Common Objects of the Microscope
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A variety of instruments has been invented for the same purpose, of which Beck’s parallel compressorium may be mentioned as the most efficient, though it is somewhat complicated, and consequently expensive, costing about twenty-five shillings.
A few glass slips and cover-glasses will also be required. The latter had better be those known as “No. 2,” since the beginner will find it almost impossible to clean the thinner ones satisfactorily without a large percentage of fractures. The safest way is to boil the thin glass circles in dilute nitric acid (half acid, half water) for a few minutes, wash well in several waters, first tap-water and then distilled, and finally to place the covers in methylated spirit. When required for use, the spirit may be burnt off by applying a light, the cover-glass, held in a pair of forceps, being in no way injured by the process.
In addition to the glass slides, the observer will find it advisable to be provided with a few glass troughs, of various thicknesses, in which portions of water-plants, having organisms attached to them, may be examined. Confined in the live-box, many of the organisms ordinarily found under such circumstances can rarely be induced to unfold their beauties, whilst in the comparative freedom of the trough they do so readily. The troughs may be purchased, or may be made of any desired shape or size by cutting strips of glass of a thickness corresponding to the depth desired, cementing these to a glass slide somewhat larger than the ordinary one, and cementing over the frame so formed a piece of thin glass, No. 3; the best material to use as cement being marine glue of the best quality, or, failing this, Prout’s elastic glue, which is much cheaper, but also less satisfactory. The glass surface must be made, in either case, sufficiently hot to ensure thorough adhesion of the cement, as the use of any solvent entails long waiting, and considerable risk of poisoning the organisms. A useful practical hint in the use of these troughs is that the corners, at the top, should be greased slightly, otherwise the water finds its way out by capillary attraction, to the detriment of the stage of the microscope.
Of optical accessories, the bull’s-eye is almost the most valuable. So much may be effected by its means alone, in practised hands, that it is well worth the while of the beginner to master its use thoroughly, and the methods of doing so will be explained in the succeeding chapter.
The substage condenser, too, even in its most simple form, is an invaluable adjunct, even though it be only a hemisphere of glass, half an inch or so in diameter, mounted in a rough sliding jacket to fit underneath the stage. Such an instrument, properly fitted, will cost about fifteen shillings, but the ingenious worker will easily extemporise one for himself.
Fig. 11.
Many plants and animals require to be dissected to a certain extent before the details of their structure can be made out, and for this purpose the naked eye alone will rarely serve. The ordinary pocket magnifier, however, if mounted as described in a preceding chapter, will greatly facilitate matters, and the light may be focused upon the object by means of the bull’s-eye condenser, as shown in Fig. 11. In the figure the latter is represented as used in conjunction with the lamp, but daylight is preferable if it be available, the strain upon the eyes being very much less than with artificial light. Two blocks of wood, about four inches high, will form convenient rests for the hands, a plate of glass being placed upon the blocks to support the dish, and a mirror being put in the interspace at an angle of 45° or so if required. A piece of black paper may be laid upon the mirror if reflected light alone is to be used.
As all delicate structures are dissected under fluid, a shallow dish is required. For this purpose nothing is better than one of the dishes used for developing photographic negatives. The bottom of the dish is occupied by a flat cork, to which a piece of flat lead is attached below, and the object having been pinned on to the cork in the required position, the fluid is carefully run in. This fluid will naturally vary according to the results desired to be obtained, but it must not be plain water, which so alters all cellular structures as to practically make them unrecognisable under the microscope. Nothing could be better than a 5 per cent. solution of formalin, were it not for the somewhat irritating odour of this fluid, since it at once fixes the cells and preserves the figure. For many purposes a solution of salt, in the proportion of a saltspoonful of the latter to a pint of water, will answer well for short dissections. For more prolonged ones, a mixture of spirit-and-water, one part of the former to two of the latter, answers well, especially for vegetable structures. When the dilution is first made, the fluid becomes milky, unless pure spirit be used, but with a little trouble the Revenue authorities may be induced to give permission for the use of pure methylated spirit, which answers every purpose. The trouble then is that not less than five gallons can be purchased, an embarras de richesses for the average microscopist, but, after all, the spirit is extremely cheap, and does not deteriorate by keeping.
