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THE
MICROSCOPE

By
SIMON HENRY GAGE

Late Professor of Histology and Embryology
in Cornell University

SEVENTEENTH
EDITION: REVISED

ITHACA -NEW YORK

COMSTOCK PUBLISHING COMPANY, INC.

J947

COMSTOCK PUBLISHING COMPANY, INC.

PRINTED IN THE UNITED STATES OF AMERICA
THE VA1L-BALLOU PRESS, BINGHAMTON, NtYT

the memory of

THEOBALD SMITH

pupil ', friend, and

master investigator who

opened new paths to the

human mind.

PREFACE

IN revising the matter for this, the seventeenth, edition of The
Microscope changes have been made in every chapter in text and often
in illustrations to render the subject more easily understood.

Attention has been called to the newly devised Electron Microscope
with its greatly increased magnifying power and resolution over the
ordinary microscope; to Polaroid for the micro- polariscope; to some
new plastics for mounting in place of Canada balsam; to the high-
pressure mercury lamps for ultra-violet radiation and the bright
mercury lines for photographing objects with the microscope.

In general, however, the book retains its former character, and it is
hoped that it will continue to serve students and users of the micro-
scope in understanding the underlying principles involved in micro-
scopical work.

As a final word, I wish to express my thanks and appreciation for
the new cuts loaned and other aid rendered by the American manu-
facturers, the Bausch & Lomb Optical Company of Rochester and
the Spencer Lens Company of Buffalo. The heads of those com-
panies, Edward Bausch and Harvey N. Ott, have been my friends
and advisers for many years and have been ever ready to help me
over the rough places in my microscopical career.

Likewise it is a pleasure to render thanks to my University col-
leagues and to my fellow microscopists for their friendly interest and
help; also to Clara Starrett Gage, Ph.D. for aid in preparing illus-
trations, making corrections and revising the index for this edition.

SIMON HENRY GAGE
May 20, 1941

CONTENTS

SECTIONS FIGURES PAGES

INTRODUCTION AND THE ELECTRON MICROSCOPE . . i-3A 1-6

CHAPTER I 1-66 4~4ia 7-50

Microscopes and Their Parts.
CHAPTER II . . 67-169 42-64 51-120

Bright-Field Microscopes; Lighting, Natural and
Artificial; Experiments with Microscopes.

CHAPTER III . . . 170-215 65-90 121-169

Dark-Field Microscopy and Its Application.
CH\PTER IV 216-272 91-119 170-221

The Polarizing Microscope; Optics of the Mi-
croscope.

CHAPTER V 273-302 120-124 222-239

Micro-Spectroscope; Pocket Spectroscope.
CHAPTER VI 303-324 125-130 240-2 s«S

Ultra-Violet Microscope; Physical Analysis.
CHAPTER VII 325-358 131-142 259-27^

Interpretation of Appearances.

CHAPTER VIII 359~398 i43~i67 279-316

Magnification and Micrometry.

CHAPTER IX 399~45o 168-199 317-363

Drawing with the Microscope and with Projec-
tion Apparatus; Class Demonstrations.

CHAPTER X 451-507 200-214 364-402

Photographing Embryos and Small Animals;
Photographic Enlargements; Photographing
with the Microscope.

CHAPTER XI 508-615 215-249 403-463

Cabinets; Slips and Cover- Glasses; Mounting,
Labeling and Storing Microscopical Prepara-
tions.

CONTENTS

SECTIONS FIGURES

CHAPTER XII 616-663 250-266

Fixing and Preservation of Tissues, Organs
and entire Organisms; Infiltrating; Imbed-
ding, Sectioning, Staining and Mounting for
the Microscope.

CHAPTER XIII .

Serial Sectioning of Organs, Small Animals and
Embryos; Preparation of Models.

CHAPTER XIV

Micro-Incinerations and the Optical Appliances
for their Examination

CHAPTER XV ...

Brief History of Lenses and Microscopes.

GENERAL BIBLIOGRAPHY .

INDEX . . ....

INTERPOLATION, TABLE OF METRIC AND ENGLISH
MEASURES . . . . ...

TABLE OF NATURAL SINES TO 90°

PAGES
464-495

664-702 267-277 496-520

703-730 278-299 521-546

300-313 547-579

616
617

THE MICROSCOPE
AND MICROSCOPICAL METHODS

INTRODUCTION

IN dealing with the possibilities and use of any method of investi-
gation, any machine or piece of scientific apparatus, the writer or
teacher will naturally proceed as seems to him best from his personal
experience, from his general theory of education, and from his con-
ception of the style and method of presentation which will render
his book most helpful and acceptable to his possible readers.

As stated in the preface to the sixth edition, this book had its
origin in the laboratory, and its purpose was, and still is, to give
the guidance by which those unfamiliar with the microscope and the
methods of work with it can gain an intelligent understanding of the
instrument, its limitations, and its possibilities for aiding one to
arrive at truth. It has also the added purpose of bringing together
the scattered information concerning new apparatus and method so
that older workers may make use of them with a minimum amount
of time and effort.

In working out the plan the following landmarks have been kept
constantly in sight:

(i) To most minds, and certainly to those having any grade of
originality, there is a great satisfaction in understanding principles;
and it is only when the principles are firmly grasped that there is
complete mastery of instruments, and full certainty and facility in
using them. The same is true of the methods of preparing objects
for microscopic study, and the interpretation of their appearances
when seen under the microscope.

Much good work can be and has been done by the rule of thumb
method, in which there is no real understanding of the underlying

2 INTRODUCTION [INTRO.

reason for any of the operations; the worker simply knows that good
results follow a certain course of action. Probably most of the work
of the world is done by rule of thumb. But for the highest creative
work from which arises real progress both in theory and in practice,
a knowledge of principles is indispensable.

(2) Need of abundant practical work to go with the theoretical
part has been shown by all human experience. In all the crafts and
in all the fine arts mastery comes only with almost endless effort
and repetition, the most common example being the attainment of
facility in music. Hence in this work there have been introduced
many practical exercises so that the worker might gain the deftness
needed. It is also a part of human experience that in successfully
going through the manipulations necessary to demonstrate principles,
there is acquired not only skill in experiment, but an added grasp of
the principles involved.

After observing the work of students in my own and in other
laboratories, the conclusion was reached, and expressed in the third
edition of this book (1891) that " simply reading a work on the
microscope, and looking a few times into an instrument completely
adjusted by another, is of very little value in giving real knowledge.
In order that the knowledge shall be made alive, it must be a part
of the student's experience by actual experiments carried out by the
student himself."

Beale, in his work on the microscope, expresses it thus: " The
number of original workers emanating from our schools will vary as
practical work is favored or discouraged. It is certain that they who
are most fully conversant with elementary details, and most clever
at demonstration, will be most successful in the consideration of the
higher and more abstruse problems, and will feel a real love for their
work which no mere superficial inquirer will experience. It is only
by being thoroughly grounded in first principles, and well practised
in mechanical operations, that any one can hope to achieve real
success in the higher branches of scientific inquiry, or to detect the
fallacy of certain so-called experiments."

And Hon. J. D. Cox, skilled alike in the arts of war, statesman-
ship, and science, in his notable address upon Systematic Instruction

INTRO.]

INTRODUCTION

in the Microscope at the University, before the American Micro-
scopical Society, in 1893, says: " I wish to urge the desirability of a
somewhat extensive course of technical training
in regard to the microscope. , . . Any one who
desires to devote himself seriously to investiga-
tion with the microscope will find great advan-
tage, as it seems to me, in devoting some time
to the study of the instrument itself in all its
parts, and the history of their development."
The study of this whole address is urged upon
the person interested in the just appreciation
of the different parts of the microscope and
their successful employment or improvement.

Sir A. E. Wright, in his book " Principles of
Microscopy," says this: " Every one who has
to use the microscope must decide for himself
the question as to whether he will do so in ac-
cordance with a system of rule of thumb, or
whether he will seek to supersede this by a
system of reasoned action based upon a study of
his instrument and a consideration of the
scientific principles of microscopical technique.
The present textbook Qiis " Principles of Micros-
copy "] has no message to those who are con-
tent to follow a system of rule of thumb, and
to eke this out by blind trial and error. It ad-
dresses itself to those who are dissatisfied with
the results thus obtained and who desire to
master the scientific principles of microscopy,
even at the price of some intellectual effort."

From the observations made during the last
fifty years I am confirmed in the belief that
for attainment in study with the microscope, as
in all other human endeavor, a person must pay for all he gets.

(3) In considering the microscope, it may be looked at as a ma-
chine composed of glass and brass complete in itself, or it may be

Un»

FIG. i. A SIMPLE
MICROSCOPE HELP-
ING THE EYE TO
FORM A RETINAL
IMAGE OF A NEAR
OBJECT.

Object The object
to be seen by the eye.

Lens The double
convex lens acting
as a magnifier or
simple microscope
to aid the eye in see-
ing a near object.

Cornea The cor-
nea of the eye.

r The single re-
fracting surface in
the schematic eye.

cl The crystalline
lens of the eye, also
the center of the re-
fracting surfaces or
the nodal point of
the eye where the
secondary axial rays
cross.

ri Retinal image;
it is inverted.

INTRODUCTION

[INTRO.

considered as an artificial aid to the eye, like a spectacle. When
complete in itself it is properly called a projection microscope,
for it produces an image wholly independent of the eye of the ob-
server. This image may be fixed on a
photographic plate or used as a basis for
a drawing (fig. 3). On the other hand,
when used as a microscope in the ordinary
way, the eye of the observer is an inte-
gral part of the optical combination, just
as integral a part as the objective or the
ocular (figs, i, 2). This being the case
the optical perfection of the eye is as in-
fluencing on the final retinal image as the
perfection of the other optical parts.

Quoting again from the preface of the
third edition: " In considering the real
greatness of the microscope and the truly
splendid service it has rendered, the
fact has not been lost sight of that the

f OBJECTIVE

r f

Object

MIRROR

FIG. 2. A COMPOUND MICROSCOPE HELPING
THE EYE TO FORM A RETINAL IMAGE OF A NEAR
OBJECT.

Mirror The plane and concave mirror to re-
flect light through the object.

Object The small object to be seen by the eye.

Objective The objective of the compound micro-
scope to form a real image of the small object.

Axis The principal optic axis of the micro-
scope.

/ Principal focus of the ocular and of the ob-
jective.

r im The real image formed by the objec-
tive.

Ocular The double convex lens enabling the
eye to see the real image formed by the objective.

cr The cornea of the eye.

rs The refracting surface of the schematic eye.

L The crystalline lens of the eye.

r i The retinal image; it is erect with reference
to the object, but inverted as compared with the
real image.

INTRO.] INTRODUCTION 5

microscope is, after all, only an aid to the eye of the observer, only
a means of getting a larger image on the retina than would be
possible without it, but the appreciation of this retinal image,

FIG. 3. PROJECTION MICROSCOPE WITH ENLARGED REAL
IMAGE ON THE SCREEN

whether it is made with or without the aid of a microscope, must
always depend upon the character and training of the seeing and
appreciating brain behind the eye. The microscope simply aids the
eye in furnishing raw material, so to speak, for the brain to work
upon."

