# Hints on the Preservation of Living Objects and Their Examination Under the Microscope

Hints
On The
Preservation of Living Objects
And Their
Examination Under the Microscope.
by
Thomas Bolton, F.R.M.S..
17, Ann Street, Birmingham.
Reprinted from the "English Mechanic and World of Science."
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Hints On the Preservation of Living Objects,
And Their
Examination Under the Microscope.
I have often been asked by my correspondents how best to examine the living objects I am sending then, and also how best to keep them alive for further examination, and to watch their continued growth, development, and reproduction; and I shall be glad (with the Editor's permission) to give all the information I can on these points to the best of my ability. The larger organisms, and such as are usually attached in their growth to weeds, I generally forward in glass tubes about half an inch in diameter, and two inches long; but unattached organisms, such as free-swimming rotifers, infusoria, volvoces, &c., I put in smaller tubes a third of an inch in diameter and 1½in, long; the latter holds half a drachm of water, and the former three times as much. Of course, larger objects, such as young newts, embryo fish, &c., which I occasionally send out, require larger tubes or bottles, which entail much extra care in package to allow for the much greater danger of breakage and leakage.
When such tubes as I have described are received by post they should be opened, uncorked, and, if they cannot be examined at once in the microscope, it is well to take a bung cork with a hole bored in by a cork borer to fit the tube. Pass the tube into the hole so that the top is level with the top of the cork, and so float the tube and its contents on the surface of water in a tumbler, basin, or still better, in an aquarium. In this way much danger to the life of the more delicate animal, organisms will be avoided from variations of temperature, which are not unlikely to occur in so small a body of water as the tubes themselves contain. In this way many of the advantages of a large body of water are obtained without any danger of the objects being lost, or diffused over too large a field to be readily found again.
Examination of Objects attached to Weeds and Root Fibres, such as Polyzoa, Hydra, Tree-Dwelling Rotifers, Vorticellidæ, &c.
Examine the contents of the glass tube with a pocket lens of about two inch focus. The tube case of a Melicerta ringens would be readily seen like a little black thorn standing erect from the surface of the leaves of the anacharis, myriophyllum, or other weed, and often attains the length of a tenth of an inch. If the tube be examined in the front of a window and held a little sideways so that the direct light of the window does not enter the eye, a little higher power of lens, such as Browning's platyscopic lens, will reveal the beautiful red colour of the tube in fine contrast with the bluish hyaline ciliatory wreath, apparently whirled round its mouth in rapid gyrations. Many other organisms may he readily detected, and some of their beauties guessed at in this way, and a little practice will soon enable the student to recognise most of the larger and many of the more minute organisms, or, which is perhaps more important, see that there is something he does not recognise, and which will require the use of the compound microscope to bring out the details and reveal its nature.
If the objects are attached to such a weed as the anacharis, after noting under the pocket lens, as above, the position of the several specimens on ​the weed, it will be best to transfer the weed by a pair of forceps from the tube to a zoophyte trough (about 2½in. long, 1½in. high, ⅜in. thick or deep), into which the water from the tube is poured, together with sufficient soft or tap water to nearly fill it. Examine again with pocket lens, and adjust the weed into a suitable position for the examination of some one or more of the specimens.
Place the trough, if convenient, at once in the microscope, and let it remain some hours at rest, and doubtless, if not before, it will now be seen to advantage.
In this position it may with advantage be examined with low powers, such as the 3in., 1½, and ⅔, and possibly occasionally, when it is peculiarly well placed, with the 4-10ths objective.
In such a trough it may be expected to live a week or so without change of water, or it may be kept longer in a small saucer, or evaporating dish, or still better in a fresh-water aquarium, in which the individual would very likely propagate and increase.
The student should carefully examine the whole of the weed under the low powers in the trough, and it is very likely he will be repaid by seeing some younger individuals just commencing the building of their tube, and he may possibly find others in a still earlier state swimming or creeping amongst the leaves.
For examination of the Melicerta under the ⅔, 4-10th, and ¼in, powers, it may be advantageously placed in a slide trough or tube cell of about 1.6th of an inch or less, covered with thin glass. To do this an individual should be noted on the weed, conveniently placed on a leaf, or, still better, on the stem. With a small pair of sharp-pointed scissors, the loaf on which the individual is place I should be cut off the weed, leaving a small piece of the stem attached, and so transferred to the trough or cell. It may sometimes be necessary, with the scissors, to pare down or split the loaf carefully without injuring the specimens, so as to reduce the leaf to a less width than the depth of the trough or cell. This being done, the leaf can be placed in the trough or cell sideways, and the piece of stem attached to it retains it in that position, otherwise the Melicerta tube, which is generally built in a position standing up from the surface of the leaf, would not be conveniently placed for examination. This manipulation may be very conveniently carried on in a deep watch-glass, under a dissecting microscope.
