. When the forms desired are mounted, the preparation should be covered in balsam. The process is by no means as easily effected as described, however.
The preparation of insects, or parts of insects, as microscopic objects is a tedious and difficult task. The main point is the trouble of softening the integument and eliminating the colour.
The latter can, in any case, be only partially effected. The beginner would do well to begin with a fairly easy form, such as the worker-ant. A good supply of these insects may be placed in a bottle of liquor potassæ, and left there for at least some days until they begin to become clear and limp. From time to time a specimen may be taken, well washed with several waters, then with acetic acid and water of a strength of about 10 per cent., then with weak spirit, about 50 per cent. An attempt may then be made to arrange the insect upon a slide, spreading out the legs so as to exhibit them to the best advantage, and when this has been done a cover-glass may be put on, supported in such a way as to prevent absolute pressure. The spirit is then withdrawn by means of a piece of filtering-paper cut to a point, and strong spirit added. This is again succeeded by absolute alcohol, then by a mixture of turpentine and crystal carbolic acid in equal proportions, and finally the cover-glass is carefully lifted, and some thick balsam solution dropped on, the limbs finally arranged by means of warm needles, and the cover-glass carefully replaced and pressed gently down by means of a clip, which may be obtained for a few pence. The whole is then set aside to harden, the deficiency caused by evaporation made good, the balsam allowed to dry, and the preparation finally painted round.
The contents of the body, in large insects, must be removed, and this is effected during the washing in water by gentle pressure with a camel-hair brush, the process being aided, if necessary, by a small incision made through the integument at the root of the tail. Sections of insects require very special methods, which will hardly fall within the scope of this work.
CHAPTER XII
Section-Cutting—Staining
No method of examination can equal, for general applicability and usefulness, that of section-work. The relations of the parts to each other being preserved, it is possible to draw conclusions as to their actual relations which no other mode allows of, and we shall devote this concluding chapter to some account of the methods to be employed to this end.
The apparatus required is not necessarily complicated. Reduced to its elements, it consists essentially only of a razor to cut the sections and a dish to receive them. It but seldom happens, however, that the relations of the parts in sufficiently thin sections can be preserved by such a rough-and-ready method, and frequently the object to be cut is of such small dimensions as to render it impossible to deal with it in this way. It is therefore necessary to “imbed” it, so as to obtain a handle by which to hold it, in such a way that it shall be equally supported in all directions. Moreover, since the human hand can only in exceptional cases be brought to such a pitch of skill as to cut a series of sections, or even single ones, of the needful delicacy, some mechanical means of raising the object through a definite distance is highly desirable. The writer has cut many thousands of sections with the “free hand,” but the personal equation is a large one, and is not always the same in the same person. For single sections the method will, with practice, succeed very well, but some means of securing a number of sections of more or less the same thickness is usually required.
Let us deal with the imbedding first.
If it be desired to imbed a tissue which has merely been fixed with formalin, the block should be immersed in strong gum (made by saturating water with picked gum arabic, white and clean) for several days. It is then taken out and, without draining, transferred to the plate of a freezing microtome, and the sections cut from the frozen block, and mounted in glycerine at once.
This plan is of limited usefulness, since it allows of very little differentiation of the tissue elements, and that only optical.
To get the best results, some plan of staining must be adopted. Perhaps the simplest, and certainly a very excellent one, is as follows. After the tissue has been passed from the hardening, or fixing, fluid into the successive alcohols, as described, it is placed in the following solution. Take about forty grains of carmine and eighty grains of borax, dissolve in about an ounce of water, add to the mixture an ounce of methylated spirit, and let it stand for some time with frequent shaking; about a week will be sufficient, and the process of solution may be hastened by gentle warming at intervals. The clear upper portion is then poured off, and into this the block of tissue is dropped, and allowed to remain until thoroughly penetrated. Perhaps the best plan is to substitute the carmine solution for the 50 per cent. alcohol, and thus to make the staining a stage in the hardening process. From the carmine solution the tissue is transferred to 70 per cent. alcohol, to each ounce of which two drops of hydrochloric acid have been added, and after remaining in it for a day (with a piece of the usual size) is placed in 70 per cent. alcohol, in two successive quantities. Sections from this material now only require treatment with the carbolic acid and turpentine above mentioned to be fit for mounting and covering in balsam. We now proceed to indicate how the sections may be cut.
