Chapter IV (4)

In mounting entomological specimens, the first thing, of course, is the dissection of the insect. This is best accomplished by the aid of a dissecting microscope, a pair of small brass forceps, and finely-pointed scissors; the parts to be prepared and mounted should first be carefully detached from the insect with the scissors, then immersed in a solution of caustic alkali (_liquor potassæ_) for a few days, to soften and dissolve out the fat and soft parts. The length of time necessary for their immersion can only be determined by experience, but, as a general rule, the objects assume a certain amount of transparency when they have been long enough in the alkali; when this is ascertained, the object must be placed in a proper receptacle and put by to soak for two or three hours in soft or distilled water. It should then be placed between two slips of glass, and gently pressed till the softer parts are removed. Should any adhere to the edge of the object, it will be necessary to wash the specimen carefully in water, a process that will be much assisted by the delicate touches of a camel’s-hair brush. Place the object now and then under the microscope to see that all extraneous matter is removed, and when this is accomplished take the specimen up carefully with the camel’s-hair brush, or a lifter, and place it on a piece of very smooth paper (thick ivory note is the best for the purpose), arrange it carefully with the brush and a finely pointed needle, place a second piece of paper over it, and press it flat between two slips of glass, and compress it by a small spring clip (Fig. 264). A dozen clips may be had for a few pence. When _thoroughly_ dry (which it will probably be in about twenty-four hours, if in a warm room), separate the glasses, and gently unfold the paper; then, with a little careful manipulation, the object may be readily detached, and placed in a little spirit of turpentine, where it should be allowed to remain until rendered transparent and fit for mounting. The time during which it should remain in this liquid will depend on the structure; some objects, such as wings of flies, will be quickly permeated, while horny and dense objects require an immersion of a fortnight or even longer. A pomatum pot with a _concave_ bottom and well-fitting lid will answer admirably for conducting the soaking process in; and it is well, in preparing several specimens at a time, to have two pots, one for large and medium, the other for very small objects, otherwise the smaller will adhere to the larger.

In mounting objects in fluid, the glass cover should come nearly, but _not quite_, to the edge of the cell, a slight margin being left for the cement, which should project slightly over the edge of the cover, in order to secure it to the cell.

_Media for Preserving Algæ._--The most useful preservative media for algæ are chrome-alum, formalin, and camphor water. The solution should consist of one per cent. of chrome-alum and one per cent. of formalin; this will render the gelatinous sheath and matrix form clear, while it will retain the colour of the algæ in most cases. The Chlorophyceæ do well in any of these media; but other species, as _Ulva Lactuca_, are rendered somewhat brittle. For such use formalin alone. The Phæophyceæ should be placed while fresh in the formalin; the larger forms are better fixed by placing them for an hour or two in chrome-alum solution. The Florideæ do well in any of the three solutions, but the more delicate species, _Griffithsia_, require a two per cent. formalin solution in sea-water; the plant preserves its natural appearance in this medium.

To preserve and mount diatomaceæ in as nearly as possible a natural condition, they should be first well washed in distilled water and mounted in a medium composed of one part of spirits of wine to seven parts of distilled water. The siliceous coverings of the diatoms, however, which show various beautiful forms under the higher powers of the microscope, require more care in preparation. The guano, or infusorial earth containing them, should first be washed several times in water till the water is colourless, allowing sufficient time for precipitation between each washing. The deposit must then be put into a test tube and nitro-hydrochloric acid (equal parts of nitric and hydrochloric acids) added to it, when a violent effervescence will take place. When this has subsided, the whole should be subjected to heat, brought nearly to the boiling point for six or eight hours. The acid must now be carefully poured off, and the precipitate washed in a _large_ quantity of water, allowing some three or four hours between each washing, for the subsidence of some of the lighter forms. The sediment must be examined under the microscope with an inch object-glass, and the siliceous valves of the diatoms picked out with a coarse hair or bristle.

Dr. Rezner’s Mechanical Finger (Fig. 265) for selecting and arranging diatoms, adaptable to any microscope, is made to slip on to the objective far enough to have a firm bearing, and so that the bristle point can be brought into focus when depressed to its limit. It is clamped in its place by a small thumb-screw. The bristle holder slides into its place, and is carefully adjusted to the centre of the field. When using the finger, the bristle is first raised by means of the micrometer screw till so far within focus as to be nearly or quite invisible, then the objective is focussed on to the slide, and the desired object sought for and brought into the centre of the field; the bristle point is then lowered by the screw until it reaches the object, which usually adheres to it at once, and can then be examined by rotating the bristle wire by means of the milled head.