(4) While the objective and ocular are the fundamental constit-
uents of a microscope, it must never be forgotten that for their most
effective use provision must be made for so lighting the objects to be
studied that their structural features may be brought out. This
involves the use of a substage condenser to insure an adequate
aperture of the illuminating light. This again necessitates a suffi-
cient source of light either natural or artificial. If it is artificial, it
must be sufficiently brilliant on the one hand, and on the other it is
a great advantage to have it of daylight quality, such as that given
by the Chalet Lamp with its daylight glass screens (figs. 46, 47).

(5) For gaining glimpses of structure and physical condition
beyond what can be gained by the microscope lighted with ordinary
visible radiation, either reflected or transmitted, one now has avail-
able dark-field illumination; illumination by polarized light, and
radiation by the invisible ultra-violet. Perhaps also in the future,

INTRODUCTION

PNTRO.

radiation by the long waves of the infra-red may reveal structural
details not hitherto known. In a word, to gain the deepest insight
into microscopic structure every possible source of information
should be utilized, and new ones constantly looked for.

An Electron
Microscope

Electron,
Source

COMPARISON OF
and

Electron r
Condenser u

Object

Electron

Objective Q
O
O

A Projection
Microscope

LiQht
Source

x100

Substage
Condenser

Object! v

X60

Light
Image

Project I on[
Ocular

Screen Image

X6000

Screen Image
xlOOO

FIG. 3A. DIAGRAMS SHOWING THAT WHILE THE REAL IMAGE OF THE OBJEC-
TIVES MAY BE EQUAL (xioo IN THIS CASE), THE SCREEN IMAGE CAN BE MUCH
GREATER WITH THE ELECTRON MICROSCOPE.

[NTBO.] INTRODUCTION 6a

ELECTRON MICROSCOPE

THE MICROSCOPE with glass lenses or glass lenses combined with
lenses of natural transparent minerals like fluorite or quartz may be
so constructed as to give almost any degree of magnification and
thus make minute objects visible to the eye. However, for the
showing of details as separate things, that is, for resolution, such a
microscope is limited by the length of the waves of light to a mag-
nification of about 1500 diameters.

Hence investigators, knowing that details beyond the ability of
the ordinary microscope to reveal are probably even more pervasive
than those visible with the best microscopes, have sought radiations
of shorter wave lengths than those of visible light, hoping thereby
not only to add to the magnification but to produce the correspond-
ing increase in resolution.

It is believed that in the newly-invented electron microscope using
electronic waves i/ioo,oooth of the wave-length of visible light,
25,000 to 100,000 magnification with accompanying resolution can
be attained. This, of course, would make possible an almost un-
believable exploration of minute objects with their details, and it
may be added, corresponding difficulties in interpreting the appear-
ances.

The possibility of making such a microscope depends upon the fact
that the electronic waves are propagated in straight lines like light
waves and that they may be concentrated and focused by electro-
magnets something as light is concentrated and focused by glass
lenses. Also, depending on the strength of the current in the electro-
magnet, the variable concentration and focusing are comparable to
the concentration and focusing in the light microscope by means of
different curvatures of the glass lenses.

As the electronic waves are wholly invisible, their images must be
made visible either by using a fluorescent screen or by means of a
photographic plate. Therefore the electron microscope is to be com-
pared with a projection microscope in which the image formed is en-
tirely independent of the eye. It is not like the microscope into
which one looks, for with this the eye of the observer forms a part of

6b INTRODUCTION [INTRO.

the optical train, and the final image is formed upon the retina of the
eye.

In the accompanying diagrams the constituents of an electron
and of a projection microscope are shown for comparison; an electron
source in one and a light source in the other; for illuminating the
object a magnetic condenser in one and a glass condenser in the
other; for producing a real image of the object, a magnetic objective
and a glass objective respectively; and finally a magnetic projector
and a glass projection ocular to form the screen image in each case.

The size of the final screen image may differ greatly in the two in-
struments, but varies most with the electron microscope, in which it
may be as great as X25o while with the light microscope it rarely
exceeds xio or xi5.

In the diagram the projection microscope is represented as pro-
ducing a screen image of xiooxio = xiooo and the electron micro-
scope xioox6o = x6ooo; but, as has been said, their respective
possibilities are as great as about xi5<Do for the light microscope and
x 2 5,000 for the electron microscope; and this magnification, in some
cases at least, might be increased to xioo,ooo in printing the nega-
tive.

There are three other differences that should be mentioned: (i) The
initial cost of the best ordinary microscope is in the hundreds while
that of the electron microscope is in the thousands of dollars. (2) All
parts of the ordinary microscope can be used in the air of any room
while all the effective parts of an electron microscope^ must be in as
complete a vacuum as possible. (3) With the ordinary microscope
the object can be of considerable size and thickness, and mounted
upon a glass slip in air or some transparent medium like glycerol or
Canada balsam. With the electron microscope the object must be
small, very thin and dry and mounted on a film of collodion, which
in turn is supported by a perforated disc of platinum or a fine wire
screen,

It will be seen from the above that at present the electron micro-
scope is not available for ordinary biological study, but is to be
welcomed for the uses to which it is adapted now and for the possibili-
ties in its future development.

INTRO.] INTRODUCTION 6c

F EFEREN ^ES

BURTON, E. F. AND W. H. KOHL. — The Electron Microscope, 233 pages,
no figures and many plates. New York, 1942.

DAVIS, WATSON. — Science News Letter, Oct. 12, 1940. Abstract in Reader s
Digest, Nov. 1940.

HALL, C. E. AND SCHOEN, A. L. — Application of the electron microscope to the
study of photographic phenomena. Journal of the Optical Society of America,
Vol. 31, pp. 281-285. This paper gives the requirements for using the electron mi-
croscope. It is also well illustrated.

HOWARD AND SCOTT. — Review of Scientific Instruments, Vol. 18, 1937.

KAEMPFFERT, VV. — New York Times, March 31, 1940, p. 8D.

LAURENCE, WM. L. — New York Times, Dec. 30, 1938, p. i.

MALOFF AND EPSTEIN. — Electron Optics, N. Y. 1938.

MARTIN, L. C. -— Nature, Vol. 142, 1938 and Journal of the Royal Microscopical
Society, Dec. 1939.

MARTON, L. — Physical Review, Vol. 56, 1939, p. 705.

The Electron Microscope. — Engineering Products Division, RCA Manufactur-
ing Co., Inc., Camden, N. J. Pamphlet with illustrations of the RCA electron
microscope and of pictures taken with it.

ZWORYKIN, V. K. — Science, Vol. 92, July 19, 1940 and Sigma XI Lectureships,
1941. (Image formation by electrons.)

ZWORYKIN, V. K. — Image Formation by Electrons. Science in Progress, third
series, pp. 69-107, 46 illustrations. 1942.

CHAPTER I

MICROSCOPES AND THEIR PARTS
§§ 1 TO 66; FIGURES 4-41

MICROSCOPES

§1. Definition of a microscope. — As the word itself indicates, a
microscope is an optical instrument with which one can see small
things, often so small that the unaided eye could not see them at all.
It is from two Greek words: /u/cp6s — mikros, small, and <rK07rcu>
— skopein, to see. The word was compounded and given a Latin
form by Giovanni Faber of the Academy of the Lincei, as shown
by a letter of his to Cesi, President of the Lyceum, dated April 13,
1625. Faber says: " As I also mention his E Galileo's] new occhiale
to look^at small things and call it Microscopium." Jour. Royal
Microscopical Society, 1889, p. 578; Carpenter-Dallinger, p. 125.
The microscope serves its purpose by increasing the visual angle.
This may be done in two ways: (i) by means of one or more lenses
used as a kind of spectacle by which the eye is enabled to form a
sharp image on the retina when optically so close to the object that
without the artificial aid a sharp image could not be produced (figs.
i, 2, 6).

(2) The second way of increasing the visual angle is by means of a
projection microscope, which, wholly independent of the eye, pro-
duces a sharp, greatly enlarged image of the object upon a white
surface or other screen. The eye then looks at this image as though
it were the object itself and of that size (fig. 3, § 445).

The fundamental difference in the two forms of microscope is that
in the first the image is formed in the eye by rays directly from
the microscope, in the second by rays from the screen.

In this book the first form of microscope is mainly considered
except in Ch. IX and X, where the projection microscope is much
used.

MICROSCOPES AND THEIR PARTS

[CH. I

Compound Mleroocopo

Convex

Lens

MAGNIFIER
Microscope
Objective
Ocular

SIMPLE AND COMPOUND MICROSCOPES

§ 2. A simple microscope or magnifier is a lens or a combination
of lenses to use with the eye. But one image is formed and that is

upon the retina. The en-
larged image has all its parts
in the same position as they
are in the object itself, that
is, the image appears exactly
as with the naked eye, except
that it is larger (figs. 5-6).

§ 3. A compound micro-
scope is one in which a lens,
or combination of lenses,
called an objective, forms a
real image, and this real
image is looked at, by the
eye and a magnifier, or
ocular. The image seen has
the object and its parts
inverted. In the compound
microscope then, two images
are formed, one by the
objective independent of the

„ TT eye, and the other on the

tie. 4. tiNE PRINT SEEN BY THE UNAIDED . .

EYE AND THROUGH A MAGNIFIER retina by the action of the

eyelens of the ocular and
the cornea and crystalline lens of the eye (fig. i).

§ 4. Real images. — A real image is one formed by a lens or other
optical instrument, like a concave mirror. It is called real because,
entirely independent of the eye, it forms a picture of an object. This
is the kind of image which makes photography possible, also the
magic lantern, and moving pictures on a screen.

§ 5. Virtual images. — In all diagrammatic drawings showing
the microscope when looking directly into it, an enlarged, imaginary
object is shown out in space. This is frequently called a virtual

Aatlgmatlem
Myopia

Preebyopla
Daylight Giaaa

Artificial Daylight
Spectre-Photometer
Section Knlvee Free Hend
PUagente Si Idea Frame
Victoria) Culturaa Alloye

Hl»toiegy Equivalent
Ciaea Demanatratlon Foe*

Mlcropolarlacope
Magic Lantern
Projection Mloroacopo
Dutch Mlcroacope
Keplorian Mlcroaeopo
Parrlfln Method Book

Mtero-Chemlatry
Cnlargemente Pointed

Opaque Metala
Photographing Large

CH.I]

MICROSCOPES AND THEIR PARTS

image. In the projection microscope there is an actual or real,
enlarged image on a screen which the observer looks at as if it were

Object

FIG. 5-6. VISION BY THE UNAIDED EYE AND BY THE AID OF A SIMPLE

MICROSCOPE.

FKI. 5. UNAIDED EYE VISION. Axis, THE PRINCIPAL OPTIC Axis OF THE
EYE EXTENDED TO THE OBJECT.

Object The object to be seen; it is at a distance of 250 millimeters from the
eye.

r i The retinal image; it is inverted.