If a slide trough or tube cell be not at hand, the individual so selected may be placed in the ordinary animalcule cage or compressor, and for the highest powers this arrangement is best.
The slide trough arrangement has a great advantage in having the object in a more natural position, and in which it will live the longer. Moreover, when not wanted for examination under the microscope, it may conveniently be transferred to a basin of river water, or still better suspended in an aquarium. In this way an individual may be kept alive for some time, and its life history watched, and possibly young ones may be propagated and attach themselves to the weed or even to the glass.
When the Melicerta is found on myriophyllum, it can be better exhibited than by taking a single leaf, placing it on a slip of glass with with a little water, (illegible text) off any little (illegible text) the leaf which might interfere with the examination of the specimen, and then covering them with glass.
In this way it can be view with this highest powers, and can be beautifully illuminated with the centrally parabolic reflector, or with the spot-lens.
​The student will find that individuals grown in confinement build their tubes of much more transparent materials, and therefore are much better adapted for examination.
The previous directions, although especially applicable to Melicerta and tube-forming rotifers, are applicable to all organisms living attached to weeds. I will next point out the best ways of examining the free-swimming rotifers and infusoria, and afterwards different manipulations applicable to both.
Examination of free-swimming Rotifers, Infusoria, &c.
First examine contents of tube, as received, with pocket-lens, in the same way as previously advised with a tube of weeds, &c., and if you have reason to expect there are objects in it with which you are not familiar, and of which it would be well to make certain before the tube is opened, or there can be any possibility of the contents being lost, I should advise that the tube be examined under the compound microscope. This is applicable, whether the organisms are free or attached to woods; and I often test the contents of the tubes in this way before I despatch them. Of course, if placed directly in the microscope, their round form is awkward to fix, and the aberration of the light is so great as to prevent the possibility of seeing anything with fair clearness, except through the centre of the tube. I have, however, of late, in great measure, overcome this difficulty. I have had troughs made in which the tubes will just go in and lie diagonally. A round tube being placed in such a trough, filled with filtered water, the aberration arising from the cylindrical form of the tube is approximately counteracted, and it is surprising how easily you can examine its contents to the very sides and bottom. If the objects are large enough to be seen without the assistance of a lens, or with a lens of only a low power, they may be picked out at once with a dipping tube and transferred to a live box or compressor. If too small, or too active, to be treated in this way, it may be well to transfer the whole contents into a zoophyte trough of about the same capacity as the tube, for examination under a low power (say 1½in, objective), but I should generally prefer to empty the tube into a watch-glass for a preliminary examination under a dissecting microscope. I work myself with one of Beck's dissecting microscopes, which is an excellently-arranged instrument for the purpose, but many cheaper ones would be quite sufficient; the only essential is a good steady stage for the support of the watch-glass with plenty of lateral movement, a diagonal mirror under to throw a good light through the object, and ready adjustment for focussing the lens, which should be supported by a long arm over the centre of the mirror and stage. If the observer can work with a watchmaker's glass held under the eyebrow it makes a very good and cheap instrument for the purpose. One of my smallest tubes could be emptied at once into a large watch-glass, but a larger tube would have to be examined in several lots. Large active organisms, such as larvæ, annelids, entomostraca, too large to be readily picked up by a dipping tube, may be removed on the point of a small sable pencil, the quantity of water in the watch-glass having first been mostly drawn up by a capillary tube. A drop of water should previously have been placed on the centre of the live-box or compressor, just sufficient in quantity to allow the animal room to move about naturally; then just touch the drop of water with the point of the sable pencil with which you have picked it up, and it will most likely free itself, or else it must be carefully pushed off the brush by a needle mounted in a short wooden handle.
