UNIVERSITY OF CALIFORNIA
A DESCRIPTION OF THE WET COLLODION
THE BEST METHODS OF MOUNTING AND PREPARING MICROSCOPIC
OBJECTS FOR PHOTO-MICROGRAPHY
A. C O WJTf R V_]V[ ALLEY,
SECOND EDITION, REVISED AND ENLARGED, WITH ILLUSTRATIONS.
H. K. LEWIS, 136 GOWER STREET, W.C.
Introduction - - - - - - l
Reflection and Refraction . 4
Aberrations - - - - 10
Microscopic Objectives - - - 13
The Microscope - - - - - - -16
Achromatic Condenser - - - 29
Illumination . ' 1 < s . . . . -33
The Dark Room ..,' '.' .... 49
Mounting Microscopic Objects .... 54
Section Cutting - - - - 65
Staining -.-..... 68
Wet Collodion Process - - - 75
Hartley's Formulae - .... 81
To wash the Emulsion - - - - -83
To flow the plates - - 84
To keep the Emulsion ..... 86
To prepare the Silver - - - - -87
Description of Washing Box - 87
Description of Apparatus for Melting and Filtering the
Emulsion ...... 88
Description of Levelling Slab ... 89
To Develop . . . 90
Formula for Developing 91
List of Articles Needed ..... 92
Cleaning and Preparing the Glass - 92
Former Methods adopted in the Arrangement of the Apparatus 101
Later Methods 103
The Method adopted in this Work - - - - 105
Dr. Dallinger's Illumination .... - 124
Defects in Negatives - - - 140
Their Kemedy - - . - < - 141
Production of Positives - - - 149
Enlarging - - - - - 159
Appendix - - 163
Preservative Media - .... 163
Bacillus Tuberculosis - - 163
Binocular Photography - . 164
Focussing at a Distance ..... 164
PREFACE TO THE SECOND EDITION.
SINCE the publication of the first edition, Photo-
micrography has become so general and the process
has so much improved, that it has been found
necessary to bring out a second edition, in which an
endeavour has been made to keep pace with the
advances of microscopy and to incorporate the more
recent improvements in photography.
The title of the first edition was chosen after much
deliberation, although I was fully aware of its liability
to criticism on account of its ambiguity. The term
now employed though less euphonious certainly pre-
It was my original intention to answer some criti-
cisms in this preface, but on further consideration
they appeared so trivial and the writers showed such
complete ignorance of both the work and the subject,
that I thought it better to abandon it, and simply
thank those from whom I received many valuable hints,
and whose friendly criticism gave me so much en-
couragement. I only hope the result will be found
worthy of acceptance and of some little use in the
advancement of those scientific subjects to which
photo-micrography has been lately applied.
A. COWLEY MALLET.
December 8th, 1884.
PREFACE TO FIRST EDITION.
THE publication of this little work has been under-
taken with a view to encourage the practice of
Micro-photography, and afford a ready means of
obtaining information without the trouble of refer-
ing to the innumerable papers on the subject, scat-
tered up and down several scientific periodicals and
As far as possible we have mentioned the sources
from which our information has been derived ; many
have no doubt been overlooked, but the absence of a
library for reference, and our isolated position, must
serve as an excuse.
Our thanks are due to several friends for their
kindness in supplying any information required, but
.especially to the Rev. Gr. B. Powell who revised all
the proofs, and whose valuable services have contri-
buted in no small measure to hasten the completion
and publication of the work.
A. COWLEY MALLEY.
BEFORE beginning a detailed description of the
apparatus employed in Photo- micrography, a few
remarks are necessary on its utility in research, and
its"application to the various branches of science.
All microscopists are aware of the fickleness with
which objects display their structure, markings
easily perceived at one time may baffle all attempts
to resolve them at another, and perhaps when seen
are beyond our power to delineate, I have before me
at present, some drawings which must have taken
the artist a week to finish, and even then, shew the
existence in his mind of preconceived notions of
Photography not only obviates the necessity of
future trouble and perhaps failure in the display of
these markings, but in a period varying from a few
seconds to as many minutes, imprints the latent
image on a sensitive surface, which after develop-
ment, can be multiplied a thousand fold, and give to
the world results, indisputably proving the truth of
some favourite theory, or showing the existence of
some doubtful structure.