The medium used for mounting diatomaceæ is of considerable importance, inasmuch as their visibility is either diminished or much increased thereby. Professor Abbe, experimenting with the more minute test objects, diatoms, &c., found monobromide of naphthaline gave increased definition to most of them. This liquid is colourless, somewhat of an oleaginous nature, and is soluble in alcohol. Its density is 1·555, and refractive index 1·6. Its index of visibility is about twice that of Canada balsam.

Taking the refractive index of air as 1·0, and diatomaceous silex as 1·43, the visibility may be expressed by the _difference_ ·43.

The following table may be constructed :--

Refractive indices Visibility of silex
(taken approximately). (Refr. index = 1·43).
Water = 1·33 10
Canada balsam = 1·54 11
Bisulphide of carbon = 1·68 25
Sol. of sulphur in bisulph. = 1·75 32
" phosphorus " = 2·11 67

These data relating to visibility must be taken in connection with the numerical aperture of the objectives and of the illuminating pencil. The effect produced on diatoms is very remarkable, the markings on their siliceous frustules being visible under much lower powers.

So that the visibility of the diatom mounted in phosphorus as compared with balsam is as sixty-seven to eleven; in other words, the image is six times more visible. Mr. Stephenson’s phosphorus medium is composed of a solution of solid or stick phosphorous dissolved in bisulphide of carbon. Great care is required in preparing the solution owing to the very inflammable nature of the materials. So small a quantity of the bisulphide of carbon is required to dissolve the phosphorus that the diatom may be said to be mounted in nearly pure phosphorus. Remarkable enough, this medium has the reverse effect upon such test-objects as podura and lepisma scales. These lose their characteristic markings.

For mounting minute objects, carbolic acid solution will be found a useful medium--the purest crystals of carbolic acid dissolved in just sufficient water to render them fluid. No more should be dissolved than may be wanted for the time being, as if left standing exposed to the light it changes colour. Small crustacean foraminifera, the palates of moluscs, after boiling a short time in liquid potash and well washing to remove all traces of alkali, may be preserved in carbolic acid solution. Should the specimens appear cloudy gently warm the slide over a spirit lamp.

_Preserving and Killing Rotatoria with cilia in situ._--Mr. C. Rousselet’s method of preserving and mounting the Rotatoria[47] has been attended with so much success that the old difficulty attendant upon the preservation of these various beautiful forms of infusorial life has been practically overcome. The process resorted to consists of four stages, namely, narcotising, killing, fixing, and preserving. In dealing with rotifers hitherto, the difficulty has been that of successfully killing them with their rotating organs fully extended. It has been found needful to have recourse in the first instance to a narcotising agent, and one that acts slowly. The most suitable is a weak solution of the hydrochlorate of cocaine, a one per cent. solution, or even weaker. This was first proposed by Mr. Weber for keeping these active little bodies quiet while under observation. Mr. Rousselet carries this agent further; he applied it to narcotise them prior to killing, and this it does most effectually. The rotifers are seen to sink to the bottom of the live-cell, and the cilia gradually to slacken in motion, and the time for killing has arrived. This is effected by Flemming’s chromo-aceto-osmic acid. A rather weak solution must be employed--consisting of 1 per cent. solution of chromic acid, 15 parts; 2 per cent. osmic acid, 4 parts; glacial acetic acid, 1 part--which is at the same time a killing and fixing medium. The word “fixing” must not be taken to imply simply fixing, as it includes rapidly _killing_ and _hardening_ and preventing further change in the tissues of the rotifers by subsequent treatment, as mounting. The animal, therefore, must remain quietly for a few minutes, and then taken out and washed in five or six changes of distilled water, and hence transferred to the preservative fluid. All this must be effected with great care. The best preservative fluid is simply distilled water, rendered antiseptic by a trace of the fixing solution (about eight drops to an ounce of water) giving the slightest tinge of yellow to the solution. This slight tinge of colour is imparted to the rotifers, otherwise they remain transparent and unchanged, while the nervous tissue throughout the body is brought out to perfection.

Some slight difference in treatment is required by certain species, as that of _Asplanchna priodonta_; after the application of the cocaine solution, which should be added slowly, that is, by letting a few drops trickle down the side of the live-trough; this, being heavier than water, sinks to the bottom, thus narcotising the rotifers, and assisting to kill them with the cilia fully expanded. They should be left quietly for fifteen minutes, then thoroughly washed with distilled water. On further experimenting, Mr. Rousselet found that a weaker solution of osmic acid alone, 1/4 per cent., answers quite as well as, if not better than, Flemming’s fluid; even this must be allowed to act for only a very short time--a minute at most; the rotifers then remain white and transparent, excepting the ova, in which a fat-like substance, _lecithene_, is secreted. If they become too much stained, they may be decolourised by passing them through peroxide of hydrogen. For narcotising the following solution has been found most useful:--Take a 2 per cent. solution of cocaine hydrochlorate, 3 parts; methylated spirit of wood naphtha, 1 part; and distilled water, 6 parts. This must be added as before directed, drop by drop, watching the effect upon the rotifers under the microscope.