When the dissection in either of these media is completed, spirit should be gradually added to bring the strength up to 50 per cent., in which the preparation may remain for a day or two, after which it is gradually brought into pure spirit, or into water again, according to the medium in which it is to be mounted.
Fig. 12.
As to the tools required, they are neither numerous nor expensive. Fine-pointed but strong forceps (Fig. 9, c), curved and straight; a couple of pairs of scissors, one strong and straight, the other more delicate, and having curved blades, and a few needles of various thicknesses and curves, are the chief ones. The latter may be made by inserting ordinary needles, for three-fourths of their length, into sticks of straight-grained deal (ordinary firewood answers best), and thereafter bending them as required. A better plan, however, is to be provided with a few of the needle-holders shown in Fig. 9, b. These are very simple and inexpensive, and in them broken needles are readily replaced by others. Dipping-tubes, such as are shown in Fig. 12, will also be extremely useful for many purposes. These are very easily made by heating the centre of a piece of soft glass tubing of the required size, and, when it is quite red-hot, drawing the ends apart. The fine tube in the centre should now be divided by scratching it with a fine triangular file, and the scratch may of course be made at such a point as to afford a tube of the required fineness. The edges should be smoothed by holding them in the flame until they just run (not melt, or the tube will close). These tubes can often be made to supply the place of a glass syringe. They may be used either for sucking up fluid or for transferring it, placing the finger over the wide end, allowing the tube to fill by displacement of air, and then re-closing it with the finger. This last method is especially useful for transferring small objects from one receptacle to another. In speaking of the dissection of objects, it should have been mentioned that the microscope itself may, under careful handling, be made to serve very well, only, as the image is reversed, it is almost impossible to work without using a prism to re-erect the image. Such a prism is shown in Fig. 13. The microscope is placed vertically, and the observer, looking straight into the prism, sees all the parts of the image in their natural positions. This appliance is extremely useful for the purpose of selecting small objects, and arranging them on slides in any desired manner. A few words may be added as to the reproduction of the images of objects.
Fig. 13.
The beginner is strongly recommended to practise himself in this from the outset. Even a rough sketch is worth pages of description, especially if the magnification used be appended; and even though the worker may be devoid of artistic talent, he will find that with practice he will acquire a very considerable amount of facility in giving truthful outlines at least of the objects which he views. Various aids have been devised for the purpose of assisting in the process. The simplest and cheapest of these consists of a cork cut so as to fit round the eye-piece. Into the cork are stuck two pins, at an angle of 45° to the plane of the cork, and, the microscope being placed horizontally, a thin cover-glass is placed upon the two pins, the light being arranged and the object focused after the microscope is inclined. On looking vertically down upon the cover-glass, a bright spot of light will be seen, and as the eye is brought down into close proximity with it the spot will expand and allow the observer to see the whole of the image without looking into the microscope. If a sheet of paper be now placed upon the table at the place occupied by the image so projected, the whole of the details will be clearly seen, as will also the point of a pencil placed upon the paper in the centre of the field of view; and, after a little practice, it will be found easy to trace round the chief details of the object. Two points require attention. The first is that if the light upon the paper be stronger than that in the apparent field of the microscope, the image will not be well seen, or if the paper be too feebly lighted, it will be difficult to keep the point of the pencil in view. The light from the microscope is thrown into the eye, and the view of the image upon the paper is the effect of a mental act, the eye looking out in the direction from which the rays appear to come. The paper has therefore to be illuminated independently, and half the battle lies in the adjustment of the relative brightness of image and paper. The second point is, that it is essential to fix one particular point in the image as the starting-point of the drawing, and this being first depicted, the image and drawing of this point must be kept always coincident, or the drawing will be distorted, since the smallest movement of the eye alters the relations of the whole. The reflector must be placed at an angle of 45°, or the field will be oval instead of circular. The simple form of apparatus just described has one drawback, inasmuch as the reflection is double, the front and back of the cover-glass both acting as reflectors. The image from the latter being much the more feeble of the two, care in illumination will do much to eliminate this difficulty; but there are various other forms in which the defect in question is got rid of. The present writer has worked with all of them, from the simple neutral tint reflector of Beale to the elaborate and costly apparatus of Zeiss, and, upon the whole, thinks that he prefers the cover-glass to them all.