FIG. 6. VISION BY THE AID OF A SIMPLE MICROSCOPE. Axis, PRINCIPAL
OPTIC Axis OF THE MICROSCOPE AND OF THF, EYE.

A1 Bl The object within the principal focus (F) of the lens.

S M A double convex lens acting as a simple microscope.

Cr The cornea of the eye.

R Single refracting surface of the schematic eye.

L The crystalline lens of the eye.

B'2 A2 The retinal image; it is inverted.

A3 B3 The virtual image projected into the field of vision at 250 milli-
meters; it is erect, and the appearance is exactly as if the virtual image were
an object as in fig. 4, and no lens were present.

a large object (fig. 3). If one keeps in mind that virtual images are
purely imaginary, and that real images are produced by actual rays
of light, it will help to avoid confusion and wrong interpretations.

MICROSCOPES AND THEIR PARTS

[CH.I

In every case where an object is seen, light rays must pass from the
object to the eye, and these rays entering the eye must form an
image on the retina. It is the retinal image which furnishes the
brain the stimulus for vision.

APPARENT SIZE or OBJECTS

Whether one is using a microscope or not, the apparent size of any
object seen depends upon the visual angle.

§ 6. Visual angle. — This is the angle made by the border rays
of light from the object to the retina, and crossing at the nodal point
or optical center of the eye (figs. 143-144).

As the visual angle depends upon the distance the object is sepa-
rated from the eye, any means by which the object can be brought
closer to the eye will result in giving a larger apparent size to the

object, or in magnifying it. The lenses
of the microscope used with the eye
enable it co get very close to the object
and thus increase the visual angle, and
depending on the closeness, finer and
finer details of the object are separated,
for they subtend an angle of one minute
or more (see § 359), and the object as a
whole has a much greater apparent size.
For further discussion see §§ 359-36°-

§ 7. Pinhole card. — Use a piece of
paper about the size of a library card.
If the slip is black or of a dark color
it makes the experiment a little easier
than when white paper is used. Make a
hole in this with a needle (fig. 7). If
now one holds the slip up close to the
eye and gets the hole in the optic
axis, the eye can see brilliantly
lighted objects very clearly. If, to start with, the object is off
about i meter, quite an extent of it can be seen, and it will

FIG. 7. PIN-HOLE CARD FOR
VIEWING NEAR OBJECTS.

CH. I] MICROSCOPES AND THEIR PARTS II

appear small. Now go up closer and closer, and still the object is
clearly seen, and constantly appears larger. The closer one gets the
smaller is the visible field, but the larger will the parts seem to be.
If the hole is quite small, one can get the object within 4 or 5 cm.
of the eye and still see the image clearly, and see details which could
not be seen at a greater distance.

As shown in the figures of the visual angle (fig. 144), the closer the
eye gets to the object the greater will be the visual angle, hence
details are shown which did not appear at a greater distance. One
of the best methods of trying this experiment is to use for object a
small mark made with ink or a glass pencil on a window or on a
milky or transparent lamp shade. Then there will be plenty of light.
The physiological explanation of the power to see clearly through
the pinhole at a distance of 5 cm., when, if the eye looks directly
at the object, it should be about 25 cm. from the eye, is that with
the pinhole the beam is so narrow that the rays entering the eye are
practically parallel. If one takes away the card, the beam gets very
wide and the eye has only a blurred impression, the diffusion circles
are so large.

In case one loses his spectacles or has the accommodation . paralyzed
by atropin for testing the eyes, it is possible to read fairly well with the per-
forated card if the print is in a brilliant light. The field which can be seen at
one time is very small, so one must move the print or the head almost constantly.

LENSES

The usual and most effective means for increasing the visual angle
when examining small objects is by the use of lenses, singly or in
combination.

§ 8. Lens. — A lens means a mass of glass or transparent mineral
substance with one plane and one curved, or with two curved sur-
faces.

The lens is usually a segment of a sphere or of two spheres (fig. 8).
In dealing with lenses mention must frequently be made of the
optical center of the lens, the principal axis, secondary axis, and the
principal focus. These are illustrated in figs. 8, 11-12, and are
briefly:

MICROSCOPES AND THEIR PARTS

[CH. I

(i) Optical center. — The point in or near a lens through which, if
rays pass, they will suffer no angular deviation, and the emerging

ray will be parallel to the incident ray
(fig. 8 c.l).

(2) Principal axis. — The axis pass-
ing through the centers of curvature
of the two spheres whose surfaces
bound the lens (fig. 8).

(3) Secondary axis. — Any axis oblique
to the principal axis, but passing
through the optical center of the lens
(figs. 11-12). A ray along a secondary
axis undergoes no angular deviation,
although it may suffer displacement as
a ray in traversing a piece of plane glass

(fig- 99)-

(4) Principal focus. — The point
where rays of light, parallel to the
principal axis, cross after traversing
the lens (fig. 10). Every lens has two
principal foci, one on each side (fig. 10.)

With concave lenses the foci are
virtual (fig. 9).

§ 9. Refraction. — By this is meant the
change of direction of oblique light in pass-
ing from one transparent medium into an- '
other of different density. The possibility
of the production of images by lenses de-
pends upon refraction. (See § 239 for non-
oblique light and refraction.)

The amount of bending of the oblique
rays depends on two things:

(1) The difference of density of the

two refracting media; the greater the difference, the greater the
refraction.

(2) The obliquity with which the light strikes the second medium.

FIG. 8. LENS WITH OUT-
LINES OF THE Two SPHERES

OF WHICH IT IS A SEGMENT.

Axis The principal optic
axis, the line joining the two
centers of curvature (c c'}.

c c' Centers of curvature,
— centers of the two spheres
from which the lens is de-
rived.

r r' Parallel radii.

/ /' Tangents at the ter-
minal points of the radii.

cl Center of the lens, —
point where the line joining
the radii at the tangential
points crosses the principal
axis.

FIG. 9. CONCAVE LENS
SHOWING VIRTUAL Focus
(F).

CH. I]

MICROSCOPES AND THEIR PARTS

The greater this obliquity, the greater the bending of the light, in
accordance with the law of sines (§ 240).

§ 10. Geometrical construction of images. — In this book the
lenses shown are thick, but the course of the rays, for simplicity, is
shown to be as if the lenses were infinitely thin, that is, they show
all the bending at one plane (the refracting plane, figs. 11-12). In
reality there is one refraction at the incident or entering surface and
one at the emerging surface. With thick lenses like those figured,
there will be no an-
gular deviation for rays 2 ^ \lf
traversing the optical
center of the lens, but
there will be a certain
amount of displace-
ment, although the
emerging ray will re-
main parallel to the
entering or incident
ray (fig. 64).

For the construction
of images it is necessary
to know the position
of the principal focus
and the optical center
of the lens.

FIG. 10.

LENS WITH A PRINCIPAL Focus ON
EACH SIDK.

Axis The principal optic axis.

F The principal focus, — the point on the axis
(at which rays parallel with the principal axis
cross.
The arrows indicate the direction of the light.

It should be remembered
in making the drawings for
the geometrical construc-
tion of images that there are two fundamental laws which must always be
obeyed.

(1) Light rays extend in straight lines in a transparent medium of uniform
density, and whenever the direction is to be changed the light must meet a
different refracting medium, or a reflecting surface. That is, the direction of a
ray of light may be changed by using a mirror, or by putting in its path a trans-
parent medium of greater or less refracting power.

(2) The second law is, that objects are always seen in the direction in which the
light reaches the eye, regardless of the actual position of the object. This will be
abundantly illustrated in the chapter on drawing; and every one knows that ob-
jects seen in a mirror are not where they appear to be in the mirror.

MICROSCOPES AND THEIR PARTS

[CH. I

v^— L— -*«

V- lm»ge ^

Fro. it-12. GEOMETRICAL C'ON-
ST RUCTION OF REAL AND OF VlR-
TUAL IMAGES.

Object, Object The object of
which an image is to be formed.

Axis, Axis The principal optic
axis extended above and below
the lens to the object and image.

S Axis, S Axis Secondary axis
passing from the object through
the center of the lens.

ft ft ft f The principal foci of
the two lenses.

r-p The plane of refraction
(the ideal plane at which all the
refraction is made to occur in
diagrams of thick lenses).

R. Image Real image.

F. Image Virtual image indi-
cated by broken lines as it has no
real existence.

o b, r m Rays of light indi-
cated by lines passing from the
extremities of the object to the
extremities of the real image,
which is inverted.

o b, i 2, 3 4, v m Lines rep-
resenting rays of light from the
object passing in a diverging
manner above the lens, and ex-
tended by broken lines below the
lens to form a virtual image at
their crossing points, v m.

§ 11. Construction of real images.

— (i) The object must be situated
outside or beyond the principal focal
point (fig. n).

(2) From some point in the object,
draw a line to the refracting plane
of the lens (§ 10) parallel to the
principal axis, and from this crossing
point at the refracting plane of the
lens to the focus of the lens, and
continue the line indefinitely (fig.

H).

(3) From the same point of the
object as in (2), draw a secondary
axis through the optical center of
the lens and extend it indefinitely
(fig. n).

The image of the point in the
object from which the two lines
were drawn will be located at the
point where the two extended t lines
cross above the lens (fig. n).

The image of all the other points
of the object may be determined
by drawing lines from them exactly
as just described.

If the image is known one can
find the object by reversing the
process just described.

§ 12. Construction of virtual im-
ages. — (i) For these the object
must be somewhere between the
principal focus and the lens.

(2) From some point in the
object draw a line to the refracting
plane of the lens, parallel to the

CH. I]

MICROSCOPES AND THEIR PARTS

principal axis, and from this point through the principal focus, and
continue it indefinitely.

(3) From the same point of the object as in (2) draw a secondary
axis through the op-
tical center of the lens
and extend it indefi-
nitely.

The two lines will
not cross above the
lens, but if they are
extended below the
lens (fig. 12) they will
cross, and the crossing
point locates the image.
But as there are no
light rays extending in
this direction the im-
age is imaginary or
virtual. That is, it
looks as if the rays
reaching the eye origi-
nated from the point
where the rays would
cross if extended back-
ward.

§ 13. Relative posi-
tion of object and im-
age. — The general law
is that the nearer the object to the principal focus, the farther
away is the image; and conversely, the nearer the image is to
the principal focus, the farther from it must be the object.
And from the law of similar triangles, the size of the image is
to the size of the object as the distance of the image from the
center of the lens is to the distance of the object from that
center. In a word, the nearer the object to the principal focus
the farther away the image from that point, and the greater the

FIG. 13-14. REAL IMAGE WITH THE OBJECT FAR
FROM AND NEAR TO THE PRINCIPAL FoCUS.

Axis, Axis The principal optic axis extended
above and below the lenses.

/> /> /» / The principal foci of the lenses.

L c, L c The same lens with the object farther
from and nearer to its principal focus.

A B, Bf A' The object and its inverted image
when the object is far from the principal focus.

A B, B' A' The object and larger inverted real
image when the object is near the principal focus.

