Transcriber’s notes:
Apart from the following corrected misspellings the text of this book
has been preserved as in the original:
xolol —> xylol
side —> slide
overstraining —> overstaining
Where appropriate, illustrations and footnotes have been positioned
adjacent to the relevant text.
SECTION CUTTING AND STAINING
A PRACTICAL INTRODUCTION TO HISTOLOGICAL METHODS FOR STUDENTS AND
PRACTITIONERS
BY W. S. COLMAN, M.D., M.R.C.P.
ASSISTANT PHYSICIAN (FORMERLY PATHOLOGIST) TO THE NATIONAL HOSPITAL FOR
THE PARALYSED AND EPILEPTIC; AND TO THE HOSPITAL FOR SICK CHILDREN,
GREAT ORMOND STREET, ETC.
SECOND EDITION
_ENLARGED AND IN MOST PART RE-WRITTEN_
LONDON H. K. LEWIS, 136 GOWER STREET, W.C. 1896
PRINTED BY
H. K. LEWIS, 136 GOWER STREET,
LONDON, W.C.
PREFACE TO THE SECOND EDITION.
In preparing this edition I have endeavoured to meet the requirements
of students, and of practitioners who desire to keep up their
histological work. Those methods are selected which have been found to
work well in practice, and it has been thought better to describe a few
in detail rather than give a short account of many similar methods.
I have again to express my obligation to the various instrument makers
for the illustrations of microtomes, &c.; to Dr. Fearnley, of Bradford,
for the description of his method for injecting blood vessels, and to
Messrs. Macmillan and Co. for permission to copy figures 10 and 11.
W. S. COLMAN.
Wimpole Street, W.
_Sept., 1896._
CONTENTS. PAGE
CHAPTER I.
Apparatus Required 1
CHAPTER II.
Hardening Processes 15
CHAPTER III.
Section Cutting 29
CHAPTER IV.
Section Mounting 55
CHAPTER V.
General Staining Methods 67
CHAPTER VI.
Special Methods for Staining the Nerve Centres 87
CHAPTER VII.
Special Methods for Staining Micro-Organisms and Blood 103
CHAPTER VIII.
Injection of Blood Vessels 120
CHAPTER IX.
Directions for Preparing Individual Tissues 129
Index 153
SECTION CUTTING AND STAINING.
CHAPTER I.
APPARATUS REQUIRED.
Probably there is nothing more perplexing to a beginner than to decide
what apparatus is required. If he consult a price list, it is difficult
for him to tell which articles will be necessary, and which will be
either luxuries, or required only for special investigation.
In the following account of requisites, those only will be described
which it is useful to have always at hand. They will be found
sufficient for ordinary work, but for special investigations a more
elaborate equipment will be required.
All staining and other reagents should be made as far as possible
by the worker himself, according to the directions given in later
chapters. This should at any rate be done at first, as the knowledge
thus gained will prove invaluable. It will also effect a great saving
if articles that are used in any quantity, such as methylated spirit,
distilled water, &c., are bought by the gallon, and not in small
quantities.
Almost all the processes described here can be carried out without the
use of a fully equipped laboratory, in fact, in an ordinary room. The
only furniture required is a firm table, and a cupboard and shelves for
storing reagents.
The following should also be procured:--
*Jars* or *bottles*, with well fitting stoppers or corks, to contain
the tissues while being hardened. They should not hold less than two
ounces. Empty drug bottles which can usually be obtained from druggists
for a few pence, serve very well.
Smaller bottles should also be procured for keeping specimens in spirit
after they have been hardened until one is ready to cut sections. After
sections have been cut from a portion of the specimen, the rest should
be preserved, in case it is wanted for further investigation. Each
specimen must be labelled, with a name or a number corresponding to a
reference in the note-book, and a large number of specimens may then
be kept in the same jar. The best way to label them is to write the
name or number on a piece of vegetable parchment in ordinary “marking
ink,” and warm it until the writing is black. The little label should
then be fixed to a corner of the piece of tissue with a stitch or by a
fine pin, and it may be identified years afterwards. The importance of
keeping tissues, sections, slides, &c., *distinctly labelled* cannot
be too strongly impressed on the beginner. The name, date, and other
particulars should be invariably written on the label at the time.
At first the student will be inclined to neglect this, as he will
recognize his pieces of tissue and sections so readily merely by their
shape and general appearance. But as time elapses and similar specimens
accumulate, he will find it most difficult or even impossible to
identify one from the other.
A number of 1 oz. and 2 oz. *stoppered bottles* for staining reagents.
The stopper of these should be fitted with a rod. This is done by
simply heating the lower end of the stopper and the upper end of a
piece of glass rod of suitable length in a blow-pipe, until they are
plastic, and then pressing them together.