All the rotatoria may be killed and preserved in the same way. For mounting, Mr. Rousselet prefers a slightly _hollowed-out_ glass cell, the advantage of which is that the rotifers are kept to the centre, and cannot move to the edge. A little difficulty at first presents itself to exclude air-bubbles, but this, with a little care, can be overcome by placing a drop of a two or three per cent. solution of formalin, just sufficient to fill the cell. Then transfer the rotifers with a dipping pipette to the cell, and lower the cover-glass down very gently, removing any excess of fluid by blotting-paper. The best cement for the cover-glass is gold-size.

_Method of Cementing._--After many years’ experience, I have arrived at the conclusion that for cementing down the cover-glass there is nothing better than either gold size or gum dammar varnish. The latter, for some preparations, will be improved by the addition of a small proportion of indiarubber dissolved in naphtha. (See Appendix.)

Should glycerine be preferred, carefully wash away any surplus quantity by gently syringing; then apply a ring of waterproof cement round the cover-glass. An inexpensive one can be made by dissolving ten grains of gum-ammoniac in an ounce of acetic acid, and adding to this solution two drachms of Cox’s gelatine. This liquid flows easily from the brush and is waterproof, rendered more so if subsequently brushed over with a solution of ten grains of bichromate of potash in an ounce of water. An especial recommendation to this cement is its adhesiveness to glass, even should there be a little glycerine left behind on the cover. After the gelatine ring is thoroughly dry any kind of cement may be employed.

A useful cement for fixing minute objects, diatoms, &c., temporarily to thin glass covers, before permanently mounting them in Canada balsam, is made as follows:--Dissolve, without heat, two or three grains of gum arabic in one ounce of distilled water, then add glacial acetic acid, three minims, and the least trace of sugar. Filter carefully through filter paper, and repeat this in the course of three or four weeks. This cement will be unaffected by the balsam.

_Mounting Chara._--It is often found difficult to preserve and mount the fruit of chara, but this can be successfully accomplished in glycerine jelly, by taking the following precautions. After cleaning the specimen place it in 92 per cent. of alcohol for several hours, then transfer it to a mixture of equal parts of spirit and glycerine for several hours longer, pour off nearly all the mixture, and add pure glycerine at intervals till the glycerine becomes concentrated. The specimen is then mounted in glycerine jelly in a cell just deep enough to take it without pressure.

There are some objects much more difficult to prepare than others, and which tax the patience of the beginner in a manner which can hardly be imagined by any one who has never made the attempt. The structure of many creatures is so delicate as to require the very greatest care to prevent mutilation, and consequent spoliation, of the specimen. The beginner, therefore, must not be discouraged by a few failures in commencing, but should persevere in his attempts, and constant practice will soon teach him the best way of managing intricate and difficult objects. The room in which he operates should be free from dust, smoke, and intrusion, and everything used should be kept scrupulously clean, since a very small speck of dirt, which may be almost invisible to the naked eye, will assume unpleasant proportions under the microscope, and not only mar, but possibly spoil a fine and delicate preparation.

Few students on commencing to work with the microscope will fully realise the fact that under medium or high powers the natural appearance of almost all objects is changed by the refractive nature of the fluid medium in which they are immersed and which enters more or less into their composition. The remarkable changes effected by the law of diffusion, when alkaloid substances enter into their composition, show the necessity of taking every precaution in the employment of preservative fluids. Glycerine affords an example of the chemical change induced, should the preparation have been passed through an alkaline solution.

_Air Bubbles_ are a constant source of annoyance both in preparing and mounting. These may be removed from the specimen by gently warming the under part of the slide over a spirit lamp, or placing the slide in the warm chamber, when the bubbles will move towards the edge of the cover-glass and ultimately disappear. The air-pump is preferred by many microscopists.

Collection of Objects.