In the domain of pathology we find many
observers differing in their descriptions of well-
known lesions. What scientific man engaged in the
investigation of the markings of the diatomaceae
agrees with others as to their true interpretation, at
the present day Agnosticism is the only name
expressing the condition of men's minds on some of
these subjects. Thousands of questions hitherto un-
settled on account of errors of description, or want
of agreement between observers, are sure to find an
easy solution if the practice of photography becomes
more universal among naturalists and our profes-
I do not wish to lead others to infer from this,
that the photographic image may not be false if im-
properly obtained, but it can neither add anything
to, nor take anything from, the structure, and rnay
be relied on to shew what was actually seen by the
In teaching Histology, Pathology, or any subject
in which the microscope plays an important part, a
photograph of the object may be thrown on the
screen in the usual manner, and the lecture proceed
uninterrupted by the use of separate apparatus, or
the time taken up by those manipulations which
must be made for every different observer.
It is unnecessary to dwell on the fascination
of the pursuit, or the recreation afforded to those
who after the arduous duties of the day, relieve the
strain by the study of microscopy, for, from what
has been said, the advantages of photography to the
Histologist, Pathologist, and student of Natural
History, are so obvious, that any tendency to extend
its domain to amusement might injure its prestige
with students of science.
In the following chapters besides the methods we
have ourselves adopted, it has been our aim to
gather together those used by others, and after ana-
lysing them to choose the parts most applicable,
when considered in conjunction with the recent ad-
vances of photography; at the same time by
showing the facility of their application, we hope to
make photo -micrography more popular, and place it
within the reach of all.
PROPERTIES OF LENSES.
PROPERTIES OF LENSES, IMPERFECTIONS OF RESULTING
IMAGES, ABERRATIONS, ETC.
ACCORDING to the undulatory theory of light an ob-
ject becomes visible, when it communicates the rapid
vibratory motion of its molecules to the luminiferous
ether, which being propagated through it in the form
of spherical waves, is transmitted to the retina.
The vibration of the molecules takes place at right
angles to the direction of the wave, for example, if a
long string of beads is shaken at one end, the vibra-
tions of the beads are at right angles to the length of
the string, while the waves propagated through it
by the shake, move in the same direction as the
Reflection. When a luminous ray falls, or is inci-
dent, on a polished surface, it is reflected at an angle
which equals its angle of incidence. (BAG and
B"AC Fig. 1.)
Refraction. "When a luminous ray passes from a
rarer to a denser medium it is bent out of its course.
The Sines of the angles of incidence BAC, and re-
fraction B'AC', (Fig. 1.), bear a constant ratio to each
PEOPERTIES OF LENSES.
This ratio is called the refractive index.
,, Crown Glass
If the medium through which the light passes has
parallel faces, the emergent B'A' and incident BA
rays are parallel, (Fig. 1), if it has not, the tendency
of the ray is to bend away from the angle at which
the faces meet.
Lenses are of six forms; double convex 1, plano-
convex 2, converging meniscus 3, double concave 4,
plano-concave 5, diverging meniscus 6.
6 PROPERTIES OF LENSES.
All rays falling on Convex lenses are refracted
towards the centres of curvature of the faces, those
incident on Concave away from the centres, (Figs. 3
In convex lenses the PRINCIPAL FOCUS (F. Fig. 3) is
that point where all rays parallel before entrance
converge after refraction, it almost coincides with
the centres of curvature of the faces.
If the rays diverge from a point A outside the
principal focus, they meet after refraction on the
other side beyond it B, these points A and B are
real foci (Fig. 3).
If they converge they meet at C within it, B and
C are called conjugate foci.
