When we have cut an exceptionally good section or when we have some specially interesting object we may wish to make a permanent slide. The exact method of doing so varies somewhat with the nature of our object and to describe all the methods of mounting microscopic objects permanently would require a book in itself. We will suppose that we wish to make a permanent slide of one of the sections floating in our watch glass of water. The first thing we must do is to get rid of the water with which our section by now is saturated. This may be accomplished by means of alcohol of various strengths, which we may put into three watch glasses. In the first watch glass we have half pure alcohol and half water; in the second three-quarters alcohol and one part water; in the third watch glass pure or absolute alcohol. The section is transferred on a brush to the first watch glass and left there for five minutes, then to the second watch glass for a similar time and finally to the third watch glass. The mountant may be either Canada Balsam or glycerine jelly. Should we decide on Canada Balsam, we put a small drop of the substance in the centre of a clean slide, place one section atop of it and then gently lower a cover slip upon it as already described. Then, without using any force, the cover slip is pressed down, but we must not fall into the all too common error of thinking that a thick section can be made thin by pressing upon the cover slip. If we have taken the correct amount of Canada Balsam it will just cover the area below the cover slip and no more, if we have used too much, it will flow out on all sides and perhaps on the top of the cover slip. A very little experience will teach how big a drop of Balsam to use.
Should we decide to use glycerine jelly, we take a small globule of the jelly on one of our mounted needles and place it in the centre of a clean slide. Then we hold the slide a little above a lighted candle, or even a match will do, to melt the jelly. Directly it is melted the section is placed upon it, and we proceed as before. Whichever mountant we have used, the slides must be put away out of the dust, and they must be flat and not placed on edge. In a few days the mountant will set, and we need take no further precautions with the slides in which Balsam was used, but those mounted in glycerine jelly must be ringed, for the reason that glycerine absorbs moisture from the air and gradually liquifies.
The process of ringing is best performed upon a turntable, which any dealer in microscope accessories will supply. It consists of a circular brass plate which revolves about its centre and to which the slide to be ringed is affixed. A bottle of ringing asphalt and a fine paint brush are essentials. With a little of the asphalt upon our brush, we revolve the turntable and place the brush against the edge of the cover slip as it revolves, in such a manner that we paint a narrow rim of asphalt over the junction of cover slip and slide. The asphalt prevents moisture from reaching the glycerine jelly. Of course we may ring our slides without making use of a turntable, but it is not easy to paint a neat ring without mechanical assistance. For square cover slips it is obvious that a turntable is useless. Specimens mounted in Canada Balsam do not need ringing, for the Balsam is unaffected by air or the moisture therein, when once it has set hard.
Several firms supply prepared microscope slides, and it is often useful to know where reliable preparations may be obtained. The slides supplied by Messrs Flatters & Garnett Ltd., 409 Oxford Road, Manchester, are models of what well-made slides should be.
It always lends greater interest to the hobby if objects are found and mounted by the microscopist himself. By going out into the fields, by the pond-side, or along the shore, in search of interesting material for examination, much will be learned of animal and plant habits which even the microscope cannot reveal. Some of us, however, have neither the opportunity to hunt for our specimens, nor the time to mount them properly, and those of us who are so situated will be glad to know where objects may be obtained.
Apparatus, useful for students of pond or sea-shore life, may be obtained from Messrs Flatters & Garnett, who always have a goodly stock of collecting jars, nets, &c. From the same firm, also, may be obtained stains and any of the limited number of chemicals required by the microscopist.
We have given a few simple directions for staining in our [chapter on Bacteria]. In many cases it is absolutely necessary to have recourse to stains in order to see the structure of the objects we are desirous of examining; in other cases it is necessary to stain when we wish to know the nature of the various parts of our object. Suppose, for example, we wish to find out whether a plant section contains starch, we then add iodine solution, and if any parts stain deep blue we know at once that starch is present. There are other stains for other plant and animal substances; stains for woody matter, stains for fats, &c., but the art of staining is a science in itself, and would require many chapters to describe fully. Most of the objects described in this book can be studied in the natural state, but, even so, they may be rendered far more beautiful by staining.
The method we described for the staining of bacteria does not apply to such objects as plant sections, &c., and we propose to describe, as briefly as possible, how to proceed with such objects. Suppose we are examining one of the common pond plants, Spirogyra for instance, and we wish to see whether it contains starch. Our specimen is in a slide, in a drop of water, and covered by a cover slip. In the first place, we must obtain some fluffless blotting paper—the ordinary filter papers sold by all chemists are excellent—from it we must cut about half a dozen pieces, about half an inch by one inch, the exact size is not important, and they need not be measured. These we must fold in the centre, so that they can be made to stand up like an inverted V. From our bottle of iodine solution we take a drop of the liquid on the end of a glass rod and place it carefully at one edge of the cover slip, avoiding allowing any of the solution to flow on to the upper surface of the slip. Now, one of the pieces of blotting paper must be placed upon the opposite side of the slide so that it will stand up; it must then be moved till it just touches the edge of the cover slip. The blotting paper will absorb the water from beneath the cover slip, and in doing so the iodine solution will be drawn along to take the place of the water. By proceeding in this manner and replacing the blotting paper with a new piece as it becomes moist, also replenishing the drop of iodine as it is used up, we act upon our object with a stronger and stronger solution, and, in Spirogyra, the object which we took as our example, we can see beautiful rosettes of starch grains, arranged at regular intervals along the green bands of chlorophyll. This method of staining may be used in most cases where we merely require a temporary stain; by reversing the process and drawing water over the object by means of blotting paper, it may be used in washing sections and parts of plants. For very small objects, such as starch grains separated from the plants in which they are formed, the method is hardly suitable, for they are liable to be drawn along in the stream of liquid and lost.
For more permanent staining processes, we must use our watch glasses, into which we pour the various liquids necessary for the operation. The precise methods of staining, the periods during which objects should remain in the staining solution, and the chemicals used for removing excessive stain vary, as may be guessed, according to circumstances. Some chemicals act very quickly, and staining takes place in a few minutes; others act slowly, and with them it is necessary to subject our specimens to their action for hours or even days. Then again, it is obvious that large specimens take longer to stain than small ones, hard objects are not so readily acted upon as soft ones. Experience alone will show what is required in various cases.
Suppose, for example, we desire to stain a section in Carmalum, a mixture of Carminic Acid 1 grain, Alum 10 grains, hot distilled water 200 c.c. We take three watch glasses, in one we place a few drops of our stain, in another water, and in the third alcohol. Our section is placed in the watch glass containing the carmalum and is left there for about two minutes, then with the help of a small brush it is transferred for a similar period to the watch glass containing water, and finally it is placed in the alcohol. From the last watch glass it may be transferred to glycerine jelly on a slide and mounted as already described; Carmalum stains our section a beautiful pink.