Hints on the Preparation of Objects--Preservative Fluids--Mounting
Media--Treatment of Special Objects.
The microscopist who relies altogether on the dealer for his permanent
preparations may expend a good deal of money, but the satisfaction
which he derives from his hobby will be very inferior to that
experienced by the worker who endeavours to secure, for exhibition or
for reference, specimens of the objects which he finds most interesting
and instructive to himself.
It will be our endeavour in the following pages to give a summary of
the elementary principles upon which reliance is to be placed, though
it must be clearly understood that the technique of the subject,
already occupying a vast amount of literature, is extending day by day,
so that it is impossible to deal exhaustively even with one single
section of it. Reference must be made, for further information, to
such publications as the _Journal of the Royal Microscopical Society_,
or that of the Quekett Club, or to the monographs on the various
departments. Davies’ work on the general subject will also be found
useful by the beginner.
Taking first the question of reagents, we may mention five which leave
the cells of a tissue as nearly as possible in the natural condition,
but fit for permanent preservation. The first of these, in order of
importance and of general applicability, is alcohol, represented for
most purposes by methylated spirit, which contains about 84 per cent.
of absolute alcohol, though, unfortunately for our purpose, there is
a certain quantity of mineral naphtha in it in addition. This last
has the effect of making it go milky upon dilution with water, which
is a considerable disadvantage, though the milkiness disappears to
some extent on standing, and it is rarely worth the while of the
ordinary microscopist to go through the formalities necessary to obtain
permission to purchase unmineralised spirit, which cannot be had in
quantities of less than five gallons (as it is only to be had from the
distillers under an Excise permit), and distillers may not supply less.
Four parts of methylated spirit with one of water forms the classical
“70 per cent.” alcohol, the most generally useful of all fluids for
hardening and preserving purposes. A considerable quantity of this
fluid should always be available.
Whatever other fluid may be used to begin with, spirit must almost
always be used to finish the process, and fit the tissue for
section-cutting and staining.
Of purely preservative, or fixative, fluids, we may mention “formalin,”
a 40 per cent. solution of formic aldehyde, which is rapidly coming to
the front, as indeed it deserves to do. It is but slightly poisonous,
if at all, and leaves in the tissue nothing which requires subsequent
removal before proceeding to harden for section-work, whilst it is an
admirable preservative of cell-form.
Another admirable but highly poisonous reagent is corrosive sublimate,
in saturated solution, with 2 per cent. of acetic acid.
A fourth is osmic acid, used in 1 per cent. solution. This is a highly
valuable reagent, but extremely expensive, very poisonous, and giving
off fumes which are most irritating to the eyes.
The fifth, a very gentle, but in many respects very satisfactory one,
is picric acid in saturated solution. Tissues preserved in this medium
must not be washed out with water, as it enters into very feeble
combination with protoplasm, and the cells swell and disintegrate as
the reagent is dissolved out.
Of mounting media we may mention glycerine, glycerine jelly (made
by dissolving starch in glycerine with the aid of heat), and Canada
balsam, dissolved in xylol or benzole. The Canada balsam must be dried
hard by evaporation over a water-bath, and dissolved as wanted. Under
no circumstances should raw balsam be used, as it takes years to set
hard, and turns of a deep yellow colour in the process.
Chloroform is frequently used as a solvent, but it has the disadvantage
of attacking and extracting a large number of the aniline dyes used for
staining structures, an objection from which the mineral solvents are
free.
We will now proceed to go through the objects already referred to, and
indicate the method of preservation.
For the study of the cell-structures of plants the portion to be
examined is to be placed in spirit of about 30 per cent. strength,
which is changed after twenty-four hours for 40 per cent., after a
further twenty-four hours for 55 per cent., and finally, as regards
our present purpose, in 70 per cent. spirit, in which it may remain
until required for section-cutting. The effect of this treatment is
to extract the bulk of the water from the tissue, with the minimum of
shrinkage of the cells, the latter being preserved in their natural
relations to surrounding parts.
In some cases, however, it is desirable to examine and preserve
delicate structures, or parts, or dissections, in a medium which allows
of the retention of the greater part of the natural moisture, and in
such a case the tissue is immersed in glycerine diluted very much in
the same way as the alcohol in the last process, but with very much
longer intervals between the alterations of strength, until it reaches
pure glycerine, in which it remains for a considerable time, as the
exchange between the tissue and the dense fluid surrounding it goes on
very slowly.