Smaller objects must be manipulated with the dipping tubes. I now generally use a short curved capillary tube, the upper end of which is blown out into a little funnel, and the end covered with a bit of stretched sheet ​india-rubber. A small orifice is pierced in the tube just below the funnel. If the tube is held between the thumb and the second finger, with the thumb (moistened) over the side orifice, the point of the tube placed under a surface of water, the first finger pressed on the stretched indiarubber to expel the air and then withdrawn, a quantity of water will return to fill the tube, and may be expelled again by a touch of the finger. In fact, I often use this means to transfer the water from the tubes to the watch-glass or trough. When it is intended to use a capillary tube to pick out organisms, the inside of the tube should always be first wet in this way, and the outside as far as possible kept dry. Now, having wet the inside of the tube, put the thumb on the orifice and drive out all the water from the point of the tube, then remove the thumb from the orifice, watch the animalcule you wish to catch in the watch-glass under the dissecting microscope, and immerse the point of the tube in the surface of the water just above the animalcule. The capillary attraction in the tube will draw up a small quantity of water with the animal included. It is well to have two or three small shallow watch-glasses at hand. Now close the orifice with the thumb, and by a slight touch of the indiarubber with the first finger you can expel this small quantity of water, with its contained animalcule or animalcules, on your animalcule cage. If at this first dip too much water has been drawn up, it may be pushed out into a watch-glass and a smaller quantity pulled up, and the process repeated till only just sufficient water is taken up as will allow the object sufficient room to assume a natural position. The advantage of this capillary tube over that of the ordinary dipping tube is that you have such perfect control over it, and can expel the small drop from its point by the mere touch of the finger whereas, in the old way, you had to blow it out with the mouth, entailing the removal of the eye from the dissecting microscope, which sometimes is very inconvenient.
I will now give a short summary of the most useful apparatus for the examination of living objects. The simple glass slip, 3in. by 1½in., or better, a ledged stage-plate 3in. by l£in., with narrow strip of glass cemented along one edge. One of these, with cover-glass, is often all the apparatus necessary to use with small infusoria and free-swimming rotifers, and is also occasionally available with a little management for larger objects, either free or attached. Manipulation with these I cannot better describe than in the words of Judge Bedwell, in his description of what I call Bedwell's rotifer-trap.
"Take a plane glass slide, on it drop one or more of the rotifers in a drop of water, about half an inch in diameter, and draw off the surplus water, if any, carefully with the empty pipette then fray out a very, very small portion of cotton wool (I always use a watchmaker's glass in the eye to do all such operations) until it is much extended, and spread out and lay this on the drop. Upon that lay the thin microscopic glass, the thinner the better, and then set up the capillary attraction by gently touching it with a needle. Draw off any superfluous water from the edges with the pocket-handkerchief, and you will have a little wilderness of wool in which the rotifer is restrained in its movements, protected from pressure, and within reach of very high powers. The amount of wool depends on the size of the rotifer. Hydatina requires more depth than rhiunps. The same plan answers equally well for all roving animals. The poduridæ in particular, when placed in deep glass cells, are easily seen by this apparatus, and it saves many a weary and vexatious five minutes with the compressorium, which, even at the best, requires with living animals extraordinary patience. The rotifers are ​easily found and secured with the pipette, and a watchmaker's glass in the eye, after a very little practice. Mr. Bolton's studio is of the greatest value to naturalists, and cannot be too well known, for to those who have not time to look for specimens it is a great privilege to be able to purchase them."
Another simple apparatus I call the Wills' compressorium. Most forms of compressorium are useless-all are expensive. Those who try the following will be surprised at the efficiency of the apparatus. Two pieces of thin glass are cemented on to a glass slip in the shape of the letter L, but with the two strokes of the letter about equal in length, and another thinner and longer one is fixed longitudinally, this L—— The L serves to retain in position a square slip of cover glass placed, of course, not on the L, but inside it; the horizontal piece, which should be ground to a bevel on its top edge before fixing it, serves to carry a fine needle, the point of which is inserted beneath the edge of the cover glass. This point being tapered, it is easy to increase or diminish the thickness of a film of water carried between the cover and the slip by pushing the needle further in or out, and so to form a cheap and effective compressorium.
Animalcule Cage and Compressorium.
The ordinary animalcule cages are often used for examination of entomostraca, larvæ, &c., with transparent light, but cannot be used for dark-field illumination, either with paraboloid or central-stopped condensers. A good compressorium is very useful; I have myself generally worked with R. and J. Beck's parallel compressorium, or their reversible compressorium. A good and cheaper form has just been made under the direction of Mr. Graham, President of our Birmingham Natural History and Microscopical Society, which will be very useful, as it gives a good range of depth, and is readily manipulated.
