In this, our concluding chapter, we propose to give a few hints upon the choice and use of the microscope and its accessories, to enlighten our readers concerning stains and staining and to add such other information as is likely to be useful, information which is better supplied in a chapter of its own than scattered about our pages and probably overlooked.
The most important question concerns the choice of a microscope. In a book of this nature it is obviously impossible to recommend the wares of any one maker. Some firms are celebrated for the good wearing qualities of their instruments, some for the delicacy—not fragility by the way—of their fine adjustments and our readers must select their maker for themselves. One thing we strongly advise, be patriotic and select a British maker, it is not only a question of patriotism but of self interest, for in many respects the British instruments lead the world. Most of the manufacturers advertise in such papers as Nature, they all print excellent catalogues, a perusal of which may be a useful preliminary.
The instrument we select will depend on the use to which we intend to put it and on the length of our purse. Perhaps the question of cost will be more important to most people. An elaborate instrument is not a desirable acquisition, till we are somewhat advanced in our work at anyrate, and one of the models which most makers call Students’ Microscopes will do everything we desire. We should have at least two objectives, three if we can afford them, a 1 inch and a 1/6 inch will enable us to examine everything described in these pages, except some of the bacteria. If we wish to add a third objective we might select a 2 inch one, for quite low-power work and later we shall probably become the proud possessors of a 1/12 inch objective, but this would be of little use to us at first. Two eyepieces will complete our optical equipment. A condenser for the stage, properly known as a substage condenser is an addition which we shall appreciate, though quite interesting work may be done without it.
Our other apparatus is inexpensive and comprises, one or two dissecting knives, some needles mounted in handles, a pair of fine scissors, a pair of small forceps, a razor, one or two camel hair brushes, three or four watch glasses, slides and cover slips, and a pipette or two. A pipette, by the way, is merely a glass tube pointed at one end and cut off square at the other; at about its centre it is much wider than at the top or bottom. It is a most useful piece of apparatus for picking up small water animals which we wish to examine. By putting the pointed end of the pipette just over an Amœba, for example, and sucking gently at the other end, we shall draw some water and the animal into the swollen part of the tube. If we then place one finger over the end of the pipette to which we have applied suction we can transfer the contents wherever we wish without it running out, as soon as we remove our finger the pipette empties itself. The dissecting knives are useful for cutting up specimens before examination, the mounted needles for teasing them, that is to say tearing them into fine shreds. The brushes we may use instead of the pipette, for picking up small objects from water, or when dry. The watch glasses are useful for examining objects such as sponges, water fleas, etc., under water. They are just as satisfactory for most purposes as the specially constructed zoophyte troughs and, of course, very much cheaper. The razor is required for section cutting concerning which we will say a few words.
For section cutting, special razors are sold, they are heavy as a rule and not hollow ground. Many microscopists aver that good sections cannot be cut with a hollow ground razor, we beg to differ, however. On this point we would advise our readers to select whichever pattern suits them best; there is no need to buy an expensive razor, for our early efforts, at anyrate, will quickly dull the cutting edge and there is no advantage in going to much expense in this direction.
To cut really good sections is not difficult, it is a question of knack rather than skill; having once acquired the knack, practice will do the rest. The great point is to begin properly, some people never take the necessary trouble in their early days and, as a consequence, they never learn to cut good sections.
There is nothing mysterious about the operation, a section is merely a very thin slice. A good section for examination under the microscope should, of course, be so thin that it is transparent; it should be equally thin everywhere, not thick in some places and thin in others and it should be cut in the proper direction. The last statement requires a little explanation; suppose we wish to make a section straight across a stem, a transverse section it would be called, then we must see to it that our section is cut straight across, and not in such a manner that the portion from which the section is cut tapers in the slightest degree. Before we begin the actual section cutting we must always trim up our specimen with one of the dissecting knives and not attempt to make the surface level with the razor. As a start, let us take some moderately fleshy plant stem for we shall find it as easy to cut as anything. Our stem must not be woody, for till we have had a fair amount of practice we shall damage our razor on its hard structures; it must not be too soft or it will be crushed out of all recognition in our fingers; it should not be hollow, for solid stems are always easier to cut. Having selected our stem and the spot upon it at which we require a section, we cut straight across it at that spot with our dissecting knife, in such a manner that the cut edge is as nearly as possible at right angles to the sides of the stem. A piece two or three inches long is the most comfortable to hold.