A combination of the two methods is also possible, the spirit-hardening
being carried out for a portion of the time, and the tissue being
thereafter transferred to glycerine, diluted or pure.
The object of using glycerine at all is merely that it has a much
lower refractive index than balsam, so that delicate structures may
sometimes be better seen in the former medium, but balsam is to be
preferred wherever it is possible to use it, _i.e._ almost always.
The writer has not mounted a preparation in glycerine or a medium
containing it for many years, nor, with proper staining, does he think
it can ever be necessary to do so, except in the case of dissections
in which the glycerine can be slowly run in without disturbing the
arrangement, as spirit would be pretty sure to do. The harder portions
of plants, woody stems, shells of fruit, or the like, require different
treatment, and must, as a rule, be allowed to dry thoroughly before
being cut.
Starch granules are somewhat troublesome to mount satisfactorily.
The writer has tried many methods, and, on the whole, prefers a
glycerin-gelatin medium, which keeps for an almost indefinite time, and
may be made as follows: Thirty grains of gelatine (Nelson’s “brilliant”
or other transparent gelatine is to be preferred) are allowed to soak
in water, and the swollen gelatine is drained, and dissolved in the
water which it has absorbed, by the aid of a gentle heat. An equal
bulk of pure glycerine is then added. In using, a small portion is
transferred to a slide with the point of a knife and melted, a small
quantity of starch granules added, and stirred into it with a needle.
The cover-glass is then laid up on the still-fluid drop, pressed gently
down so that the drop is extended to the margin of the cover, and the
whole allowed to cool. It is then to be painted round with several
layers of Brunswick black, or Hollis’s glue, or zinc-white cement, to
prevent evaporation,--Hollis’s glue being perhaps the best medium for
the purpose.
Petals, or other parts of which it is desired to obtain a surface view,
must be mounted in cells, which may be made by the use of button-moulds
of suitable size, cemented to the glass slide with marine glue. The
slide must be free from grease, as the tissue must be fixed in position
by the use of gum, and allowed to dry thoroughly before closing the
cell, or the cover-glass will be bedewed with moisture when the cell
is closed. The best plan is, after air-drying for a couple of days,
to place the preparation on a metal plate over a beaker of boiling
water for an hour or more, and then to close the cell immediately with
Brunswick black, maintaining the heat at first to ensure rapid drying,
and then slowly withdrawing it. When cool, another coat should be
given, and rather thick covers should be used, as these preparations
are never required to be examined with high powers.
To mount pollen-grains, they should be sprinkled upon the surface of a
slide which has been previously moistened with thin gum, and allowed
to dry until it has become just “tacky”; the drying is then completed
by gentle heat and a drop of balsam placed upon the grains, with a
cover-glass over all. Bubbles will probably form, but with Canada
balsam this is not of the slightest importance, as they always come
out of their own accord, and balsam mounts should never be closed with
cement of any kind until thoroughly dry.
Air-bubbles in other media may be eliminated by the use of the
air-pump shown in Fig. 16, which may be obtained from Baker at a very
reasonable rate, and which is useful not only for that purpose, but for
accelerating the drying of moist tissues. To do this, there is placed
upon the plate of the pump a porcelain dish containing strong sulphuric
acid, and upon this is placed a little triangle of platinum wire, which
serves to support the preparation. The air is now exhausted; the tissue
parts with moisture to supply its place, and this moisture is in turn
greedily absorbed by the sulphuric acid, so that drying is rapid and
continuous, as well as very thorough, whilst the process has the great
advantage of dispensing entirely with the use of heat.
[Illustration: Fig. 16.]
Portions of many of the delicate algæ may be mounted in glycerine,
having previously been soaked in it as already described; whilst the
unicellular forms, such as desmids and diatoms, may be preserved in
almost exactly the natural condition by simply mounting them in a
saturated solution of picric acid.
Probably formalin, in a solution of 10 per cent. strength, would answer
the purpose equally well, but the writer has not tried it. It is
hardly necessary to say that, with such extremely fluid media, great
care is required in closing the cell. A thin layer of Hollis’s glue
should be first painted on, to secure the cover in position, and when
this is thoroughly dry, several successive layers must be added in the
same way.