*Watch glasses.*--At least a dozen watch-glasses, in which to perform
the operations of staining, clarifying, &c. Those with a flat bottom
should be employed as they are less easily upset than the others.
Plenty of *filter papers*.
Both coarse ones, for use in the manufacture of reagents, and small
fine white ones (2-1/2 inch) for filtering the staining fluids
immediately before using them, should be procured. Before using them
a few drops of alcohol or distilled water should be placed in them to
saturate the paper. This not only allows the fluid to pass through
more rapidly, but prevents a portion of it being wasted through being
absorbed by the pores of the paper.
Several *needles* mounted in handles.
They must be kept very bright and smooth, and care must be taken that
the point does not get turned up.
A large and small *funnel*.
Several *pipettes* consisting of pieces of glass tube with an internal
diameter of 1/8″ and about ten inches long, drawn out almost to a point
at one end.
*Section lifter.*--This instrument is required for transferring
sections from one reagent to another, or from oil of cloves, &c., to
the slide. The most convenient form is Woodhead’s, made of thin sheet
copper, which allows the blade to be bent at any angle to the stem. The
stem or handle is about six inches long, and continuous with, and at an
angle to it, a flat blade about 3/4 in. square with the corners rounded
off. Larger ones can be obtained for mounting sections of large size,
_e.g._, kidney, medulla oblongata, &c. The surface of the blade should
be brightly polished, and kept scrupulously clean.
Ordinary dissecting *forceps*.
One or two *scalpels*.
A pair of fine *scissors*.
A *razor* or other instrument for cutting sections.
A smooth *oil stone* for keeping the razors and knives properly
sharpened.
A *spirit lamp* for warming the staining fluids.
A few *test tubes*.
A *minim measure*.
*Scales* and small weights.
A gross of ground glass slides 3 x 1 in.
Half a gross of ground glass slides 3 x 1-1/2 in.
Half an ounce of thinnest coverslips, 7/8 in. diameter.
Quarter of an ounce of thinnest coverslips, 1-1/4 in. diameter.
*Microscope.*--This is not the place for a description of the
microscope as an optical instrument, but some hints as to the selection
of one may be found useful.
Showy microscopes with much brass work should be avoided, simplicity of
construction being a great recommendation. The microscope should have
a large heavy base, either of the horse-shoe or tripod pattern, large
enough to afford a firm base when the microscope is tilted.
Mechanical stages are unnecessary and they add greatly to the expense,
and very little to the utility of the instrument for ordinary
histological work. Binocular arrangements also are of little use for
this purpose.
The microscope should be provided with a *coarse and fine adjustment*,
which should be most carefully tested before purchasing the instrument.
They should work freely and smoothly, and the slightest turn in either
direction should at once alter the focus.
There should be a *reversible mirror*, one side being concave and the
other plane. The concave surface is the one usually employed, the
plane surface being chiefly used in conjunction with the sub-stage
condenser for the examination of micro-organisms. There should be an
*eye-piece* of moderate magnifying power. Very powerful eye-pieces
do not reveal additional details, but merely enlarge the image, and
with it any defects that may be produced there by faults in the
objective. Eye-pieces II. and IV. of most makers will be ample for most
requirements.
*Objectives.*--These are the most important parts of the microscope,
and the student will be well advised if he spends a little extra money
to secure good lenses.
Most objectives and stands are now made with a universal thread, so
that any objective will fit any make of stand. Many workers provide
themselves with a cheap stand such as that supplied by Leitz, and then
fit it with lenses by Zeiss, or other first class maker.
The most useful lenses are the 1 in. low power lens, and 1/5 in. or
1/6 in. high power, or No. 3 and No. 7 of Continental makers, or
Zeiss’s A and D. A 1/2 in. lens will also be found very useful.
For minute work, such as bacteriology and blood investigations,
higher powers will be required, 1/8 or 1/12 *immersion lenses*. These
objectives come extremely close to the object, and very thin cover
glasses must be employed. In order to avoid the refraction caused
by the rays traversing the air between the coverslip and lens some
immersion fluid is placed between the two. With some lenses water
is employed, but usually an oil having the same refractive index as
glass is used, and the one most generally employed is cedar oil (Zeiss
prefers the oil from the species Juniperus virginiana). A spot of
oil is placed with a rod just over the object to be examined and the
objective carefully lowered by the coarse adjustment till it comes in
contact with the droplet of oil. The focussing should then be managed
with the fine adjustment only.
When the section has been examined the oil must be removed from the
lens. For this purpose a soft silk handkerchief or a special piece of
chamois leather may be employed, and used very gently. If all the oil
cannot be removed, the handkerchief may be moistened with a little
absolute alcohol, and the lens hastily wiped. The alcohol must not be
allowed to remain in contact with the lens as it is a solvent of Canada
balsam with which the lenses are often cemented in position.