Eays diverging from a point C Fig. 3, within the
principal focus of a convex lens, can never meet at
the opposite side, but only become less divergent,
they therefore cannot form a real focus. But if the
emergent rays are prolonged backwards they will
meet at V Fig. 3. The point V is called the virtual
PROPERTIES OF LENSES.
If an image of an object be received on a screen
placed opposite a small hole in the shutter of a dark-
ened room it will appear inverted, because the rays
from different points of the object cross at the aper-
The size of the image varies inversely as the dis-
tance of the object, and directly as the distance of
If a convex lens be placed in the aperture, inverted
and diminished images of distant objects will be
formed on a screen placed at the principal focus.
If an object (Fig. 4), be placed at twice the focal
distance of a convex lens, a real inverted image, the
same size as the object, will be formed on a screen
placed at a similar distance on the opposite side.
If nearer, a real inverted magnified image (Fig. 5),
is formed at increasing distance until the principal
focus is reached, when no image is formed on the
screen, because the rays can never come to a focus
PROPERTIES OF LENSES.
The human eje consists of a convex lens, the
crystaline lens placed behind the aperture of the iris,
and a screen, the retina, behind the crystaline lens,
C and E (Fig. 6).
A real inverted image of objects is formed on the
retina, (how they appear erect to us has not yet been
shown), and as the crystaline lens can alter its form,
so as to bring convergent or divergent rays to a
focus on the retina, the eye can percei7e clearly the
same object when placed at different distances.
There is a limit to the accommodation of the eye,
in normal eyes eight or ten inches is the shortest
distance an object can be clearly perceived, without
giving rise to an unpleasant feeling owing to our
efforts to increase the curvature of the lens, and to
enable it to bring the divergent rays to a focus on
the retina, but if a convex lens L Fig. 6 be placed
between the object and the eye it assists in bringing
the rays to a focus on the retina.
The size of the retinal image varies with that of
the angle AOB Fig. 6. This angle is called the
PROPERTIES OF LENSES. 8
As the image of an object is only a collection of
the foci of the different points of an object, these
images will be real or virtual as the image is situated
without or within the principal focus.
A real magnified and inverted image of an object
is seen by the eye if the object is situated outside the
principal focus of a lens, and at less than twice the
focal distance, because the angle of intersection of
the rays from opposite sides of the object, i.e. the
visual angle, is increased.
A virtual erect and magnified image is seen by the
eye if the object is situated within the principal focus
of the lens, because the crystaline lens of the ob-
server C Fig. 6 has the power of bringing the rays
to a focus and forming a real image on the retina R,
on this principal the magnified image in the simple
Concave lenses can only form virtual images no
matter what the distance of the object. (Fig. 7).
Up to the present the passage of light through
lenses has been considered in its simplest form, it
remains now to investigate the imperfections in the
PROPERTIES OP LENSES.
resulting images, their causes, and the various
methods of correcting them.
When a ray of light passes from one substance
through another a part is reflected at the incident
surface, a second absorbed in its passage, and a third
transmitted. The greater the thickness and number
of the transparent media through which the light is
transmitted, the greater is the quantity reflected and
absorbed. Since Microscopic Objectives are com-
posed of several lenses the loss of light is conse-
quently very great.
Spherical Aberration (Fig. 8), shows itself in want
of distinctness in an image at the margins when
sharp at the centre, and vice versa ; it is caused by the
unequal refraction of the rays owing to the spherical
shape of the lens, those at the margins coming to a
focus before those at the centre.
Chromatic Aberration (Fig. 9), a ray of white light
PROPERTIES OF LENSES. 1 L
is composed of rays of different colours, if it pass
through, a medium (such as a lens or prism), Fig. 9,
whose sides are not parallel, it is divided into its
component colours, OP dispersed.
Now since all the different rays into which a pen-
cil of white light can be divided, have different foci,
that is, the point where the red rays come to a focus is
more distant than that of the violet, the intermediate
colours forming other foci between them, V 1 2 3 4 R
Fig. 9, the rays are unequally dispersed and the result-
ing image will be coloured at its edges.