Holding our razor firmly in the right hand we take our specimen in the thumb and index finger of the left hand. Place the side of the stem against the first joint of the index finger and slightly bend the tip of the finger round the object. The end of the stem, from which we propose to cut our section, should be slightly above the level of the index finger. With the thumb, the stem is pressed firmly against the first joint of the index finger and the first joint of the thumb must not be bent upwards. This last point is very important, as we shall see in a moment. From our description the method of holding the stem may seem somewhat cramped; it should not be so, however, and an easy position will help towards success but let it be the correct position. We may play the violin, in a manner, without learning the correct method of bowing but we can never become expert violinists unless we hold our bow properly, in the same way we can never cut really fine sections unless we hold our specimen in the correct way. We now lay our razor along the second joint of the index finger in such a manner that the heel of the razor, or a point near the heel comes gainst the end of the stem. For the first time we realize the importance of holding our specimen correctly, for the index finger forms a convenient platform on which to steady the razor. To cut our section, we, of course, press the razor towards us and, at the same time draw it outwards from heel to toe. We must never attempt to press the razor straight through the specimen, however soft the latter may be, we must always draw the razor along, at the same time as we press it through the specimen. Never cut outwards as in sharpening a pencil and never move the razor backwards and forwards as though using a saw. The outward cut will never result in a good section, a sawing movement will give a section alternately thick and thin. Should the section be somewhat tough we may find the razor slip suddenly over its surface, an event which will impress itself upon us painfully if we have forgotten the injunction not to bend the thumb, for the certain result of the slip will be the loss of a goodly portion of skin upon one’s joint. A straight thumb will be out of the danger zone of a slipping razor.
By the courtesy of Messrs. F. Davidson & Co.
1. A Fly’s Eye
A fly’s eye highly magnified, showing some of the many lenses of which it is composed.
2. Images Seen by a Fly
Photographs of a statue taken through a fly’s eye. Instead of seeing one image of an object, the fly sees as many images as there are lenses in its eyes.
Having cut a section it is hardly likely that, at this our first attempt, it will be good enough to put under the microscope, so we will continue to cut section after section till we have a dozen or more from these we can select the most transparent for examination. The sections of such material as a stem should be kept moist and to do so we will place them in a watch glass containing water. It is often easier, also, to cut our sections if the razor be moistened with water, at anyrate the moisture prevents the sections from adhering to the razor. The sections should be removed from razor to watch glass and from watch glass to slide, by means of a brush, never by means of the fingers. The razor of course, should be well dried with a soft rag before it is put away; rust, besides being unsightly, ruins the cutting edge. If we are really anxious to cut our own sections, and every good microscopist does so, we shall return to the operation again and again, even cutting objects which we have no desire to examine, for the sake of the practice. We shall soon reach a stage where our razor will be dulled and require stropping and the efficient stropping of a razor is, to many people, a more difficult operation than the cutting of sections.
We may point out here, that all sections are not quite so easy to cut as the one we have taken as our example. Some objects are so soft that they need hardening with chemicals before they can be cut, some are so hard that to attempt to cut them would ruin the edge of the razor, though it is wonderful what hard substances may be cut when we have had a little experience; some are so delicate that they must needs be buried in melted wax, then object and wax are cut together and later the wax is separated from the section.
Leaves and very small stems may be sectioned by the beginner, as easily as larger stems. For such objects little sticks of pith are sold as holders. Having cut of our piece of leaf from which we wish to derive a section, we make a slit, with our dissecting knife, down the middle of one end of a piece of pith. The piece of leaf is then placed in the slit and by holding the pith at the sides our piece of leaf is held firmly. Sections are cut through pith and leaf, and the two are floated in water, when the thin slices of pith will float away from the leaf sections.