A very simple plan, not so mechanical as the last-named, consists in the use of “drawing-squares,” which are delicate lines ruled upon a piece of thin glass, and dropped into the eye-piece so that the lines rest upon the diaphragm of the eye-piece, and therefore are in focus at the same time as the object. By the use of these, in combination with paper similarly ruled, a diagram of any required size can be drawn with very great facility. The squares, if compared with a micrometer, will furnish an exact standard of magnitude for each object-glass employed. The micrometer is a piece of thin glass upon which are ruled minute divisions of an inch or a millimeter. Suppose the micrometer to be placed under the microscope when the squares are in the eye-piece, and it be found that each division corresponds with one square of the latter, then, if the micrometric division be one one-hundredth of an inch, and the squares upon the paper measure one inch, it is clear that the drawing will represent the object magnified a hundred “diameters”; if two divisions of the micrometer correspond to three squares, the amplification will be a hundred and fifty diameters; if three divisions correspond to two squares, sixty-six diameters, and so on. If a draw-tube be used, it will be necessary to know the value of the squares at each inch of the length, if they are to be used for measuring magnification.
CHAPTER III
Examination of Objects—Principles of Illumination—Mirror and its Action—Substage Condenser—Use of Bull’s-eye—Opaque Objects—Photography of Microscopic Objects.
So much depends upon a right method of employing the microscope, as regards both comfort and accuracy, that we propose to devote a little space to the consideration of the subject.
Let us first warn the intending observer against the use of powers higher than are required to bring out the details of the object. Mere magnification is of very little use: it increases the difficulties both of illumination and of manipulation, and, as already said, interferes with that grasp of the object which it is most desirable to obtain. Rather let the beginner lay himself out to get the very most he can out of his lowest powers, and he will find that, by so doing, he will be able far better to avail himself of the higher ones when their use is indispensable.
The essential means to this end is a mastery of the principles of illumination, which we now proceed to describe.
We suppose the microscope to be inclined at an angle of about 70° to the horizontal, with a low-power objective attached to it, a one-inch by preference. Opposite to the microscope, and about a foot away from it, is a lamp with the edge of the flame presented to the microscope, the concave mirror of which is so arranged as to receive the rays from the flame and direct them up the tube of the microscope. Upon the stage is placed a piece of ground-glass, and the mirror-arm is now to be moved up or down upon its support until the ground-glass receives the maximum of illumination, which it will do when the lamp-flame is at one conjugate focus of the mirror and the ground-glass at the other. The focus will not be an image of the flame, but a bar of light.
If an object be now placed upon the stage, instead of the ground-glass, and the objective focused upon it, it will, if the mirror be properly adjusted, be brilliantly illuminated.
