SECTION MOUNTING.
1. By *flotation*.
In this method the section whether stained or unstained is placed in a
bowl of water, or normal salt solution (p. 53). A clean slide is then
introduced into the water at an angle of about 60°, a little more than
half of its length being submerged. The section is then brought up by
the needle and floated as far as possible into position on the slide.
One corner is then fixed by the needle, and on gently withdrawing the
slide the section should lie flat. If any folds are left no attempt
should be made to smoothen them out with a needle, but the slide should
be re-immersed until the folded part of the section is under water. It
should then be gently withdrawn, when the fold will disappear. This
manœuvre must be repeated in different directions until the section
lies quite smoothly on the slide. Stained and unstained sections are
floated out in this way before being mounted in Farrant’s medium, and
unstained sections previous to staining in picrocarmine.
2. By *transference* with a *section lifter*.
This method is employed in mounting in Canada balsam in order to
transfer the section from the clarifying agent (p. 63) to the slide.
The lifter is polished, and insinuated under the section. The section
being held in position by the needle is now raised from the fluid,
excess of which is removed by holding the section in position with a
mounted needle, and tilting the lifter so as to allow it to drain off.
*Removal of air bubbles from sections.*--When sections contain many
air bubbles, the best plan is to leave them in methylated spirit for a
time. The bubbles then coalesce and escape from the section.
For delicate structures and for fresh sections the transference to
spirit, and the subsequent flying out of the section when returned to
water are risky, and the best method of treating these is to put the
vessel containing them under the receiver of an air pump, if one is
available, and slightly exhausting the air.
The most frequent cause of air bubbles in mounted specimens, however,
is the employment of cover-glasses which have not been thoroughly
cleansed. Proper cleansing is best effected by placing the covers when
bought in a shallow wide mouthed stoppered bottle containing strong
nitric acid, and leaving them in this fluid for twenty-four hours. The
acid should then be drained off and water run through the vessel from
a tap, until the washings no longer give an acid reaction with litmus
paper. The water should then be drained off, and the glasses covered
with absolute alcohol. They can be removed one by one and rapidly dried
as required. With cover-glasses properly cleansed in this manner, not
only will air bubbles be avoided, but the covers will be dried much
more easily with the cloth, and fewer will be broken in the process.
Another very frequent cause is the transference of air bubbles with the
mounting medium on the glass rod. This occurs especially if the rod be
fused to the stopper. The proper bottles to use, both for Farrant’s
medium and balsam are “balsam bottles” which have no stopper, but the
mouth is closed by a glass cap which fits accurately (fig. 9). A short
glass rod is attached to the cap, and is used to transfer the medium to
the slide.
[Illustration: Fig. 9.--Balsam bottle.]
*Treatment of folded sections.*--The folding may be due:--
(1.) To the section having creased through being cut with a knife whose
surface was not perfectly smooth. This is best remedied by placing the
section in methylated spirit for a minute, and then transferring it to
a bowl of clean water, when the section will rapidly rise to the top,
and spread itself out flat on the surface of the water, in consequence
of the alcohol rapidly diffusing out at the edges into the surrounding
water.
(2) To the section containing a large amount of *fat*, as in those of
the skin and subcutaneous tissue. The fat may be removed from the fat
cells without materially altering the appearance of the section. This
is done by dehydrating the section in alcohol, and then transferring
to a watch glass containing ether or chloroform to extract the fat.
The tissue should be washed free from ether in the alcohol and then
transferred to the bowl of water, and allowed to float out. This
process does not interfere with subsequent staining operations.
MOUNTING MEDIA.
*Farrant’s solution*:--
Gum Arabic (picked, colourless) }
Glycerine } equal parts.
Water }
In making this solution the best gum arabic must be used, and only the
clearest pieces of this. “Powdered gum acacia” should be avoided, as
though it looks white it often yields a brown mucilage, and besides is
frequently adulterated with starch, &c.
The glycerine and water should be mixed and the gum arabic added.
The mixture should be allowed to stand for some weeks, with frequent
stirring until the whole of the gum is dissolved. Then allow it to
stand for a week or two longer in order that the dirt may subside,
and the bubbles rise to the top. The scum should be removed and the
clear fluid decanted from the sediment into a “Balsam bottle” (p. 58)
containing a few drops of a saturated solution of arseniate of sodium
and a small lump of camphor.
If properly made it is an extremely useful mounting reagent. It does
not clarify the tissues too much, and in consequence of its containing
gum it dries at the edges and cements the cover-glass more or less
firmly in a week or two. If this is not the case the medium contains
too much glycerine and more gum must be added to compensate for this.
This drying at the edge prevents any further evaporation while the
glycerine keeps the section permanently moist.
The camphor and arseniate of sodium prevent the formation of fungi.
Sections preserve their original appearance in this medium for many
years. After a long time they are apt to become a little cloudy and
granular.
Unstained sections should always be mounted in Farrant’s medium, as the
Canada balsam process renders them quite transparent. It is suitable
for almost any tissue stained or unstained, but sections of the nervous
centres require to be mounted in Canada balsam, owing to the opacity of
myelin when mounted in glycerine.