There are several methods of remedying these
aberrations, some of them diminish Spherical and
Chromatic aberration at the same time.
Spherical Aberration may be destroyed by giving
the lens a decreasing curvature towards the edges,
in fact an ellipsoidal form, the difficulty of this in
practice was found so great that it was abandoned,
and if the form was such that its performance was
perfect for parallel rays, it would be inaccurate for
all others. It is much more easily corrected by altering
the curves of the two sides. For instance if the
plane side of a plano-convex lens be turned towards a
very near object, or the convex side towards a very dis-
tant one, the resulting aberration would be about one-
fourth of that which would occur if the respective
positions of the sides of the lens were reversed
A method which has been frequently adopted for
photographic objectives deserves mention, namely
lessening the aperture, by placing a diaphragm in
front of or behind the lens which admits the rays at
12 PROPERTIES OF LENSES.
the centre, but cuts off those at the edges, it will be
evident that this lessens the chromatic aberration at
the same time.
The diaphragm has other advantages, it enables
objects lying at different distances from the lens to
appear distinct, in other words, the penetrating
power is increased. Its disadvantages are that it
produces distortion, if placed in front of a lens the
image of a square becomes barrel-shaped, if behind
the reverse. It lessens the light, and diminishes the
Angular Aperture, upon the extent of which the
most important qualities of an objective depend.
The aberration of a concave lens being exactly
opposite to that of a convex lens, one may be made
to correct the other without decreasing the magnify-
ing power to any great extent. The lens A Fig. 10
completely destroys the spherical aberration of the
lens B, for it does not change the position of the
rays situated near its centre as much as it does those
at the edges, owing to its increasing prismatic form.
If advantage be taken at the same time of the
different relations between the refractive and disper-
sive powers of different kinds of glass, and if the
lenses which correct the spherical aberration by their
PROPERTIES OF LENSES. 13
shape are formed from different kinds, chromatic
aberration will be destroyed. If the lens A (Fig. 10)
of high dispersive power, concave form, and low cur-
vature, be joined to B of lower dispersive power and
greater curvature, it is evident that the dispersion
caused by the latter is neutralysed, whilst its refrac-
tive power is only decreased by the opposite refrac-
tion of the concave.
The convex lens of crown is generally corrected by
a concave of flint, it has been found that no two
lenses can be made to correct each other perfectly,
so opticians overcome the difficulty by combining
Before leaving this subject we should mention that
in photography errors frequently arise, especially
when using low powers, owing to the difference be-
tween the visual and chemical foci. A point of
white light may appear perfectly distinct at E
Fig. 9, when surrounded with a violet areola, and if
a photograph be taken it will be quite indistinct;
while at V nearer the lens, the image may not ap-
pear so distinct, but will be surrounded by a red
areola, and if a photograph be taken at this point, it
will be found perfect, as the violet rays are the most
chemically active. This is owing to the different
refrangibility of the red and violet rays, the latter
coming to a focus sooner than the former. When
photographing, the point V should be chosen where
the object is surrounded by a red areola, this being
the most accurate for the violet rays.
Microscopic objectives have of late years arrived at
14 PROPERTIES OF LENSES.
such perfection that it seems impossible to improve
them. They are designated from four inches up to one
fiftieth of an inch, according to their magnifying
power : for example, a one inch objective is supposed
to magnify as much as a single lens of one inch focus,
although its own focal length may be different.
This rule is only approximately correct, as glasses
of the same designation by different makers vary in
Low powers up to two inches generally consist of
a single combination, but as it has been found im-
practicable to construct objectives of high power free
from chromatic and spherical aberrations in this way,
all good makers have adopted the plan of correcting
one combination by another, so that objectives from
two inch to one inch generally consist of two, while
those of quarter inch and upwards generally consist
of three combinations, formed perhaps of as many as
eight different lenses.