It will be understood that every concave mirror has a focus, and converges the rays which fall upon it to this focus, behaving exactly like a convex lens. The principal focus of a concave mirror is its radius of curvature, and this is not difficult to determine. Place side by side a deep cardboard box and the lamp, so that the concave mirror may send the rays back, along a path only slightly inclined to that by which they reached it, to the bottom of the box. The lamp and box being equidistant from the mirror, it is evident that when the mirror forms an image of the former upon the latter equal to the flame in size, we have the equivalent of the equal conjugate foci shown in Fig. 2. Now move the box to the distance from the mirror which corresponds to the distance of the stage of the microscope from the mirror when the latter is in position upon the microscope, and then move the lamp to or fro until the mirror casts a sharp image of the flame upon the bottom of the box, which is not to be moved. The lamp distance so found will be the correct one for working with the concave mirror. The writer is led to lay special stress upon this matter, from the fact that he almost invariably finds that the mirror is arranged to be used for parallel rays, i.e. for daylight, and is therefore fixed far too close to the stage to be available for correct or advantageous working with the lamp, unless, indeed, the bull’s-eye condenser be used, as hereinafter described, to parallelise the rays from the lamp.
Work done with the concave mirror can, however, under the most favourable conditions, only be looked upon as a pis aller. The advantages gained by the use of some substage condenser, even the most simple, in conjunction with the plane mirror, or even without any mirror at all, are so manifold that the beginner is strongly urged to provide himself with some form or other of it, and we now proceed to describe the way in which this should be used to produce the best effect.
To reduce the problem to its most simple elements, turn the mirror altogether out of the way, and place the microscope upon a block at such a height as shall be convenient for observation, and shall allow the rays from the lamp, placed in a line with it on the table, to shine directly into the tube of the microscope. Ascertain that this is so by removing both objective and eye-piece and looking down the tube, when the flame should be seen in the centre, edgewise. Now replace the eye-piece, and screw on to the tube the one-inch combination or objective. Place upon the stage an object, preferably a round diatom or an echinus-spine, and focus it as sharply as possible. Now place the substage condenser in its jacket, and slide it up and down until the image of the object is bisected by the image of the flame.
The centre of the object will now be brilliantly illuminated by rays travelling in the proper direction for yielding the best results. The object is situated at the common focus of the microscope and the condenser, and, whatever means of illumination be adopted, this is the result which should always be aimed at.
Satisfactory as this critical arrangement is, however, from a scientific point of view, it has its drawbacks from an artistic and æsthetic one. It is not pleasant, for most purposes, to have merely the centre of an object lighted up, and we have now to consider how the image of the edge of the flame may be so expanded as to fill the field without sacrificing more than a very small fraction of the accuracy of the arrangement just attained.
Referring to Fig. 1, we see that if we place the lamp at the principal focus of a lens, it will emit a bundle of parallel rays equal in diameter to the diameter of the lens. This is the key of the position. We cannot place the lamp at an infinite distance from the substage condenser, but we can supply the latter with rays approximately parallel, so that it shall bring them to a focus upon the object at very nearly its own principal focus. This we do by means of the bull’s-eye condenser. Place the latter, with its flat side toward the edge of the flame, and at its principal focal distance (the method of determining which has already been described) from the latter, so that the bundle of parallel rays which issue from it may pass up to the substage condenser. On examining the object again, it will be found that, after slight adjustments of the position of the bull’s-eye have been made, the object lies in the centre of an evenly and brilliantly lighted field.
It may be necessary to place the bull’s-eye a little farther from or nearer to the lamp, or to move it a little to one side or the other, but when it is at the correct distance, and on the central line between the lamp and the substage condenser, at right angles to this line, the effects will be as described. It may help in securing this result if we mention that when the bull’s-eye is too far from the lamp, the image of the flame is a spindle-shaped one; whilst, when the distance between the two is too short, i.e. less than the principal focal length of the lens, the field is crossed by a bar or light, the ends of which are joined by a ring, whilst on either side of the bar there is a semi-circular dark space.