MICROSCOPES AND THEIR PARTS

[CH. I

FIG. 15. TRIPOD

MAGNIFIER.

relative size of the image. This is equally true of real and of virtual
images (figs. 13-14)-

§ 14. Mounting of simple microscopes. — Magnifiers are arranged

in mountings to be held in the
hand; for example, reading glasses
and pocket magnifiers. The tripod
magnifier (fig. 15) may be held in
the hand or supported by its legs
over the object to be seen. Some-
times there is a special support with
arrangements for focusing as well as
holding the magnifier in any desired
position (fig, 17). This arrangement
is especially desirable when mag-
nifiers are used for dissection. For
the purposes of dissection and examin-
ing objects under a small magnification, binocular arrangements like
spectacles are very convenient, as one can move the head and bring
the object into view at will.

COMPOUND MICROSCOPE AND PARTS

§ 15. Compound microscope. — This, as shown in figs. 2 and 18,
and explained above, aids the eye in obtaining an enlarged retinal
image by two steps, viz., the for-
mation of a large real image by the
objective and a retinal image of
this real image by means of the
microscope ocular, and the cornea
and crystalline lens of the eye, the
ocular acting in general like a
simple microscope (§2).

For holding the objective and oc-
ular and focusing the microscope, FlG l6 TRIPOD MAGNIFIER WITH
there are a number of mechan- A SECTION REMOVED TO SHOW THE

v Two COMPONENT, CONVEX LENSES
ical arrangements necessary, .tor AND INTERVENING DIAPHRAGM.

illuminating the object there is

CH.I]

MICROSCOPES AND THEIR PARTS

usually a mirror and often a condenser. It is customary and con-
venient to divide the parts of a compound microscope into two
groups: (i) the optical parts, and (2) the mechanical parts (fig. 26).

- OPTICAL PARTS OF A COMPOUND MICROSCOPE

§ 16. Objective. — This is a lens, or combination of lenses, which,
under the proper conditions, produces an enlarged, inverted image
of some object (figs, n, 18).

FIG. 17. ADJUSTABLE LENS HOLDER WITH JOINTS.
Base The heavy base supporting the lens holder.
Coarse Adjustment The rack and pinion for focusing the lens.
Joint, Joint The joints enabling one to put the lens in any desired position.
Lens This is held in a spring fork or in a socket.

Practically all microscopic objectives are composed of one or of
several combinations of lenses. The purpose of combining the

MICROSCOPES AND THEIR PARTS

[Cn. I

lenses is to produce an image as nearly as possible like the object
itself, by doing away with certain defects or aberrations inherent in
simple lenses (fig, 19-21).

§ 17. Optical designation of objectives. — As will be seen in
sections 20-34 objectives are designated in various ways to indicate
one or more of their special qualities. They have also been merely
lettered or numbered. This method is purely arbitrary, and gives

no infcrrration.

In striving to find some
method of designation which in
itself would give some definite
information to the user, micro-
scope makers adopted the plan
of engraving the equivalent/oca/
length or focus (E.F.) upon
each objective, thus indicating
that at any given distance the
objective composed of several
lenses would give an image of
the same size as a simple lens
of the designated focal length.

The given distance agreed
upon by practically all makers
at which to measure the image
is 10 inches or 250 millimeters,
as this distance is assumed to
be the one giving to normal
adult human beings, the most
perfect vision for near objects.

FIG. 1 8. DIAGRAM OF A KEPLEKIAN,
COMPOUND MICROSCOPE AND THE EYE
OF THE OBSERVER TO SHOW THE DIF-
FERENT IMAGES, TUBE-LENGTH, AND
THE IMAGE DISTANCE OF THE PRO-
JECTED VIRTUAL IMAGE.

/ Focal point of the objective.

r i Real image formed by the objective.

r t Retinal image in the eye.

cr Cornea of the eye.

When the long-tubed microscopes
were in use it was natural to adopt 10
inches or 2 50 millimeters for the tube-
length, then the virtual image (fig. 18)
would appear to be at about the level of the stage of the microscope where the
actual object is situated, and the appearance to the observer is as if the object
itself were of that size. From the optico-physiological standpoint this was a very
logical tube-length to adopt; but with the study of living things and of objects in
liquids it is almost imperative that the microscope be vertical and thus give a

CH. I] MICROSCOPES AND THEIR PARTS 19

horizontal stage (fig. 26). With the microscope vertical, however, the top of
the ocular was so high that the observer needed to use a very high chair for
the ordinary table, or an especially low table. To overcome the difficulty, the
tube of the microscope was shortened and is now almost universally 160 mm.
from the level where the objective is inserted to the top of the tube. With the
250 mm. or lo-inch tube-length the special or initial magnification of the ob-
jectives and the oculars was found in the usual way by dividing 250 mm. or
10 inches by the equivalent focus (e.f.). For example, if the objective has an
equivalent focus of 25 mm. (i in.), its power would be rated as 250/25 — 10,
or 10/1 = io. With an ocular of the same equivalent focus the magnification
would be found in the same way, and would likewise be io. Then the total
magnification of the microscope with a 25 mm. objective and a 25 mm. ocular
would be 10x10 = 100. Working on this plan, it was necessary merely to mark
the objective and ocular with their equivalent foci (e.f.), and the observer could
make any desired combinatipn.

When the short or 160 mm. tube came into use, while the magnification of a
given ocular remained as before, the magnification of the objective x of any
given e.f. was proportionally lessened by the shortened tube. For some time,
however, the rating of the objective was not changed, and the total power of the
microscope was made correct by giving the ocular a rating sufficiently below its
actual power to compensate for the error introduced by shortening the tube.
This was not a satisfactory solution, and finally in 1901-2 the Spencer Lens
Company of Buffalo introduced the plan of giving the objectives their true
magnifying power on the short tube, and the oculars their true rating; and
instead of marking only the equivalent focus on the objectives and oculars, the
initial magnification was added. For example, an objective of 16 mm. e.f. was
given an initial magnification of lox with the short tube, and the 25 mm. e.f.
ocular was marked tox also. With this combination the total magnification of
the microscope would, then, be 10x10 = 100.

With this method of designation it is very simple to make any desired com-
bination by selecting the initial magnifications of objective and ocular which
when multiplied together give the power needed. It should be remarked, how-
ever, that one must not rely on this if exactness in a given case is necessary, for
even when one makes the combination indicated there is considerable variation
as one can see by consulting the table on page 67. In any special case one must
determine the exact magnification of the combination used by one of the methods
given in Chapter VIII.

§18. Determination of the initial magnification of an objective with the
160 mm. tube-length. — The tube-length is made exactly 160 mm. This is
most easily and accurately determined by inserting into the open body and
draw-tube of the microscope a slender rod precisely 160 mm. long. A block of
wood or other flat object is held against the opening for the insertion of the
objective to stop the rod at the right point. Then the draw-tube is adjusted
until the upper end of the rod is just on the level with the top of the draw-tube.
It should be noted that with some of the earlier microscopes the mark of 160
mm. on the draw- tube indicated the proper tube-length without the revolving
nose-piece; hence, to get the proper 160 mm. tube if the nose-piece is used,
the draw-tube must be pushed in as many millimeters as the nose-piece adds
at the lower end. With the newer microscopes the 160 mm. mark on the draw-
tube indicates the tube-length with the nose-piece in place. All of which em-
phasizes the need of making a special determination in each case if real accuracy
is desired by the worker.

After the tube-length is made correct, the given objective whose initial mag-
nification is to be found is screwed in place and a lox ocular is put into the tube.

MICROSCOPES AND THEIR PARTS

[Cn. I

For object a stage micrometer in i/ioth and i/iooth mm. is placed on the
stage and sharply focused. The lox ocular is then removed and a icx positive
ocular with micrometer rulings in i/ioth mm. is inserted. The ocular mi-
crometer is sharply focused by raising or lowering the eyelens or combination,
and then, without changing the focus of the objective in the least, the draw-tube
is pulled out or pushed in until the lines of the stage micrometer are perfectly
sharp upon the ocular micrometer. Make the lines of the two micrometers
parallel and see how many of the spaces in the ocular micrometer are required
to measure one or more of the spaces on the stage micrometer.

FIG. 19. Low OBJECTIVE IN SECTION.

Axis The principal optic axis of the objective.
fl The front lens of the objective.

be The back combination composed of a concave and a convex lens.
Stage The stage of the microscope in section.

Mirror The mirror is above the stage in this case and reflects light down upon
the object.

rl Reflected light from the object.

$/, sp The glass slide and the specimen on the slide.

eg Cover-glass over the specimen.

FIG. 20. HIGH POWER OBJECTIVE IN SECTION.

Axis The principal optic axis of the objective.

be Back combination of a double convex and a plano-concave lens.

me Middle lens combination.

fl Front lens of the objective.

eg, s/>, si The cover-glass, specimen, and slide.

Stage The stage of the microscope in section.

Mirror The mirror reflecting parallel rays up through the specimen.

FIG. 21. HIGH-POWER OBJECTIVE OF FOUR COMBINATIONS.
i The front lens.

2, 3, 4 The three combinations of lenses, the back combination (4) composed
of three lenses.

CH. I] MICROSCOPES AND THEIR PARTS 21

§ 19. Names applied to parts of objectives. — As objectives have
usually two or more combinations of lenses (figs. 19-21) it is con-
venient to have a name for each combination.

(1) Front combination. This is the part of the objective nearest
the object.

(2) Back combination. The combination of lenses farthest above
the object, and, hence, nearest the ocular.

(3) Intermediate or middle combination. The lenses between the
front and back lenses. Sometimes there are two or more inter-
mediate combinations (fig. 21).

KINDS OF OBJECTIVES

Depending on their construction or iranner of use, objectives have
received special designations or names.

§ 20. Dry objectives. — These are objectives in which air is be-
tween the objective and the object or cover-glass (fig. 43).

§ 21. Immersion objectives. — With these there is some liquid
between the front of the objective and the object or the cover-glass
(fig. 20). Immersion objectives are usually designated by the name
of the liquid used.

§ 22. Water immersion objectives. — With these there is water
between the cover-glass or the object and the front lens.

§ 23. Homogeneous or oil immersion objectives. — The immer-
sion liquid in such objectives has the same refractive index (see § 269)
as glass, hence the light suffers no refraction in passing from the
glass slide and cover-glass into the immersing liquid, and from that
into the objective. As the liquid used with these objectives is nearly
always thickened cedar-wood oil, they are mere frequently called oil
immersion than homogeneous immersion objectives.

§ 24. Achromatic objectives. — These are objectives in which the
image is practically free from rainbow colors. They are composed
of one or mere combinations cf convex and of concave lenses (see
§ 257, under chromatic aberration). All good microscope objec-
tives are achromatic.

§ 25. Aplanatic objectives, etc. — These are objectives or other

22 MICROSCOPES AND THEIR PARTS [CH. I

pieces of optical apparatus (oculars, illuminators, etc.) in which the
spherical distortion is wholly or nearly eliminated, and the curva-
tures are so made that the central and marginal parts of the objec-
tive focus rays at the same point or level. Such pieces of apparatus
are usually achromatic also.