Various devices have been adopted to lessen the
aberrations, increase the angular aperture, and at the
same time reduce the number of lenses. For an ex-
ample of the ingenuity displayed in overcoming these
difficulties, we refer the reader to the description of
Mr. Wenham's new objective published in the Pro-
ceedings of the Royal Society, vol. xxi. p. Ill, and
content ourselves with mentioning that it consists of
a single front of the usual form, a single plano-convex
back whose focus is four and one half times that of
the front, and a middle triple in which a single con-
cave lens of flint, three times the focal length of the
PROPERTIES OF LENSES. 15
front, corrects all the others. In this combination
only five lenses are used, and the errors arising from
the sixteen surfaces of glass in the older forms re-
duced to ten.
A good objective should possess the Standard
screw, adopted by the various societies, and now
fixed by the best makers to their lenses.
Its definition should be clear, the field flat, objects
at the edge of the field should be as free from colour,
and their definition as perfect as those at the centre.
The illumination should be white, not yellow, as is
the case with inferior glasses, especially those of
If of low power, that is of one inch or more
nominal focal length, opaque objects should be
clearly shown without the use of any apparatus for
condensing the light upon them.
The rotundity of certain objects should come out
well, the more distant parts being in as good focus
as those nearer the objective.
Transparent objects possessing a certain thickness,
should be clearly defined to a certain depth. This
power of penetration is difficult to form a correct
opinion of, as we shall find that a glass of given
focus magnifies the depth of an object in the pro-
portion of the square of the magnification of the
diameter laterally. It is in inverse ratio to the
magnifying power of the objective. Therefore low
powers are most suitable for the display of those
objects in which different points, lying on different
planes require to be in focus at the same time.
16 PROPERTIES OF LENSES.
It has been found that the thin glass used for
covering microscopic specimens, produces by refrac-
tion of the rays passing through it a sufficient nega-
tive aberration (Fig. 11) to destroy the adjustment
of an objective perfect in its performance on uncovered
objects. This may be remedied, as has been shown
by Mr. A. Ross, by under-correcting the front andover-
correcting the two back combinations, at the same
time making the distance between them susceptible
of alteration by means of a screw collar, this arrange-
ment by bringing the front and back combinations
nearer together, enables us to give the objective an
excess of positive aberration sufficient to counteract
the negative produced by the thin glass.
The screw collar adjustment at the same time cor-
rects the errors caused by the difference in the
refractive indices of the various media employed for
the preservation and mounting of specimens. We
therefore consider it an indispensible adjunct to all
objectives of higher power than one half inch. It
must not be forgotten that these adjustments affect
the magnifying power considerably, and allowance
PROPERTIES OF LENSES. 17
must be made for this when accurate measurements
of a covered object are required.
Many methods of practically making these cor-
rections are mentioned in the various manuals,, but I
think the following will be found the easiest, and
sufficiently correct for all purposes. Turn the index
(A Fig. 12) to Zero, in other words adjust for un-
covered, the object is now brought into focus. We
next turn the screw collar till some dust or scratches
on the cover-glass come into focus. The object is again
brought into focus by means of the fine adjustment.
If now on turning the fine adjustment an equal
expansion of the outlines of the object takes place
when within and without the focus, the correction is
perfect. If the greatest expansion occurs when the
object is outside the focus we have gone too far, the
screw collar must be turned and the index moved
back towards or uncovered, and vice versa. When
the expansion is the same on both sides it will be
perceived that the minutest details become visible,
at the same time, and with the same distinctness, as
the outlines of the object.
The number to which the index on the screw
collar points is now written on the slide for refer-
ence in cases of future examination.
18 PROPERTIES OF LENSES.
The angular aperture is the capacity, which a lens
has for receiving rays from the object and transmitting
them to the image, and is determined by the ratio
between the diameter of the opening and the focal
The apparent depth of focus of an objective, is
in inverse ratio to its magnifying power, and as the
utility of the higher powers depends upon their capa-
bility for showing the minute surface-structure of
markings, and as this increases with the power
the objective has of collecting a large number of
rays from an object, in other words on its angular