We have hitherto supposed the objects viewed to be transparent, but there are many, of great interest, which are opaque, and call for other means of illumination. Of these there are several. The simplest and, in many ways, the best is to use the bull’s-eye condenser to bring to a focus upon the object the rays of light from some source placed above the stage of the microscope. If light can be obtained from the sun itself, no lens will be needed to concentrate it; and indeed, if this were done, there would be considerable risk of burning the object. The light from a white cloud, however, with the help of the bull’s-eye, answers admirably. At night-time an artificial source of light, the more intense and the more distant the better, is required. For most cases, and with powers not higher than one inch, a good paraffin lamp, placed about two feet away from the stage, and on one side of it, so as to be about a foot above the level of the object, will give all that is needed. Such a lamp is shown in Fig. 14. Low magnifications are, as a rule, all that is called for in this method.
Lieberkuhn’s condensers are useful aids, but are somewhat expensive. They are concave mirrors, which are so adjusted to the objective that the latter and the reflector come into focus together, the light being sent in from below, or from one side.
One other method of illumination must be mentioned before leaving the topic, and this is the illumination of objects upon a “dark field.” With suitable subjects, and when carefully managed, there is no method which gives more beautiful effects, and it has the great advantage of allowing the object to be brilliantly lighted, without the strain to the eyes which is involved in such lighting by the usual method of direct illumination.
Fig. 14.
It consists essentially in allowing the light to fall upon the object from below, at such an angle that none of it can enter the objective directly. Thus the concave mirror, turned as far as possible to one side, and reflecting on to the object the rays from the lamp placed upon the opposite side, will give very fair results with low powers; this plan, however, is capable of but very limited application. Again, a disc of black paper may be stuck on to the middle of the bull’s-eye, and the latter be placed below the stage between it and the mirror. In this case everything depends upon the size of the disc, which, if too small, will not give a black ground, and if too large will cut off all light from the object.
The best and only really satisfactory plan is to arrange the illumination with the substage condenser, as previously described, and then to place below the lens of the latter a central stop of a suitable size, which can only be determined by trial. When this has been done the object will be seen brilliantly illuminated upon a field of velvety blackness. Such stops are supplied with the condenser.
We have devoted a considerable portion of space to this question, since it is, of all others, the most important to a successful, satisfactory, and reliable manipulation of the microscope; but even now, only the main points of the subject have been touched upon, and the worker will find it necessary to supplement the information given by actual experiment. A few failures, rightly considered, will afford a great amount of information, but those who desire to go thoroughly into the matter are recommended to consult the present writer’s Guide to the Science of Photomicrography, where it is treated at much greater length, as an essential part of the subject-matter of the book.
It may be added here, that no method of reproducing the images of objects is on the whole so satisfactory as the photographic one; and whilst a lengthened reference to the topic would be out of place in a work of the character of the present one, the one just mentioned will be found to contain all that is necessary to enable the beginner to produce results which, for faithfulness and beauty, far excel any drawing, whilst they have the additional advantage that they can, if required, be exhibited to hundreds simultaneously.
CHAPTER IV
Vegetable Cells and their Structure—Stellate Tissues—Secondary Deposit—Ducts and Vessels—Wood-Cells—Stomata, or Mouths of Plants—The Camera Lucida, and Mode of Using—Spiral and Ringed Vessels—Hairs of Plants—Resins, Scents, and Oils—Bark Cells.
We will now suppose the young observer to have obtained a microscope and learned the use of its various parts, and will proceed to work with it. As with one or two exceptions, which are only given for the purpose of further illustrating some curious structure, the whole of the objects figured in this work can be obtained without any difficulty, the best plan will be for the reader to procure the plants, insects, etc., from which the objects are taken, and follow the book with the microscope at hand. It is by far the best mode of obtaining a systematic knowledge of the matter, as the quantity of objects which can be placed under a microscope is so vast that, without some guide, the tyro flounders hopelessly in the sea of unknown mysteries, and often becomes so bewildered that he gives up the study in despair of ever gaining any true knowledge of it. I would therefore recommend the reader to work out the subjects which are here mentioned, and then to launch out for himself on the voyage of discovery. I speak from experience, having myself known the difficulties under which a young and inexperienced observer has to labour in so wide a field, without any guide to help him to set about his work in a systematic manner.