[Illustration: Fig. 1.--Double or Triple Nose-piece.]
*Double or triple nose-piece* (fig. 1).--This mechanical arrangement
is placed on the lower end of the tube. Two or three objectives
of different magnifying power are attached to it. The nose-piece
rotates round a central pivot in such a way that the objectives can
successively be brought accurately into position above the object on
the stage. It is, therefore, a moment’s work to replace a high power
objective by a low power one and _vice versa_. It is an extremely
convenient time-saving appliance, and by its use the risk of dropping
and injuring the objectives when screwing them on and off frequently
is avoided. Those whose microscopes are not already fitted with this
appliance can easily have one fitted on at a cost of about a sovereign.
*Substage condenser.*--This mechanism for concentrating light on the
object is a necessity for bacteriological work. The most convenient
form is *Abbe’s illuminating apparatus* (fig. 2).
This consists of a system of short focus lenses which collects the
light received by the mirror, and throws it on the object. The amount
of light received from the mirror is controlled by an “iris diaphragm,”
the aperture of which can be dilated or contracted by moving a small
lever at the side. It can be fitted on to most microscope stands, but
it is better to get a stand in the first instance which is constructed
to carry one.
The cost of a microscope varies from two guineas to two hundred. There
are many excellent microscopes in the market, and of these several may
be mentioned which the writer has found to work satisfactorily.
[Illustration: Fig. 2.--Abbe’s Illuminating Apparatus.]
Of the cheaper student’s microscopes the “Star” microscope made by
Messrs. R. and J. Beck, of Cornhill, E.C., will be found a safe
investment. It may be obtained with coarse and fine adjustment,
nose-piece, and 1 in. and 1/4 in. objectives, for about £5. Those who
require a better instrument will find Beck’s “Pathological” microscope
fitted with nose-piece, Abbe’s illuminator, &c., for £16, meet all
requirements.
Leitz of Jena, supplies two good and cheap microscopes for £3 10_s._
and £5. They are not, however, of uniform excellence, and they should
be carefully tested by some competent judge before the purchase is
completed. Leitz immersion lenses are cheap, and often extremely good,
but should be carefully tested beforehand, as their quality is not
quite uniform. The microscopes can be obtained from Mr. A. Frazer,
Teviot Place, Edinburgh.
The “Bacteriological” microscope, made by Messrs. Swift, of Tottenham
Court Road, is one with which no one can be disappointed. It is sold
with Abbe’s condenser, triple nose-piece, 1/6 in., and a 1/12 in.
immersion objective, for just under £20. Both stand and lenses are
turned out in Swift’s first-class style, and those who can afford the
initial outlay will not regret it. Or the stand may be purchased, and
the objectives and accessories added singly from time to time.
Among Continental makers, excellent microscopes for histological work
are turned out by many makers. Zeiss’s lenses stand deservedly high in
reputation, as no faulty Zeiss lens ever leaves the works, and their
optical properties are nearly perfect. For this guarantee, however,
the purchaser has to pay somewhat higher prices, but the money is well
invested. Zeiss’s agency is at 29 Margaret Street, Regent Street, W.
Reichert, of Vienna, sells microscopes and lenses which are modelled on
the lines of those of Zeiss, and though cheaper are often equal to them
in excellence, but the quality is not quite uniform. His instruments
can be obtained through any optician, but his agent in this country is
Mr. A. Frazer, Teviot Place, Edinburgh.
Before buying a microscope the student should obtain an illustrated
price list from any of the firms mentioned above, and, having selected
an instrument, he should test it very carefully, or better get some
experienced friend to test it for him, before deciding to purchase it.
Delicate test objects such as diatoms, scales of butterfly’s wing,
or a stained specimen of micro-organisms should be employed. The
coarse and fine adjustments should be tried. They should work freely
and smoothly and without any delay. The definition of the lens must
be tested with the fine objects mentioned. The field should be quite
flat, _i.e._, every part should be in focus at the same time, and the
definition should be perfectly sharp and accurate, and the test objects
without double contour. The field should be totally free from prismatic
colours. If there is a halo of colour around the objects it indicates a
defect in the optical properties of the objective, and another should
be selected.
A microscope must always be treated with the greatest care. Jars and
falls tend to slightly loosen and shift the lenses, and to permanently
impair its optical properties. Dust must be most carefully excluded.
This is best effected by keeping the instrument under a glass bell jar
when not in use. The lenses should be wiped as little as possible, and
when it is necessary, very soft chamois leather should be employed.
The microscope must be kept in a dry room, or the brass work will soon
tarnish and the steel parts will tend to rust.