Section-Cutting--Staining
No method of examination can equal, for general applicability and
usefulness, that of section-work. The relations of the parts to each
other being preserved, it is possible to draw conclusions as to their
actual relations which no other mode allows of, and we shall devote
this concluding chapter to some account of the methods to be employed
to this end.
The apparatus required is not necessarily complicated. Reduced to its
elements, it consists essentially only of a razor to cut the sections
and a dish to receive them. It but seldom happens, however, that the
relations of the parts in sufficiently thin sections can be preserved
by such a rough-and-ready method, and frequently the object to be cut
is of such small dimensions as to render it impossible to deal with it
in this way. It is therefore necessary to “imbed” it, so as to obtain
a handle by which to hold it, in such a way that it shall be equally
supported in all directions. Moreover, since the human hand can only
in exceptional cases be brought to such a pitch of skill as to cut a
series of sections, or even single ones, of the needful delicacy, some
mechanical means of raising the object through a definite distance is
highly desirable. The writer has cut many thousands of sections with
the “free hand,” but the personal equation is a large one, and is not
always the same in the same person. For single sections the method
will, with practice, succeed very well, but some means of securing
a number of sections of more or less the same thickness is usually
required.
Let us deal with the imbedding first.
If it be desired to imbed a tissue which has merely been fixed
with formalin, the block should be immersed in strong gum (made by
saturating water with picked gum arabic, white and clean) for several
days. It is then taken out and, without draining, transferred to the
plate of a freezing microtome, and the sections cut from the frozen
block, and mounted in glycerine at once.
This plan is of limited usefulness, since it allows of very little
differentiation of the tissue elements, and that only optical.
To get the best results, some plan of staining must be adopted. Perhaps
the simplest, and certainly a very excellent one, is as follows. After
the tissue has been passed from the hardening, or fixing, fluid into
the successive alcohols, as described, it is placed in the following
solution. Take about forty grains of carmine and eighty grains of
borax, dissolve in about an ounce of water, add to the mixture an ounce
of methylated spirit, and let it stand for some time with frequent
shaking; about a week will be sufficient, and the process of solution
may be hastened by gentle warming at intervals. The clear upper portion
is then poured off, and into this the block of tissue is dropped, and
allowed to remain until thoroughly penetrated. Perhaps the best plan is
to substitute the carmine solution for the 50 per cent. alcohol, and
thus to make the staining a stage in the hardening process. From the
carmine solution the tissue is transferred to 70 per cent. alcohol, to
each ounce of which two drops of hydrochloric acid have been added, and
after remaining in it for a day (with a piece of the usual size) is
placed in 70 per cent. alcohol, in two successive quantities. Sections
from this material now only require treatment with the carbolic acid
and turpentine above mentioned to be fit for mounting and covering in
balsam. We now proceed to indicate how the sections may be cut.
A mixture of wax and almond oil, in proportions varying with the
heat of the weather, usually about equal ones, is prepared. The
piece of tissue is freed from superfluous spirit by being placed on
a bit of blotting-paper for a minute or two, and is then immersed in
a quantity of the wax-and-oil mixture contained in a little box of
paper or lead-foil. The tissue is held on the point of a needle, and
lifted up and down until it is coated with the mixture, and, before
solidification of the mass sets in, is lowered into the box and left
to cool. The block now furnishes a handle, and this should be wrapped
round with paper, the sections cut with the keenest possible razor,
and as thin as possible, and placed in spirit as cut. From the spirit,
which must be the strongest obtainable, they are placed in the clearing
liquid, carbolic and turpentine, and then slid on to the slide, a drop
of balsam placed on the section, and the cover over all. Of late years
all sections of ordinary soft tissues, animal or vegetable, have been
cut by one of the infiltration methods, in which the interstices of the
tissue are filled up by some material which prevents the relations of
the cells from being altered during the process of cutting. To enter
fully into this matter would occupy too much space, and would serve no
useful purpose, for the worker who requires to make use of such means
will find it indispensable to obtain Bolles Lee’s _Microtomist’s Vade
Mecum_, in which the whole matter is exhaustively treated.
The simple method above detailed will answer most ordinary purposes,
provided that a few precautions be attended to. The chief are as
follows. The outside of the block of tissue must be sufficiently dry
for the wax-and-oil to adhere to it. The razor must be extremely
sharp, and must be kept so by application to a Turkey stone during
the section-cutting. The blade must be drawn across the tissue from
heel to point, and kept wetted with spirit the whole time, so as to
prevent any dragging of the section. The transference of the section to
the slide must be effected by means of a section-lifter, which may be
made by beating out a piece of stout copper wire to a thin flat blade;
or a small palette-knife, or German-silver lifter, may be purchased
for a few pence. The carbolic turpentine is best used by placing a
little in a watch-glass, and floating the sections on to it by lifting
them singly with the lifter, freeing them from superfluous spirit
by draining on to blotting-paper, and allowing them to float on to
the surface of the liquid in the watch-glass, so that the spirit may
evaporate from above, and be replaced by the clearing agent from below
The balsam solution should be thin, and the cover-glass must be allowed
to settle down into place without pressure.
The question of staining sections is a very large one, and is becoming
of daily increasing complexity.