§ 26. Apochromatic objectives. — By this is meant objectives in
which by means of special forms of glass and a natural mineral (cal-
cium JIuor id, Jluor tie, fluor-spar) the color and the spherical correc-
tions have been made especially perfect, that is, rays of three spec-
tral colors are combined into one focus instead of rays of two colors
as with the ordinary achromatic objectives.

§ 27. Fluorite objectives. — These are objectives containing one
or more fluorite lenses with lenses of the new kinds of glass. They
are more perfect than the achromatic objectives, approximating the
apochromatics, and are good for photography.

§28. Non-adjustable or unadjustable objectives. — Objectives
in which the lenses or lens systems are permanently fixed in their
mounting so that their relative position always remains the same.
Lower power objectives and those with homogeneous immersion are
mostly non-adjustable. For beginners and those unskilled in mani-
pulating adjustable objectives (§ 29), non-adjustable ones are more
satisfactory, as the optician has put the lenses in such a position that
the most satisfactory results may be obtained when the proper thick-
ness of cover-glass and tube-length are employed.

§ 29. Adjustable objectives. — An adjustable objective is one in
which the distance between the systems of lenses (usually the front
and the back systems) may be changed by the observer at pleasure.
The object of this adjustment is to correct or compensate for the
displacement of the rays of light produced by the mounting medium
and the cover-glass after the rays have left the object. It is also to
compensate for variations in tube-length (§ 149). As the displace-
ment of the rays by the cover-glass is the most constant and im-
portant, these objectives are usually designated as having cover-glass
adjustment or correction. (See also practical work with adjustable
objectives, § 149).

§ 30. Variable objective. — This is a low power objective of 36 mm.

CH. J] MICROSCOPES AND THEIR PARTS 23

(4x) to 26 mm. (6x) equivalent focus, depending upon the position of
the combinations. By means of a screw collar the combinations may
be separated or brought closer together. If they are separated the
power is diminished; and if brought closer together, the power is
increased.

§ 31. Illuminating or vertical illuminating objectives. — These
are designed for the study of opaque objects with good reflecting
surfaces, like the rulings on metal bars and broken or polished and
etched surfaces of metals employed in «inicro-metallography. The
light enters the side of the tube or objective and is reflected verti-
cally downward through the objective and thereby is concentrated
upon the object. The object reflects part of the light back into the
microscope, thus enabling one to see a clear image.

§ 32. Dark-field objectives. — Objectives for the microscope con-
structed with a numerical aperture low enough so that no light from
the dark-field condenser can enter the objective directly. For ho-
mogeneous immersion objectives this is accomplished by inserting
a reducing diaphragm, or by a special cons true tion or mounting of
the objective. (See also § 183.)

§ 33. Ultra-violet objectives. — Objectives constructed of quartz
or ultra-violet transmitting glass.

§ 34. Low and high objectives. — A low objective is one that
magnifies relatively little, and a high objective is one that magnifies
much (figs. 19-21). By looking for the equivalent focus or the initial
magnification of an objective one can tell precisely concerning its
magnification. The mounts of objectives are, for the most part,
so nearly alike that it is not easy to tell them apart at a glance.
It is possible so to mark them with different colored rings that no
confusion need arise when one is deeply immersed in thinking about
the structures being examined. The author has adopted the follow-
ing color scheme: The colors chosen are those of the sun spectrum.
The color of longest wave-length, red, is used for the objective of
longest focus (40 or 32 mm.); for the 16 mm., pink; for tjie 8 mm.,
orange-yellow; for the 4 mm., green; for the 3 mm., blue; and for
the oil immersion, black. Colors avoid this confusion or loss of time
in trying to see the equivalent focus or the initial magnification.

MICROSCOPES AND THEIR PARTS

[CH. I

Perhaps a better method would be for the manufacturers to make the
mountings of the objectives so different that there could be no confusion.

OCULARS AND THEIR DESIGNATION

§ 35. An ocular or eyepiece for the microscope consists of one
or more converging lenses or lens systems next the eye. Its main
purpose is to act with the eye as a magnifier of the real image
formed by the objective (fig. 18). Incidentally the ocular also serves
to correct some of the defbcts of the objective.

Oculars may be divided into groups according to their construction
or action.

§ 36. Positive oculars. — With these the real image of the objec-
tive is formed below all the lenses of the ocular (figs. 22, 23) hence all
the lenses of the ocular, together with the eye, form a real image on

the retina, of the real image formed
by the objective.

§ 37. Negative oculars. — In
these the real image formed by
the objective is between the lenses
(figs. 24, 25).

In a negative ocular the lower
or field lens acts with the objective
to form the real image, while the
upper or eyelens acts with the eye
to form a retinal image of the real
image (figs. 24, 25).

FIG. 22. RAMSDEN OCULAR WITH
THE REAL IMAGE BELOW AND THE
EYEPOINT ABOVE.

Axis The principal optic axis of the
ocular,

(/, ri The ocular diaphragm and the
real image formed by the objective below
all the lenses of the ocular.

Fl The field lens of the ocular.

El The eyelens.

Eycpoint The eyepoint in section
and in face view, looking at the upper
end of the ocular.

CH. I]

MICROSCOPES AND THEIR PARTS

Positive and negative oculars can be readily distinguished by in-
spection, as the ocular diaphragm, at the level where the real image
of the objective is formed, is between the lenses of the negative type,
and below all the ocular lenses of the positive type (figs. 22, 23, 24).

Eye- Point

Ey«- Point

FIG 23. POSITIVE COMPENSA-
TION OCULAR.

Axis The principal optic axis of the ocular.

d> ri The ocular diaphragm and the real image.

FL The field combination composed of three lenses.

EL The eyelens.

Eye- point The eyepoint in section and as seen by looking down upon the enc
of the ocular.

FIG. 24. LOW-POWER HUYGENIAN OCULAR IN SECTION.
Axis The principal optic axis of the ocular.
FL Field lens of the ocular.

d, ri Diaphragm and real image between the ocular lenses.
EL Eyelens of the ocular.

Eyepoint The eyepoint seen in section and by looking down upon the
end of the ocular.

MICROSCOPES AND THEIR PARTS

[CH.I

Eye- Point

§ 38. Huygenian ocular. — A negative ocular devised by the
Dutch astronomer Huygens is the most common ocular used on the
microscope, and consists of a plano-convex field lens and a similar
but higher power, eyelens, the convex surfaces of both facing down-
ward (figs. 24, 25). Theoreti-
cally the focal length of the field
lens is about three times that
of the eyelens, but in practice
the ratio varies with the power,

.»^^^^^^_y being i to 1.5 or i to 2 with low

il (/ ~"t fi^^BI^^^ powers and nearer i to 3 with

the high powers. The ocular
diaphragm is placed approxi-
mately at the focus of the eye-
lens.

§ 39. Ramsden ocular. —
This is a positive ocular com-
posed of two plano-convex
lenses with the convex faces
turned toward each other,
and so arranged that the
real image is formed below
both lenses (fig. 22), not be-
tween them, as with the
Huygenian ocular. In the
best mod*ern forms of Rams-
den ocular the simple lenses

FIG. 25.

HIGH-POWER HUYGENIAN
OCULAR.

Axis The principal optic axis.

FL Field lens.

d, ri The diaphragm and real image
between the ocular lenses.

EL Eyelens.

Eye-point The eye-point in section
and face view, looking down upon the
upper end of the ocular.

are not used, but achromatic
combinations. The Ramsden
form is often used for ocu-
lar micrometers (§378).
§ 40. Compensating oculars. — These are either positive or nega-
tive oculars chromatically overcorrected to compensate and correct
the residual color defects in the extra-axial portion of the visual
field due to the non-achromatic front lens of the objective (fig. 23).
They are regularly used with apochromatic objectives, and may be

CH. I]

MICROSCOPES AND THEIR PARTS

OCULAR

Coarse
Adjui

used to advantage with high-angled objectives of the ordinary type.
(See further, § 260.)

§ 41. Telaugic oculars. — These, as the name indicates, have a
high eyepoint making it possible for persons who wear spectacles
for eye defects to keep the glasses on while looking into the micro-
scope. Most oculars of the usual
form have the eyepoint so close
to the ocular that one cannot
wear spectacles and get the eye
close enough to the eyelens to
see the entire microscopic field
(§ 145). Besides the high eye-
point, these o,culars give a large,
flat, brilliant field. The lox
used by the author is positive,
and is composed of two com-
binations with the convex faces
inward, and the plane faces
outward as in fig. 22.

§ 42. Projection oculars. —
These are oculars in which the
upper combination of lenses is
movable to enable one to focus
the real image upon different
distances of the receiving screen.
They are especially useful in
photographing with high powers
(§ 474b).

§ 43. Ultra-violet oculars. —
Oculars whose lenses are com-
posed of quartz or ultra-violet
transmitting glass.

OBJECTIVE

:NSER

FIG. 26. LABORATORY COMPOUND MI-
CROSCOPE WITH THE PARTS 'NAMED.
Mirror, Condenser, Objective, Ocular
The optical parts of the microscope.

Tube-length This is the space between
the insertion of the objective below and
that of the ocular above. It is most com-
monly 1 60 millimeters.

Mechanical parts These are named in
order from the base.

Trade names for oculars. — These are very numerous as: holoscopic,
hyperplane, orthoscopic, planoscopic, etc. In these the simple Huygenian and
Ramsden forms are usually somewhat modified with the purpose of improving
the optical qualities.

28 MICROSCOPES AND THEIR PARTS [Cn. I

So-called demonstration oculars are also available by which two
persons can look into the same microscope at once. This recalls the
devices of Harting and Nachet by which two, three or four persons
could look into the same microscope. (Harting, vol. 3, 1866, figs.
120-127).

§ 44. Designation of oculars. — Formerly, oculars were, and to
some extent at present still are, lettered or numbered A, B, C, D;
i, 2, 3, 4; I, II, HI, IV; etc. This is a purely arbitrary designation
except that the earlier the letter or the lower the number, the lower
the magnification.

At present the progressive manufacturers give the equivalent focus
or the initial magnification or both. If the magnification is given, it
shows how much the ocular increases the magnification of the ob-
jective. If the power of the objective is given (§ 18), one can get
the magnification of the combination approximately correct by
multiplying the power of the objective by the power of the ocular, but
see the table on page 67. However, the exact magnification in any
given case must be especially determined (§§ 367, 396).

BINOCULAR MICROSCOPES

Very early in the history of the telescope and of the compound
microscope, as nature has endowed us with two eyes, it was insisted
upon that both eyes should be used in examining objects instead of
using only one eye. This required two similar microscopes or
telescopes side by side and the right distance apart for the two eyes.
There still persists in the common opera-glasses the original binocu-
lar Dutch telescope-microscope.

§ 45. Binocular microscopes with two objectives and two oculars.
— These are in principle like the original binocular microscope of
Cherubin d'Orleans (1677) except that his had no erecting prisms.