_Infusorial Life_, with all its fascinations, was fully unveiled to naturalists by the celebrated Ehrenberg. It was he who termed it infusorial, because he first met with the more interesting forms of minute life in infusions of hay and other vegetable substances. Since his day it is a well-known experience of those who take up the microscope that the most interesting objects to commence with are infusorial living creatures of sufficient dimensions to be easily understood and seen with moderate magnifying powers. Moreover, infusoria are more readily found in almost any pool or running stream of water, either near the surface or clinging to the under surfaces of aquatic plants. At one time all the small shallow pools in the neighbourhood of London--Hampstead Heath, Clapham, Wandsworth, and other commons--abounded in the most interesting forms of life, were famous hunting grounds for the marvellous volvox, the charming dismid and diatom, the wonderful budding and self-dividing hydra. A few hours’ ramble furnished the microscopist with a bountiful supply of these and many other forms of life. Now all is changed; our commons have been devoted to other purposes, and with the general _levelling_ up all the little pools have disappeared, and the microscopist has been warned off and driven further afield, or seeks the good offices of a country friend for an occasional peep into pond life.[48]

A teaspoonful, however, judiciously taken from a well-chosen locality will often be found to contain a variety of living forms, every one of which will deserve a careful and patient study.

Of the microscopic organisms, the collection of which requires no other methods than those ordinarily pursued by the naturalist, most of them must be sought for in pools or running waters, basking in the sunshine, clinging to leaves and rootlets of all aquatic plants; some freely moving about, others clinging to stones or pieces of wood at the bottom. Dismids congregate in shallow waters or rise to the surface in a quiet nook, while the diatomaceæ are seen covering the bottom of clear water, to which they give a yellowish-brown tinge of colour.

Infusorial animal life, as vorticellæ, stentors, rotifers, and various polyzoa, cling, as also do hydra, in colonies to vallisneria, duck-weed, frogbit, or small branches dipping down under water; and if some of the water-weed is brought home the little creatures will live and thrive for several weeks. No waters, however, are so full of minute animal life as the sphagnum bog. A number of species of diatoms, as well as protozoids and the smaller molluscs, will be found in all peat bogs. It is remarkable, too, that the same species, everywhere, are associated with this kind of moss. Lord Sidney Godolphin Osborn supplied his friends with moss growing in a damp part of the garden walk of his rectory; this always furnished the same species of rotifers. These proved to be most interesting objects to my friends, and in an early communication I described them as _indestructible_, since they will bear any amount of desiccation; nevertheless, they were revived when a drop of water was introduced into the glass-cell.

The Thames mud always furnishes a number of beautiful forms of triceratum. Lower down the river, as brackish water is reached, greater varieties of diatoms appear. But to secure them the collector must be provided with a collecting stick. A convenient form is furnished by Messrs. Baker (Fig. 266). This consists of an ordinary walking-stick, together with a lengthening rod, a cutting hook to clear away weeds, ringed bottles with screw tops, and a net with a glass tube attached. Their uses are too obvious to need further description.

The siliceous skeletons of diatoms are met with in the fossil state. Among the first discovered of the infusorial strata were the polishing slates of Bilin and Tripoli, the berg-mehl or mountain meal, the entire mass of which is composed of the siliceous skeletons of different species of diatoms. Richmond, Virginia, is rich in the same organisms, while the great mass of our chalk cliffs are composed of foraminiferous shells, xanthidiæ, &c. One remarkable fact in connection with fossil infusoria is that most of the forms are still found in the recent state. The beautiful engine-turned discs, _Coscinodisci_, so abundant in the Richmond earth, may be met with in our own seas, and in great profusion in the deposits of guano on the African and American coasts, and in the stomachs of the oyster, scallop, and other salt-water molluscous animals common to our shores.

A great number of infusorial earths may be mounted as dry objects, while others require careful washing and digesting in appropriate media. The finer portions of the sediments will be found to contain the better and more perfect siliceous shells.

Preparing and Mounting Apparatus.

1.--Ross’s instrument for cutting thin covering-glass for objects. This apparatus consists of a bent arm supporting the cutting portion of this apparatus, which consists of a vertical rod with a soft cork at one end. A brass arm at right angles carries the diamond parallel with and close to the main rod.

2.--Covering-glass measurer. To measure the thickness of covering-glass, place it between the brass plate and the steel bearing; the long end of the lever will then indicate the thickness on the scale, to 1/50-th, 1/100-th, or 1/1000-th inch.

3.--Brass table on folding legs, with lamp for mounting objects.

4.--Whirling table with eccentric adjustment for making cells and finishing off slides.

5.--Air-pump with glass receiver, 3-1/2-inch brass plate for mounting objects and withdrawing air-bubbles.

6.--Improved table with knife for cutting soft sections. This consists of an absolutely flat brass table, with a square hole to receive the wood, or other matter, on a movable screw, which adjusts the thickness of the section.

7.--Smith’s holder with spring and screw for adjusting pressure when mounting objects.

8.--Cutting diamonds for cell-making and cutting slips of glass.

9.--Writing diamonds for cutting thin covering-glass and naming objects.

10.--Page’s wooden forceps, for holding glass slips or objects when heated, during mounting.]