We cannot go into it here, further than to say that most sections cut
from unstained tissue will yield excellent results if stained first
with Delafield’s logwood solution (to be obtained at Baker’s) to a
very slight extent, and then with a solution of safranin. The sections
should be washed with tap-water after the logwood stain, and should be
of a pale violet colour. If over-stained, the colour may to a great
extent be removed by washing with a very weak solution of hydrochloric
acid, about two drops of acid to each ounce of water, and repeated
washing in tap-water to remove the acid, and restore the violet. The
safranin stain should be weak, and should be allowed to act for some
time. From this last the sections are transferred to strong spirit,
the latter being renewed until the sections cease to give up the red
dye; and they may then be mounted as described. The results with most
tissues are superb, every detail of the structure being splendidly
brought out. Safranin alone is also an admirable stain for many
purposes.
Further information must be sought in the book already mentioned.
Let us, in closing, warn the beginner of two things which are of
general application in practical microscopy. The first is, not to be
discouraged by failures. The manipulations are in many cases very
delicate, and premiums must be paid to experience for insurance against
failure in every one of the processes.
The second is, that the most scrupulous cleanliness will hardly suffice
to prevent contamination of preparation by the all-pervasive dust
which, invisible to the eye, assumes colossal proportions under the
microscope, and the particles of which have an unpleasant habit of
collecting on the most interesting or most beautiful portion of the
preparation. This can only be guarded against by careful filtration of
all fluids, and constant watchfulness.
A preparation properly made is a thing of beauty, and a joy for
ever,--or if not for ever, at any rate for many years; and one such
will repay an infinitude of pains taken in its production.
INDEX
PAGE
Air-pump, 174
Algæ, 78
" marine, 92
Anemones, sea, 159
Antennæ, 96
Ants, 97
Bacillaria, 87
Balancers of Fly, 112
Bark, 61
Blights, 89
Blood, circulation of, 129
" corpuscles of, 128
Bone, 123
Breathing-tubes, 109
Bull’s-eye, use of, 32
Camera lucida, 25
Canada Balsam, 170
Cartilage, 124
Cells, animal, 122
" circulation in, 40
" mounting dry in, 173
" pigment, 121, 165
" spiral, 46
" vegetable, 37
Ceramidia, 93
Chlorophyll, 40
Compressorium, Beck’s, 18
Condenser, bull’s-eye, 19
" substage, 19
Confervæ, 84
Conjugation, 82, 84
Convex lenses, 7
" foci of, 7, 8
" image formed by, 10
" virtual image, 11
Corallines, 164
Corrosive sublimate, 170
Cover-glasses, 18
Desmids, 81
Diatoms, 85
" preparation of, 175
Dipping-tubes, 22
Dissection, 20
" instruments, 21
" under microscope, 24
Drawing, 25
" squares, 26
Echinoderms, 162
Entomostraca, 152
Epidermis, animal, 122
" vegetable, 68
Extemporised apparatus, 5
Feathers, 119
Fish, scales of, 118
" parasites of, 153
Fixation of cell-forms, 171
Focus of mirror, 29
Foraminifera, 156
Formalin, 164
Frog-plate, 129
Gills of mussel, 122
Gizzard of insects, 109
Glycerine-gelatine, 172
" jelly, 171
Hairs, animal, 116
" vegetable, 53
Heads of Insects, 104
Illumination, correct, 31, 32
" dark-field, 34
" for opaque objects, 33
Imbedding, 180
" by infiltration, 182
Infusoria, 135
Injection, 164
Insects, 97
" mounting of, 177
Jelly-fish, 158
Larva of _Chironomus_, 152
Light, arrangement of, 29
Live-box, 17
Logwood solution, 183
Magnification, to measure, 27
Mare’s tail, 91
Marine life, 155
Microscope, Baker’s, 14
" " “portable”, 15
" primitive, 5
" simple, 12, 13
Mildew, 89
Mirror, concave, 29
Mollusca, 161
Mounting, 168
" dry, 173
" foraminifera, 157
Mosses, 96
Muscle, 127
Nails, 124
Needles, 22
Nerve, 127
Net, 133
Nucleus, 40
Objectives, 16
Objects, drawing of, 24
" photography of, 36
Oil-cells, 58, 61
Oscillatoriæ, 84
Osmic acid, 170
Parasites, 153
Petals, 69
Picric acid, 170
Pigment, 121
Pocket magnifiers, 13
Polariscope, 166
Pollen, 71
Polyzoa, 147
Pond-hunting, 132
Preservatives, 169
Radiolaria, 157
Rotifers, 147
Safranin stain, 183
Sap, 128
Scent-glands, 57
Sea-weeds, 92
Section-cutting, 178
Seeds, 75
Skin, 120
Spiracles, 102
Sponge, fresh-water, 135
" spicules, 155
Sporangia, 92
Stage-forceps, 116
Starch, 63
" mounting, 172
Stomata, 49
Suckers, 108
Teeth, 125
Troughs, glass, 18
Water-bears, 152
Wings, 110
Wool, 116
Worms, fresh-water, 14
" marine, 160
Yeast, 89
Zoœa, 161
Zoophytes, 157
Zygnemaceæ, 85
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End of Project Gutenberg's Common Objects of the Microscope, by J. G. Wood