These instruments have been greatly improved in every way, and
with the devices for quickly changing the paired objectives are
indispensable in a biological laboratory, especially where much dis-
section under the microscope must be undertaken, and where objects
ire to be seen in relief, like the villi of the intestine, etc. The

CH.I]

MICROSCOPES AND THEIR PARTS

mountings of these binoculars are as varied as the uses to which
they are put. One of the simple forms of binocular dissecting
microscopes is shown here (fig. 27).
The reader is advised to secure cata-
logues of the manufacturers in which
are illustrated all the different forms.
He can then select the one best adapted
to his purpose.

§ 46. Binocular microscopes with
two oculars, but with a single objective.
— The double microscope with two
complete tubes, two objectives, and
two oculars is not available for high
powers, for the two objectives cannot
be close enough together to bring an
exceedingly small object into the field
of both microscopes at the same
time. Naturally, therefore, an effort
was made to use a single objective and
to divide the light passing through it
so that half should go to the right and
half to the left eye. The first success-
ful binocular of this kind was invented
by Riddell of New Orleans in America
in 1851. In this, four prisms are used
just above the objective which serve to
divide the light equally and to pass it
on to the two eyes through two parallel
tubes, each with its own ocular. Later
a satisfactory form was invented by
Mr. Wenham of England in which
there is but a single prism (fig. 28).
Neither of these forms permitted of very high powers.

The light from two sides of the objective was separated and sent
to the two eyes just as if the single objective were divided into a
right and left half. This gave to each eye half the aperture and half

FIG. 27. DOUBLE OBJECTIVE
BINOCULAR OF THE GREEN-
OUGH TYPE

(Outline drawing based on
Microscope KA, of the Bausch
& Lomb Optical Co.)

1-2 The drums containing
the Porro erecting prisms.
These may be rotated to give
the correct interpupillary posi-
tion to the oculars.

3 Focusing wheels.

4-5 The two similar objec-
tives. 4 is fitted with a fine
adjustment to compensate for
difference of focus of the eyes
of the observer.

6 Hand-rests when dissect-
ing an object on the stage.

30 MICROSCOPES AND THEIR PARTS [Cn. I

the diffracted light from any one point, and therefore gave only the
resolution, brilliancy and clearness of image of half the aperture. It
was believed, however, that since the two eyes receive all the aper-
ture, the brain in fusing the two images would give the impression
that would be received by one eye receiving the full aperture, and
besides would give a stereoscopic effect. These binoculars were
rather large and cumbersome, and were not much used for serious
investigation.

§ 47. Necessary qualities of binocular microscopes (§§ 45-46). —
i. The light to each eye should be of the same color and of the
same intensity.

2. The real image formed by the objective in each tube must be
of the same size, then similar oculars can be employed (§ 49).

3. The full aperture and equal diffracted light should be supplied
to each eye.

4. The ocular tubes should be laterally adjustable so that the eye-
points of the oculars may correspond with the pupillary separation
in the eyes of the observer.

5. The entire microscope should be focused by a coarse and a fine
adjustment as with monocular microscopes.

6. There should be a special focusing device on one side to com-
pensate for slight differences in the two eyes.

7. For single-objective binoculars, objectives of all powers should
give good results.

§ 48. Advantages and disadvantages of binocular microscopes. —
The advantage of using both eyes is unmistakable. Both are trained
and stimulated alike as in naked-eye vision. The advice is common
to keep both eyes open and to use the eyes alternately with the
monocular microscope, but this advice is easier to give than to fol-
low. As it is easier to see with the naked eye, the eye outside the
microscope is likely to dominate the situation and the microscopic
image disappears, consequently most users of the microscope shut
one eye when looking into the instrument. It is easy to keep both
eyes open by means of the screen or shade close to the unused eye
(fig. 36); but the experience of many people who have used the
monocular microscope during many years has been that the eye

CH. I]

MICROSCOPES AND THEIR PARTS

most used gains in ability to see fine details, but loses in sensitive-
ness to light. This is easily demonstrated by using a high power on

FIG. 28. WENHAM'S BINOCULAR MICROSCOPE.
(From Carpenter).

A Section of the microscope with the two converging tubes. By pulling
out draw-tubes the oculars are separated for the correct pupillary distance of
each observer.

L R The axes of the left and right tubes.

a The prism which divides the light from the object.

c b The field lenses of the two oculars.

B Enlargement of the dividing prism.

o, 6, c, d Path of the light in the prism for the left eye.

As shown, the light to the right eye extends straight upward. This arrange-
ment is limited to rather low powers.

fine details, and looking first with one eye and then with the other.
No matter how sharply the image is focused, the " microscopic eye "
can see the most detail, but the other eye sees a brighter image, but
less detail. No doubt beginners can get the sense of depth, that is,

32 MICROSCOPES AND THEIR PARTS [Cn. I

the stereoscopic effect, more easily with a binocular than with a
monocular. For those who have learned to judge of the relative
vertical position or depth of objects by focusing up and down, no
great help is given by the binocular, and even with the binocular
the final test of relative depth must be by focusing up and down.

Among the disadvantages of the binocular may be mentioned its
greater cost, and for many at least, the increased light necessary for
illumination. The tube-length must be varied in lateral adjustment
for the pupillary separation of different observers, and this changes
the magnification which may interfere with the optical corrections
(§ 143). For micrometry, photography, the micro-spectroscope and
micro-polariscope it is not so well adapted as is a monocular instru-
ment. To overcome these difficulties the manufacturers have pro-
vided a device for pushing the prisms aside and thus making a
monocular instrument, or the entire binocular tube arrangement is
removed and a monocular tube put in place — that is, in all modern
forms provision is made for converting the binocular microscope
into a monocular one (figs. 35, 36). Several manufacturers now pro-
duce binocular eye pieces for monocular microscopes.

While it is a great advantage to use both eyes in vision, as far as
the microscope is concerned this is largely outweighed by the weari-
ness that conies from holding the head so rigidly to keep the eyes
over the eyepoints of the two oculars. In some cases workers in
industrial plants have asked to go back to monoculars on account
of the tiring effect of the rigid position. Of course, the advantage of
using both eyes as in natural vision is gained by using some form of
projection microscope like the Euscope described under drawing and
demonstration (§ 444). Finally, it should be remembered that the
more unlike the two eyes are, the less the usability of binocular in-
struments other than correcting spectacles.

§ 49. Modern single-objective binocular microscopes. — In 1902
Mr. Frederick E. Ives, in a paper before the Franklin Institute of
Philadelphia, showed how it was possible to construct a binocular
microscope using one objective in which each eye received the full
aperture from each point of the object and also shared equally the
diffracted light. This microscope could be used for all powers from

CH. I]

MICROSCOPES AND THEIR PARTS

the lowest dry to the highest immersion objective. At that time he
had constructed and used such a microscope (fig. 29). Instead of
dividing the light reaching the objective into two halves, each half
with half the aperture, he utilized a half-silvered prism which
allowed half the entire light of every beam to pass through the tube
to one eye, and reflected half to the other eye. ' In this way each

Ocular I

•pa

FIG. 29. IVES BINOCULAR ARRANGEMENT FOR ALL POWERS.
(Journal of the Franklin Institute, Dec. 1902).

Objective The single objective.

pb The prism box at the lower end of the tube.

a, b, c The prisms dividing the light equally from each point to the two eyes.

a, b The transparent silvered surface m the prism allowing half the light to
pass through and half to be reflected to the right.

c Prism at the right reflecting the light upward to the right eye; as, adjust-
ing screw to tilt the prism c, at the correct angle for the position of the right
ocular.

apd Adjustment for the pupillary distance.

Ocular i, Ocular 2 The oculars for the right and the left eye.

Axis i The principal optic axis for the left eye.

Axis 2 The principal optic axis for the right eye.

Due to the length of the prism c, this axis is optically of the same length as
Axis i for the left eye.

24 MICROSCOPES AND THEIR PARTS [Cn. I

eye receives the full aperture of light from each point of the object
and also an equal share of the diffracted light. Furthermore, he
showed that by a proper extension of the glass in the reflecting prism
of the second tube the two optical paths were made equal, hence
gave equal magnifications, and similar oculars were used for each
tube, (fig. 29, Axis i, Axis 2.)

Every point in which the new forms of binoculars are superior
in optical performance over the original forms of Riddle and Wen-
ham was clearly stated by Ives, except that now the half-silvered
prism is half-coated with platinum instead of with silver. The
platinum gives a more equal color to the two images.

It is evident that the actual path in millimeters is greater for Axis 2 of fig. 29
than for Axis i. This would result in the magnification being greater in the tube
with the longer axis but for the optical device of extending the glass prism on
that side sufficiently to elevate the position where the real image is formed with-
out increasing the magnification. As in fig. 52 A, B, C the object seems to be
raised by the thickening of the cover-glass, so extending the glass prism in the
binocular raises the real image, and when the right thickness is used, the two
tubes of the microscope are made optically equal. The practical opticians speak
of this as a shortening of the optical path by means of the extra thickening of
the glass prism (figs. 29-31), For Mr. Ives' original paper see the Journal of the
Franklin Institute, vol. 154, Dec. 1902, pp. 441-445- See also Conrad Beck,
Jour. Roy. Micr. Soc., 1914, pp. 17-23.

§ 50. Converging or parallel tubes for binocular microscopes. —

The first binocular microscope (fig. 289) and all erecting binoculars
at present have converging tubes. This is mechanically necessary
to bring the two objectives close enough together and to separate
the oculars sufficiently for the two eyes.

The single-objective binoculars of Riddle, Harting and Nachet had
parallel tubes. That of Wenham had converging tubes. The mod-
ern forms are also divided on the arrangement of the two tubes.
That of Ives had converging tubes (fig. 29), and those of the English
opticians and of one American firm are also converging. The Con-
tinental opticians and one American firm have the tubes parallel.

The adherents of the converging form urge that as naturally the
eyes converge for distinct vision at the near point, the tubes should
converge accordingly. Those who use the parallel tubes urge that
in microscopic work the eyes should be at rest as for viewing distant
objects and therefore that the eye axes should be parallel.

CH. I]

MICROSCOPES AND THEIR PARTS

In considerable experience with students and with others not
especially familiar with optical instruments, it was found that with
the converging tubes the binocular effect was more easily obtained
than with the parallel ones. For some, however, the effect was
quickly and easily gained with either form indifferently. Most
observers can learn to use either form. Occasionally a person can
never get the binocular effect with the parallel tubes, and not very
satisfactorily with the converging ones.

FIGS. 30-31. PRISM ARRANGEMENT FOR Two FORMS OF BINOCULARS FOR

ALL POWERS.

(Conrad Beck, Jour. Roy. Micr. Soc., 1914.)

In Fig. 30 the arrangement is for parallel tubes, and in fig. 31 for converging
tubes.

Object The object.

06 The objective.

/, r; I, r The right and left beams of light emanating from the same point of
the object.

As these beams extend through the objective and into the prisms they are
equally divided so that half the right beam goes to the left and half to the right
eye, and so with the left beam. This is indicated by the heavy and light broken
lines by which the two beams are indicated.

i, 2, 3, 4; i, 2 The four prisms in fig. 30, and the two prisms in fig. 31. The
prisms are of the necessary length to make the optical path of the light equal for
the two tubes, hence the magnification is equal for the two eyes.