Having cut a satisfactory section, let us proceed to describe the method of making a slide thereof. We will suppose that we do not wish to make a permanent preparation but one for temporary use. A clean slide must be selected, all through our pages we emphasise the cleanliness of slides, at or near its centre we put a drop of water and, lifting it with a brush, we place our section in the drop of water. If our examination is to be with a low magnification, we need not use a cover slip, nevertheless it is worth while to cultivate the habit of using one. The cover slip is not only a protection for our object but for our objective. Water you may argue cannot harm the lenses of the objective. Perhaps not, we will not argue the point but, when the water dries on the objective, it leaves a certain amount of deposit on the glass and this deposit must be rubbed off. The less often the lenses are rubbed the better for them, glass especially very highly polished optical glass, is far more easily scratched than many people imagine and a scratched lens is an inefficient lens. When using high magnifications a cover glass should always cover our object and the same remark applies to objects examined in Canada Balsam. This substance is likely to cause serious trouble if it finds its way on to an objective. It must be removed, that is obvious, but it sets hard, it must not be scraped away for fear of damaging the optical glass and, as it is used to cement the lenses together, there is the great danger that any solvent used to remove the Balsam from the face of the objectives, may also dissolve their setting. Our digression may seem somewhat unnecessary, but the very great importance of keeping all chemicals and even water, from coming into contact with the lenses of our instrument cannot be insisted upon too strongly.
In whatever substance we examine our object, water, glycerine or Balsam, there is a right and a wrong way of applying the cover slip. It must not be dropped or laid down flat upon the object, if we do this we shall certainly imprison a number of air bubbles and that must be avoided. One edge of the cover slip must be laid against the edge of the mountant, as the liquid used for mounting our object is called, then placing a needle beneath the cover slip it must be gently lowered into position. We shall now find that all air bubbles are driven out as the cover slip is lowered.
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.
There is a temptation to buy a large stock of stains of all hues and of varied composition. The temptation should not be allowed to get the better of us. In our early days at anyrate, we shall do far better if we use but few stains and learn to understand their peculiarities.
The most useful selection for the beginner will comprise Haematoxylin, Safranin, Eosin, Carbol-Fuchsin, Methylene Blue, and Carmalum. These should all be obtained ready for use: this will save errors in compounding, and, in certain cases, will save time; some of the stains, Haematoxylin for example, are not fit for use till months after they have been mixed with the other ingredients which form the complete stain.
Frequently when examining small but lively water animals we may feel the necessity of some method of sobering them. All the little organisms, both plants and animals, which are provided with the little whip-like structures mentioned so often in our pages, are difficult to examine whilst they are in motion. There are many substances, we might add, which would kill them, but in doing so we shall nearly always find that they contract to such an extent as to lose all semblance of their natural shape, and become useless as objects for our microscope. Should our inclinations lead us in the direction of much study of these little beings, we shall do well to keep handy a small bottle of Rousselet’s solution; it is composed of 2 per cent. solution of hydrochlorate of cocaine, 3 parts; Methylated Spirit, 1 part; water, 6 parts. Any chemist will mix the solution for us. When we have occasion to examine a too lively specimen, we simply run a drop of the solution under the cover slip, as described in our remarks on staining with iodine, and the creature we are examining will abandon its frolics and conveniently remain in a fully expanded state.
Many of our readers will be anxious to carry their microscopic investigations to a more advanced stage than we have reached in our pages. Some will have the advantage of a teacher, and it is a great advantage to have someone who can show rather than merely explain what should be done; others not so well placed may like to know a few really useful books which will help them in their work. “The Microscope and its Revelations,” by B. H. Dallinger, is a large and therefore costly work, but it contains a rare fund of information for the microscopist. Smaller and eminently suitable for the general worker is “Modern Microscopy,” by Messrs Cross & Cole. Those who wish to specialize in one of the sciences, such as Botany, Zoology, or Geology, will find no lack of books dealing with the subject that most appeals to them. For photographers who are also microscopists, we know of no better books than “Practical Principles of Plain Photomicrography,” by G. West, or the more advanced “Handbook of Photomicrography,” by Messrs Hind & Randles.
Neither books nor teacher will be able to reveal to any of us all the secrets of the microscope. By its means a new world is unfolded before the eyes of mankind, a world of unlimited possibilities. No man will ever see all that the microscope can show him, each day some fresh wonder is looked upon for the first time.
As a hobby, microscopy can hardly be excelled. It is a sensible hobby and, after the initial outlay, need cost us but a few pence each year. We hear someone say that he prefers an outdoor hobby, but surely the microscope, if used intelligently, will take us out of doors. The search for specimens is one of the attractions of microscopy. As an instrument for the serious worker the microscope compares favourably with any other invention of mankind.
Think for a moment what we owe to the microscope in a hundred and one walks of life, and then you will realize the romance attached to the instrument, and its revelations from the days of Euclid to the present time.
FOOTNOTE:
[1] Humour is here used in its original sense, meaning moisture or a liquid.