It may be said here, that it is advisable in all cases to use circular
cover-glasses, as far as possible, as they lend themselves with great
facility to a mechanically accurate closure. This slide is placed
upon a turn-table, carefully adjusted until the cover is seen to be
central when rotated, and a brush, preferably a small camel-hair
pencil, charged with the desired fluid, but not in large excess, is
held against the junction of the slide and cover, whilst the table
is rapidly spun. A little experience will teach better than any
description what amount of fluid there should be in the brush, and how
thick the cement should be. If too thick, it will drag off the cover;
if too thin, it will flow over the latter and over the slide.
The preparation of diatom-skeletons as permanent objects is easy.
Consisting, as they do, of pure silex, or flint,--_i.e._, practically
glass,--they resist long boiling in acids, so that there is little
difficulty in isolating them from any organic matter with which they
are mingled. It is generally recommended to treat them with strong
nitric acid. This is a mistake. The acid acts much more powerfully
and less violently when diluted with an equal bulk of water, and it
is in an acid so diluted that portions of water-plants, or other
diatomaceous material, should be boiled in a glass beaker until all the
organic matter is dissolved. The beaker should be covered with a glass
plate, to prevent dissipation of the acid fumes. When the process is
complete, usually in about half an hour, the contents of the beaker are
thoroughly stirred with a glass rod, poured rapidly off into a larger
bulk of cold water, and allowed to settle for another half-hour. The
process is then repeated with a smaller bulk of water, several times,
to allow the removal of the last traces of acid, and finally with
distilled water. The separation of the diatoms into grades is effected
by settlement. The final result is poured into a tall glass vessel,
and allowed to settle for, at first, a minute, the supernatant fluid
again poured off, and allowed to settle for two minutes, and so on, the
period being gradually increased, and each sediment preserved apart.
The first will probably only be sand, but the proportion of diatoms
will increase with each separation, though there will always be a
certain proportion of sand of such a size as to settle at the same rate
as the diatoms. Marine plants especially will furnish a rich harvest by
treatment as described.
Solid diatomaceous deposits, such as kiesel-guhr, mountain-meal, and
especially the famous Oamaru deposit from New Zealand, demand different
treatment, and perhaps the best way is to disintegrate the mass, either
by boiling with Sunlight soap (though the alkali attacks the flint to
some extent) or to mix the mass with a super-saturated solution of
acetate of soda (made by saturating water with the crystals whilst
boiling), and by successive coolings, heatings, and stirrings to cause
the process of crystallisation to break up the mass, which it will do
very thoroughly. The diatoms are then separated by sedimentation, as
above described.
A small portion of the deposit may now be spread thinly on a glass
slide, allowed to dry thoroughly, be treated with balsam, and covered.
If it be desired to select individual diatoms, this must be done under
the microscope, by means of a bristle fixed in a handle either with
glue or sealing-wax. The diatom selected will adhere to the bristle
if the latter be slightly greasy, and should then be transferred to
a slightly adhesive slide, coated either with thin solution of white
shellac, or with thin gum nearly dry. When the forms desired are
mounted, the preparation should be covered in balsam. The process is by
no means as easily effected as described, however.
The preparation of insects, or parts of insects, as microscopic objects
is a tedious and difficult task. The main point is the trouble of
softening the integument and eliminating the colour.
The latter can, in any case, be only partially effected. The beginner
would do well to begin with a fairly easy form, such as the worker-ant.
A good supply of these insects may be placed in a bottle of liquor
potassæ, and left there for at least some days until they begin to
become clear and limp. From time to time a specimen may be taken,
well washed with several waters, then with acetic acid and water of
a strength of about 10 per cent., then with weak spirit, about 50 per
cent. An attempt may then be made to arrange the insect upon a slide,
spreading out the legs so as to exhibit them to the best advantage, and
when this has been done a cover-glass may be put on, supported in such
a way as to prevent absolute pressure. The spirit is then withdrawn
by means of a piece of filtering-paper cut to a point, and strong
spirit added. This is again succeeded by absolute alcohol, then by a
mixture of turpentine and crystal carbolic acid in equal proportions,
and finally the cover-glass is carefully lifted, and some thick balsam
solution dropped on, the limbs finally arranged by means of warm
needles, and the cover-glass carefully replaced and pressed gently down
by means of a clip, which may be obtained for a few pence. The whole
is then set aside to harden, the deficiency caused by evaporation made
good, the balsam allowed to dry, and the preparation finally painted
round.
The contents of the body, in large insects, must be removed, and this
is effected during the washing in water by gentle pressure with a
camel-hair brush, the process being aided, if necessary, by a small
incision made through the integument at the root of the tail. Sections
of insects require very special methods, which will hardly fall within
the scope of this work.