MICROSCOPES AND THEIR PARTS

[CH. I

FIG. 32. THE BAUSCH & LOME OPTICAL Co.'s MODEL HA-8 WITH MECHANICAL
STAGE FOR STUDENT LABORATORIES.

(Courtesy of the Bausch & Lomb Optical Co.)

CH. I]

MICROSCOPES AND THEIR PARTS

TM EN T

0 U

,-:;:a«l?f%i|tif

' ' '

'BO R K "If PIJ

FIG. 33. THE SPENCER LENS Co.'s MODEL 13 MAH WITH MECHANICAL STAGE
FOR STUDENT LABORATORIES.

(Courtesy of the Spencer Lens Co.)

38 MICROSCOPES AND THEIR PARTS [Cn. I

CHARACTER OF COMPOUND MICROSCOPES

§ 51. Student microscopes. — A great deal of beginning work
with the microscope can be done with relatively simple and inexpen-
sive apparatus. Fortunately, the manufacturers now furnish all their
microscopes with excellent objectives and oculars so that the achro-
matic objectives and Huygenian oculars on their cheapest instruments
are of the same quality as those with the more expensive outfits.

For student laboratories in colleges, medical and technical schools,
junior colleges, and the more advanced preparatory and high schools
the microscope should have at least the character and parts shown
in figure 26. Such microscopes are now (1941) supplied by practically
all the great microscope manufacturers. Examples of two American-
made ones are illustrated in figures 32-33.

§52. Microscope stand. — As seen in the figures 32-33, the
mechanical parts are rugged. This renders them stable and gives
the durability called for by the hard usage they will be likely to meet
in actual laboratory use by beginners.

There should be a triple or a quadruple nose-piece with parfocalized
objectives. A mechanical stage is often convenient.

§ 53. Optical equipment for laboratory microscopes. — (i) There
should be a plane and a concave mirror.

(2) There should be a substage condenser of 1.20 to 1.25 N.A. with
iris diaphragm. The top element should be removable; then the
lower element alone can light the whole field of the lowest objective.
Furthermore, if the top is removable, a dark-field element can be put
in its place. This will serve for much dark-field work. (See § 181.)

(3) There should be three achromatic objectives: (a) a 16 mm.
divisible. When the front combination is removed or turned aside
the upper combination forms a serviceable 32 mm. objective; (b) a
4 mm. objective, and (c) an oil-immersion objective.

(4) There should be two Huygenian oculars, one of $x or 6.4X and
one of i ox.

§ 54. Royal Microscopical Society standards. — Down to 1857 and
even much later with many microscope manufacturers, each one had
a different screw for his objectives and a special size for oculars and

CH. I]

MICROSCOPES AND THEIR PARTS

substage condensers, tube-length, etc. In the year 1857 the Royal
Microscopical Society of London urged standardization and began in
earnest by designing a standard screw for objectives. Later, in 1896
and 1899, the Society again worked on standards, perfecting the
" Society Screw" and recommending standard sizes for oculars and
substage condensers. It is a great advantage to have all the parts
standardized, then one can use on the same microscope stand the
optics of any manufacturer. (See Beck, 1938 ed., pp. 18-19.)

§ 65. Pointer in the ocular. — This
is a slender rod of some sort situated
at the level of the real image in the
microscope, and it appears with the
specimen in the field of view (fig. 34).

A pointer may be inserted in any
Huygenian ocular as follows:

Remove the eyelens and with a little
mucilage or Canada balsam, fasten a
hair from a camel's-hair or other fine
brush to the upper surface of the
ocular diaphragm, and let it project
toward the center of the free opening.
The pointer will then appear in the
field with the image, and by moving
the specimen or rotating the ocular any
particular structure can be pointed
out just as one indicates the part of a magic lantern image on the
screen or on a chart.

If one uses positive oculars, a pointer can be put in the same way
upon the top of the ocular diaphragm.

The ocular pointer was devised by Quekett. His was movable and
was made of steel. It was called an indicator and the whole ocular
was called an index eyepiece (Carpenter-Dallinger, p. 381 ; Carpenter,
6th ed., 1881, p. 112).

§ 56. Research microscopes (figs. 35-36). — For over fifty years
our country has been especially strong in the manufacture of the
best stands and objectiyes, and during the last twenty-five years our

Fro, 34. POINTER OCULAR AND
MICROSCOPIC FIELD.

P P The pointer attached to
the diaphragm of the ocular and
extending out into the free space.

At the right a field of blood
corpuscles with the pointer indi-
cating the position of a leucocyte.

MICROSCOPES AND THEIR PARTS

[CH. I

FIG. 35. THE BAUSCH & LOME OPTICAL Co.'s MODEL GGDE RESEARCH
MICROSCOPE WITH CIRCULAR REVOLVING AND MECHANICAL STAGL.

CH. I]

MICROSCOPES AND THEIR PARTS

f'M'^V f ', 7 > ' > -.

ffi^^v-i:^^' '
T^SgS^V^aM

^'^Vr,',;'^;'''1;'!^

-^p-;"'-' :!;"'S

''•" "'**^i'J ^'* '* - sr''ViJ
~ ! , r* ' "/ f *i 4

^«i«

V,'; -t'iM'^'1

^WV'iS

^§;:'^4^*'//»^

^w'HaV/W11

0«l;f|

'%& %i-ir ^ ', ?'

?*®^

;;^-. :,ti

iV ^/v-J,

:,v,; -^"!,=
"'-,''"•-;*:',

FIG. 36. THE SPENCER LENS Co.'s MODEL 3]} WITH CIRCULAR REVOLVING
AND MECHANICAL STAGE.

42 MICROSCOPES AND THEIR PARTS [Off. I

microscope makers have exerted all their skill to meet the demands
from the biologist, the physiologist and the workers in the chemico-
physical sciences. Fortunately, now almost any required arrangement
of the mechanical parts and range in the optical parts can be obtained
from American manufacturers, whose products are of the highest
quality. Fortunately also, optical glass of the widest range is now
produced here in any desired quantity. Furthermore, the microscope
makers are ready and anxious to make apparatus of all kinds which
shall meet a real demand.

While it is true that most of the fundamental discoveries that have
been made by the aid of the microscope have been made with rela-
tively simple apparatus, still for ease and certainty of accomplishment
the more elaborate and precise modern instruments are desirable.
But after all, it must be held in mind that it is the brain of the ob-
server and not the elaborate apparatus that determines the final
outcome of research.

When one is ready to buy a microscope, it is wise to get the latest illustrated
catalogues of the various makers and select the form within one's means which
seems best adapted to one's needs. Students, teachers and investigators are
strongly urged to visit some great optical works like those of the Bausch & Lomb
Optical Company in Rochester or the Spencer Lens Company in Buffalo and see
with their own eyes the many and complicated processes that are necessary to
produce a microscope. It is amazing that they can be made so well and so cheaply.

§ 57. Stand of a research microscope. — It should be rugged so
that there will be the minimum give with the various manipulations.
The foot should be large so that the microscope can be inclined to any
angle without losing its balance.-

Commencing below, the equipment should consist of a strong
mirror fork with set-screw to hold the mirror in any desired position.
The substage fitting should be centerable so that the substage con-
denser can be put in accurate axis alignment with the objective and
ocular. It should be on a rack and pinion so that it may adjusted up
and down; and if there is a special fine adjustment, it is of great
advantage in the most critical work.

The stage should be of the circular, revolving type to enable the
observer to put the object in any desired orientation for photography
and for polarization.

CH. I]

MICROSCOPES AND THEIR PARTS

There should be a mechanical stage which may be removed when a
broad, free stage is demanded. It should have a wide excursion so
that entire slides of serial sections can be studied.

The revolving nose-piece should be
quadruple so that a battery of objectives
may be used successively when necessary.
It should be fixed to the movable focusing
block, then the monocular and binocular
bodies can be changed without disturbing
the objectives or the focus of the micro-
scope (fig. 37).

The monocular body should have a
graduated draw-tube with a Society Screw
at its lower end for very low objectives and
for use with the apertometer (fig. 117).

A coarse and a fine adjustment are in-
dispensable.

§ 58. Optical equipment of a research
microscope. — There should, first of all,
be a good mirror, concave on one side and
plane on the other. If the microscope is
to be used for fluorescence effects, the
mirror should be of the first-surface type
with the newly devised chromium-alumi-
num vapor film. Such a mirror is good
also for all microscopic work. It reflects
the ultra-violet and is much more con-
venient than the quartz prism reflector

(§ 304).

Second, it should have an aplanatic,
1.40 N.A. condenser with a removable top
element and central stops (fig. 282).

Third, unless one has a dark-field microscope, there should be avail-
able one of the special dark-field condensers (figs. 74, 77, 84) to use in
place of the aplanatic condenser.

Fourth, there should be some form of polarizing outfit unless one
has a special polarizing microscope (fig. 92).

FIG. 37. DEVICE FOR
CHANGING FROM BINOCULAR
TO MONOCULAR BODIES WITH-
OUT DISTURBING THE RE-
VOLVING NOSE-PIECE WITH
ITS OBJECTIVES.

It was suggested by W. B.
Carpenter in 1875 for the
Stephenson binocular, and
put in practice by Browning's
modification in 1881, and
Swift's in 1887.

i, i Bar attached to the
movable focusing block for
holding the nose-piece.

2 Screw to clamp the body
in place.
• 3 Coarse adjustment.

4 Fine adjustment.

0 Objectives in the nose-
piece. Their position is not
disturbed in changing bodies.

T Large body-tube.

160 Draw-tube at the 160
mm. tube-length mark.

MICROSCOPES AND THEIR PARTS

[CH. I

Fifth, one should be supplied with at least four dry objectives:
a 40 or 32 mm., and a 16, 8, and 4 mm. There should be also an oil
immersion, the 8 and 4 dry; and the oil immersion objectives should
be supplied with iris diaphragms so that the aperture can be adjusted

Front View

Side View

FIG. 38. THE BAUSCH & LOMB MODEL DDE RESEABCH AND
PHOTOGRAPHIC MICROSCOPE.

With this microscope the observer sits in front of the instrument, the light
reaching the mirror from behind. A monocular body goes with it for photography.
It is the first of the modern Mega-Microscopes.

(Line cuts through the courtesy of the Arthur H. Thomas Co.)

at will (Ch, III and XIV), As to the quality of these objectives, the
achromatic modern objectives are excellent for general work and are
relatively inexpensive. If one wishes to use objectives for the most

CH. I] MICROSCOPES AND THEIR PARTS 45

correct color value and for photomicrography, the fluorite and
apochromatic objectives are superior but more expensive.

Sixth, the ordinary Huygenian oculars are good for the achromatic
objectives. There should be one of 5x and one of lox. If one must

FK;. 39. THE NKW RESEARCH MICROSCOPE No. 8 OF THE SPENCER LKNS Co.

The observer sits in front of the stage, and the light source is behind the mi-
croscope. There is a single tube for photography.