Hollow Cells.
These are ordinary slips of glass, 3in. by 2in., with a slight hollow ground in the centre, in which a drop of water can be placed and covered with thin glass.
Tube Cells.
These are very useful, and can be made by cementing with marine glue or gold size, on ordinary glass slips, either vulcanite, tin, or glass rings. These are filled with water containing the objects for examination, covered with a thin glass cover, which will be retained in its position by capillary attraction.
Tube-Cell Troughs.
Tube cells may be conveniently converted into most useful little zoophyte troughs by cementing a semicircle of thin glass on the half of the cell.
Thin Glass Slide Troughs.
These are made of glass slips, 3in. by 1½in., in which are cemented slips of thin glass 2in. by 1in., out of which a semicircle of ¾in. radius has been cut, and then covered with another thin glass 2in. by 1in. It is well to have an assortment of these, of different thicknesses or depths, and for those of greater depths it is more convenient to make the distance-plates of vulcanite instead of glass. Those troughs should always be washed out directly after use, but if dirty are best cleaned under a stream from a water-tap, with a wooden smoking-spill, the thickness of which can be readily reduced, if required, with a knife. In using these troughs, great care should be taken to have the top edge, face, and side, quite dry, as, if in fitting them any moisture is allowed to ​remain outside, capillary attraction is very apt to commence and cause the water to escape over the edge, especially if any fine fibres of algæ are present, which will often act as syphons. By omission of this precaution such troughs are often condemned as leaking when the fault is in the manipulation; but if a trough is found to leak it should be carefully dried, and the outside edges carefully painted over with one or two coats of gold size. One or two of the ordinary large Zoophyte troughs will also be occasionally wanted for large objects.
Glass Syringes.
In addition to the aforesaid apparatus, a small glass syringe would be found useful for transferring the water to and from the troughs, and another still smaller syringe is easily made by slipping a baby's sucking-bottle test on the end of a glass tube, the other end of the glass tube being drawn out into a capillary tube.
In examining rotifers and infusoria the student should never omit the use of carmine or indigo. It a little of either of these colours be rubbed up in a little water in a watch-glass, and a little taken up in a brush, and the brush run along the top of the water in a trough, sufficient will he added to barely tinge the water with the colour, which will gradually subside over the rotifers. It will be seen in the microscope like a rising cloud of dust, which as soon as it comes near the rotifers, is whirled round in definite curves, showing at once the action of their wonderful ciliary wreaths, This colouring matter is also greedily devoured by these animals, and may be followed with the microscope down their digestive canal from the mouth to the anus.
If the rotifers or infusoria are in a cell or under a thin cover, a drop of the mixed colour may be placed at the edge of the cover glass, and a piece of blotting-paper touched at the other side will draw a current through the cell.
The cilia and fine flagella on many of the small protophytes and infusoria, which are very difficult to see in their full activity, are easily seen when dying or afterwards from the effect of iodine. Its effect on Volvox globator, Euglena viridis, and Protococcus pluvialis is very interesting, and besides showing the cilia, it brings out many histological characters which are totally invisible without its help.
Aniline dyes are also occasionally useful for the same purpose. Osmic acid is now also often used for killing infusoría quickly in their expanded condition, and they may afterwards be stained advantageously with picrate of carmine (see Journal of the Royal Microscopical Society, Sept. 1878, page 189.)
The most useful aquaria for preserving and breeding microscopical organisms I find to be the ordinary confectionery cake-glasses inverted. I have a square block of wood (5in. square) with a hollow turned in the centre to receive the knob. If this is black varnished it looks very well. It should be covered with a round glass to exclude the dust. and is best raised slightly from the edge of the cake-glass by three thin slices of cork cemented with marine glue to the cover to allow a little circulation of air.
Common plain finger-glasses do very well for smaller ones, and I occasionally use the little glass cups made to hold the food and water in bird-cages. The management of the aquarium I cannot enter into; it can only be learnt by experience. The great thing is to hit the medium of enough vegetable and not too much animal life. I can recommend the handbook on the subject published by Mr. Bogue, the "Aquaria." by the Editor of Science Gossip.
Illustrations and prices of several parts of the apparatus above referred to will be found inside the cover of my Portfolio of Drawings, No. 2.
This work was published before January 1, 1930, and is in the public domain worldwide because the author died at least 100 years ago.
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