A mixture of wax and almond oil, in proportions varying with the heat of the weather, usually about equal ones, is prepared. The piece of tissue is freed from superfluous spirit by being placed on a bit of blotting-paper for a minute or two, and is then immersed in a quantity of the wax-and-oil mixture contained in a little box of paper or lead-foil. The tissue is held on the point of a needle, and lifted up and down until it is coated with the mixture, and, before solidification of the mass sets in, is lowered into the box and left to cool. The block now furnishes a handle, and this should be wrapped round with paper, the sections cut with the keenest possible razor, and as thin as possible, and placed in spirit as cut. From the spirit, which must be the strongest obtainable, they are placed in the clearing liquid, carbolic and turpentine, and then slid on to the slide, a drop of balsam placed on the section, and the cover over all. Of late years all sections of ordinary soft tissues, animal or vegetable, have been cut by one of the infiltration methods, in which the interstices of the tissue are filled up by some material which prevents the relations of the cells from being altered during the process of cutting. To enter fully into this matter would occupy too much space, and would serve no useful purpose, for the worker who requires to make use of such means will find it indispensable to obtain Bolles Lee’s Microtomist’s Vade Mecum, in which the whole matter is exhaustively treated.
The simple method above detailed will answer most ordinary purposes, provided that a few precautions be attended to. The chief are as follows. The outside of the block of tissue must be sufficiently dry for the wax-and-oil to adhere to it. The razor must be extremely sharp, and must be kept so by application to a Turkey stone during the section-cutting. The blade must be drawn across the tissue from heel to point, and kept wetted with spirit the whole time, so as to prevent any dragging of the section. The transference of the section to the slide must be effected by means of a section-lifter, which may be made by beating out a piece of stout copper wire to a thin flat blade; or a small palette-knife, or German-silver lifter, may be purchased for a few pence. The carbolic turpentine is best used by placing a little in a watch-glass, and floating the sections on to it by lifting them singly with the lifter, freeing them from superfluous spirit by draining on to blotting-paper, and allowing them to float on to the surface of the liquid in the watch-glass, so that the spirit may evaporate from above, and be replaced by the clearing agent from below The balsam solution should be thin, and the cover-glass must be allowed to settle down into place without pressure.
The question of staining sections is a very large one, and is becoming of daily increasing complexity.
We cannot go into it here, further than to say that most sections cut from unstained tissue will yield excellent results if stained first with Delafield’s logwood solution (to be obtained at Baker’s) to a very slight extent, and then with a solution of safranin. The sections should be washed with tap-water after the logwood stain, and should be of a pale violet colour. If over-stained, the colour may to a great extent be removed by washing with a very weak solution of hydrochloric acid, about two drops of acid to each ounce of water, and repeated washing in tap-water to remove the acid, and restore the violet. The safranin stain should be weak, and should be allowed to act for some time. From this last the sections are transferred to strong spirit, the latter being renewed until the sections cease to give up the red dye; and they may then be mounted as described. The results with most tissues are superb, every detail of the structure being splendidly brought out. Safranin alone is also an admirable stain for many purposes.
Further information must be sought in the book already mentioned. Let us, in closing, warn the beginner of two things which are of general application in practical microscopy. The first is, not to be discouraged by failures. The manipulations are in many cases very delicate, and premiums must be paid to experience for insurance against failure in every one of the processes.
The second is, that the most scrupulous cleanliness will hardly suffice to prevent contamination of preparation by the all-pervasive dust which, invisible to the eye, assumes colossal proportions under the microscope, and the particles of which have an unpleasant habit of collecting on the most interesting or most beautiful portion of the preparation. This can only be guarded against by careful filtration of all fluids, and constant watchfulness.
A preparation properly made is a thing of beauty, and a joy for ever,—or if not for ever, at any rate for many years; and one such will repay an infinitude of pains taken in its production.