*Canada balsam solution*:--The medium is made thus:--
The ordinary Canada balsam which is of a treacly consistence is heated
gently in a water bath for some hours, to drive off turpentine and
other volatile oils. It is then allowed to cool to a yellow vitreous
mass. Take of
Dried Canada balsam } equal parts.
Xylol }
Leave till dissolved, stirring occasionally.
Unless the solution be perfectly clear, it must be filtered through a
very thin paper, previously wetted with xylol. If the medium be too
thick more xylol should be added, if too thin, the xylol should be
allowed to evaporate until the medium is of the consistence of a thin
syrup.
If the medium is made too thin much annoyance will be caused by its
evaporating at the edge of the cover-glass, leaving an air-space, which
will increase daily until the section is left quite dry. This should be
remedied by putting another drop of balsam at the edge of the coverslip
and allowing it to run in and displace the air. A ring of cement should
be put on as early as possible afterwards.
The bottle in which the balsam is preserved must be very carefully
dried before being filled and then rinsed out with absolute alcohol,
and afterwards with xylol. Turpentine or benzol are often used instead
of xylol in the preparation of the medium, and in the same proportion,
but the latter is less apt to dissolve out the aniline colours from the
sections.
To mount sections in Canada balsam they must be transferred first to
a watch glass containing absolute alcohol or an alcoholic solution
of some staining reagent, _e.g._, eosine (p. 72) and left in it, no
attempt being made to spread it out, until it is perfectly dehydrated,
_i.e._, in about two minutes. It should then be transferred to the
clarifying oil on a mounted needle, or on a section lifter, which must
be perfectly dry as any spot of moisture that gets on to the section
will resist the clarifying action of the oil, and will cause unsightly
opaque areas when the section is mounted. Even breathing on the section
on its way to the clarifying agent will prevent uniformity of clearing.
Should white spots appear in the section while in the oil it must be
taken out with as little oil as possible, and again dehydrated in
absolute alcohol.
The process of *clarifying* must be performed in some medium in which
Canada balsam is readily soluble, and which is also readily miscible
with alcohol. Those most frequently employed are oil of cloves, xylol,
oil of bergamot, oil of cedar, and origanum oil. The first named has
always been much used because of its agreeable odour, its cheapness,
and the ease with which it can be obtained. But it has the disadvantage
of dissolving out many important staining reagents, especially eosine
and the various aniline colours. In addition as it dissolves celloidin,
sections cut in this medium tend to fall to pieces when transferred to
oil of cloves, and one of the other oils (which have no solvent action
on celloidin) should always be employed with celloidin sections. Oil
of bergamot is the most generally useful, but rather expensive. Where
there are special reasons for employing other dehydrating agents, they
will be indicated in the special directions for particular staining
methods in Chapters VI. and VII.
As soon as the section is plunged into the oil, the alcohol rapidly
diffuses out, so that the edges of the section fly out with it, and
the section floats quite flat on the surface of the oil. When it is
completely clarified (in about a minute), as shown by its sinking in
the oil, it should be transferred to the slide by the section lifter,
and the oil drained off. Excess of oil may be removed by pressing
gently on the section with a flat piece of filter paper folded several
times. If carefully performed this manœuvre will not injure the
section, but it requires practice.
If the tissue is very delicate, and likely to be injured by changing
from one vessel to another, or if it is larger than the section lifter
will conveniently carry, it should be floated out on a glass slide,
and, as much water as possible having been removed by blotting paper,
should be dehydrated by adding a little alcohol from a pipette once or
twice. Most of the alcohol should then be removed by tilting the slide,
and before the remainder has evaporated, some oil of cloves or bergamot
should be added from another pipette. The section will float on the oil
at first, but the latter will gradually come through and appear on the
top of the section. When this occurs the clarification is complete, and
the oil may be run off by tilting the slide and the section mounted in
Canada balsam.
*Cementing of cover-glasses.*--The cover-glasses may be cemented down
to prevent their shifting and spoiling the specimen. If the cover-glass
be circular, a Shadbolt’s turntable should be used. It consists
simply of a horizontal heavy brass disc, rotating easily on a pivot.
There are a number of circles traced on the disc concentrically. The
slide is then fixed on the disc by means of the clips, so that the
circumference of the cover-glass corresponds to one of the circles.
The disc is then rotated and the cement applied to the edge of the
cover-glass with a brush.
Many materials are employed. The most suitable are:--(1) Canada balsam,
which is almost colourless and transparent and looks very neat. (2)
Gold size. (3) Marine glue.
When these are dry a finished appearance may be given to the slide by
laying on a ring of zinc white. This is made as follows:--
Oxide of zinc 1/2 drachm.
Benzole } half an ounce
Gum dammar } of each.
*Preservation of sections.*--They should be kept _flat_, and preserved
from both light and dust. Very useful cardboard trays are now sold by
almost all dealers in boxes made to contain twenty-four dozen slides
for about eight shillings, or suitable cabinets may be constructed by a
carpenter.