(Courtesy of the Spencer Lens Co.)

wear glasses, oculars with high eye-points, like the telaugics (§ 145)
or the wide-field oculars of various manufacturers, are desirable. If
apochromatic objectives are to be used, one must have a group of
compensating eye-pieces; sx, lox and isx are to be especially recom-
mended. The i5x is particularly effective for dark-field work with
all types of objectives.

Seventh, for micrometry one must possess some form of ocular

46 MICROSCOPES AND THEIR PARTS [Cn. I

micrometer. The one with drum and movable scale is most accurate
and easily used (figs. 158-159; §§ 376~378)- of course, a stage mi-
crometer is a necessity (§ 366).

§ 69. Mega-microscopes (figs. 38-39). — During the last few years
most elaborate research instruments have been devised by various
manufacturers. These from their size and complexity may properly
be called mega-microscopes. They have certain advantages in that
there are present all the modern devices in one instrument. However,
their size makes them unhandy to move about, and their very ex-
cellencies make them rather confusing; they are also very expensive.
Finally, the possessor will probably find that they will not of them-
selves produce great discoveries; that, as stated above, depends on
the brain of the observer.

Instead of one elaborate instrument to serve for all the needs of
an investigator, it seems to the writer from much experience that it is
better to have several instruments of rather simple fundamental
design especially equipped for his various needs. Then each one
can be kept in perfect adjustment and is available at a moment's
notice. For example, there should be one fully equipped for bright-
field work, one for the dark-field, one for polarization, and one for
fluorescence effects. These four would not cost more than the single
mega-microscope and could be purchased as needed in one's researches.

§ 60. General care of the microscope. — The microscope should
be handled carefully and kept clean. The oculars and objective
should never be allowed to fall, for that might injure or displace their
lenses. When not in use, the microscope should be covered or kept
in a place as free as possible from dust. All parts of the microscope
should be kept free from liquids, especially from acids, alkalies,
alcohols, xylene, turpentine and chloroform.

§ 61. Care of the mechanical parts. — To clean the sliding me-
chanical parts put a small quantity of some fine oil (olive oil or
petrolatum and xylene, equal parts) on a piece of gauze or lens paper
and rub the parts well; then with a clean, dry piece of cloth or lens
paper wipe off most of the oil. If the sliding parts are kept clean in
this way a special lubricator is rarely needed. In cleaning lacquered
parts xylene alone answers well, but it should be quickly wiped off.
Do not use alcohol, as it dissolves the lacquer.

CH. I] MICROSCOPES AND THEIR PARTS 47

§ 62. Care of the optical parts. — These must be kept scrupulously
clean in order that the best results may be obtained. Glass surfaces
should not be touched by the fingers, for that almost invariably
clouds them. Whenever an objective is left in position on the mi-
croscope or when several are attached to a revolving nose-piece, an
ocular should be left in the upper end of the tube to prevent dust,
lint, etc., from falling down upon the back lens.

As pointed out by Wright (p. 93), one of the surest ways to detect anything
wrong with the objective is to examine the eyepoint with a magnifier. The field
should be lighted well and the aperture of the objective filled about f full of light.
If there are any defects, as smears of balsam or liquids on the front lens, unsealing
of the combinations, or dust on the upper face of the back lens, the defect can
be seen in the eyepoint.

Another and very certain method of detecting imperfections is to rotate the
different elements while looking into the microscope. If the defects are in the
mirror, they will change in position when the mirror is moved, and so with all
the other elements. Defects in the ocular are strikingly shown by rotating it.

§ 63. Lens paper. — The so-called Japanese filter paper, which
from its use with the microscope I have designated lens paper, has
been used in the author's laboratory since 1884 for cleaning the lenses
of oculars and objectives, and especially for removing the fluid used
with immersion objectives. Whenever a piece has been used once it
is then thrown away. It has proved more satisfactory than cloth
or chamois because dust is not present and because of its bibulous
character it is very efficient in removing liquid or semi-liquid sub-
stances. Some other workers with the microscope have found that
absorbent cotton has the desired qualities for cleaning optical parts.

§ 64. Removal of dust, etc. — (i) Dust may be removed with a
camel's-hair brush, then the lens wiped with lens paper.

(2) Cloudiness may be removed from the glass surfaces by breath-
ing on them, then wiping quickly with a soft cloth or the lens
paper.

Cloudiness on the inner surfaces of the ocular lenses may be re-
moved by unscrewing them and wiping as directed above. A high
objective should never be taken apart by an inexperienced person.

If the cloudiness cannot be removed as directed above, moisten
one corner of the cloth or paper with 95% alcohol, wipe the glass
first with this, then with the dry cloth or the lens paper.

48 MICROSCOPES AND THEIR PARTS [Cn. I

(3) Water may be removed with soft cloth or the lens paper.

(4) Glycerin may be removed with cloth or lens paper saturated
with distilled water; remove the water as above.

(5) Blood or other albuminous material may be removed while
fresh as in (4). If the material has dried on the glass, it may be
removed more readily by adding a small quantity of ammonia to the
water in which the cloth is moistened (water 100 cc., ammonia i cc.).

(6) In general, to remove any foreign substance from a glass sur-
face a solvent of the foreign material must be used. For example,
Canada balsam, damar, clarite, or cedar-wood oil is best removed
from the front lens of an objective by wiping it with lens paper or a
soft cloth moistened with xylene, toluene, or chloroform, and then
wiping it dry with a fresh piece of lens paper or gauze.

(7) It frequently happens that the upper surface of the back com-
bination of the objective becomes dusty. This dust may be removed in
part by a brush but more satisfactorily by using a piece of the lens
paper loosely twisted. When most of the dust is removed, some of the
paper may be put over the end of a pine stick (like a match stick)
and the glass surfaces carefully wiped. Sometimes it is necessary to
moisten the wiper with water and then wipe dry.

§ 66. Care of the eyes. — Keep both eyes open, using the eye-shade
if necessary (fig. 40), and divide the labor between the two eyes, using

. ^^

FIG. 40. EYE-SHADE FOR THE TOP OF THE MICROSCOPE xc
ENABLE THE OBSERVER TO KEEP BOTH EYES OPEN.

(Devised by Lister; Quekett, pp. 170-171.)

CH. I] MICROSCOPES AND THEIR PARTS 49

one eye for a while and then the other. It frequently happens that
one eye is much more perfect than the other, then, of course, the more
perfect eye is used all the time (see Quekett, pp. 170-171).

The binocular microscope has certain advantages in that one uses
both eyes all the time as in naked-eye observation. If a binocular is
used, however, one must adjust it accurately so that each eye sees
an equally sharp image (§ 163).

FIG. 41. LABORATORY TABLE AND ADJUSTABLE STOOL.

This table is 122 cm. long, 61 cm. wide, and 73 cm. high (2x4 feet on top,
and 29 inches high).

The corners and edges are rounded and the top is stained with aniline black.
The front of the rail is cut out, and the drawer is at the right so that it can be
opened without moving the stool.

In the beginning it is not advisable to look into the microscope continuously
for more than half an hour at a time. One never should work with the micro-
scope after the eyes feel fatigued. After one becomes accustomed to micro-
scopic observation he can work for several hours with the microscope without
fatiguing the eyes. This is due to the fact that the eyes become inured to labor
like the other organs of the body by judicious exercise. It is also due to the
fact that but very slight accommodation is required of the eyes, the eyes

MICROSCOPES AND THEIR PARTS

[Cn. I

remaining nearly in a condition of rest as for distant objects. The fatigue
incident upon using the microscope at first is due partly at least to the con-
stant effort on the part of the observer to remedy the defects of focusing the
microscope by accommodation of the eyes. This should be avoided and the
fine adjustment of the microscope used instead of the muscles of accommoda-
tion. With a microscope of the best quality, and suitable light — that is,
light which is steady and not so bright as to dazzle the eyes nor so dim as to
strain them in determining details — microscopic work should improve rather
than injure the sight.

If artificial light is used, give it daylight qualities by placing a piece of day-
light glass between the source of light and the microscope. This will give
one a very soft light like that from a white cloud (§76).

§ 66. Position and character of the work-table. — The work-table should
be very firm and large (61 X 122 cm. on top, and 73 cm. high; 24 X 48 X 29
in., figs. 4 1-41 a), so that the necessary apparatus and material for work may
not be too crowded. The table should also be of the right height to make
work by it comfortable. An adjustable stool, something like a piano stool,
is convenient; then one may vary the height corresponding to the necessities
of special cases.

FIG. 4ia. MICROSCOPICAL LABORATORY DESK WITH MICROSCOPIC
AND CHALET LAMP.

(Desk designed by Dr. V^A. Moore; about one-twentieth natural size.)

The size of the top and the height are the same as for the laboratory table (fig.
41, §66). ^

At the right there is a cabinet with combination lock (10, cl) for a microscope,
and above a drawer with combination lock (a, cl}.

At the right is a writing shelf (s) above the four drawers (6, c, d, e).

Near the bottom is a brace (br) which also serves as a foot rest.

M Compound microscope with the Chalet Microscope Lamp in front of it.

OTHER READING

NELSON, E, M. — On the Origin of the Society Screw. J.R.M.S., 1910.
RIDDELL, J. L. — On the Binocular Microscope. Amer. Jour. Science, vol. 65,
1853, p. 68; Quarterly Jour. Micr. Science, vol. ii, 1854, pp. 18-24, 4 figs.

CHAPTER II

BRIGHT FIELD MICROSCOPES: LIGHTING, NATURAL AND

ARTIFICIAL: EXPERIMENTS WITH MICROSCOPES.

§§ 67-169; FIGURES 42-64

§ 67. Bright-field lighting. — With the great majority of micro-
scopic work the objects are viewed on a light field, the general
appearance being like dark or colored letters on a white sheet of
paper. The light may be directed upon the surface, as in all ordi-
nary vision with the naked eye, or the light may be made to shine
through the support and the object from behind as in the glass
signals for automobilists, or commercial signs on glass. When the
microscope is used \dth a light field, it is called a bright-field micro-
scope in contradistinction to a dark-field microscope where the
object is bright and the field dark (§ 170).

§ 68. Lighting ^ ith daylight. — Full sun-
light is not used in ordinary work. North
light is best and most uniform. When the
sky is covered with white clouds, the light
is most favorable. To avoid the shad-
ows produced by the hands in manip-
ulating the mirror, etc., it is better to face
the light; but to protect the eyes and to
shade the stage of the microscope some
kind of screen should be used. The one
shown in fig. 4.2 is cheap and efficient. If
one dislikes to face the window or lamp it
is better to sit so that the light will come
from the left, as in reading.

It is of the greatest importance and ad-
vantage for one who is to use the micro-
scope for serious work that he should

jo cm

FIG. 42. SCREEN FOR
SHADING THE MICROSCOPE
AND THE OBSERVER.

It is composed of heavy
paper hung over a bent
wire, which in turn is an-
chored in a small tin dish
filled with lead.

comprehend and appreciate thoroughly the various methods of illu-

THE BRIGHT-FIELD MICROSCOPE

[Cn. II

ruination, and the special appearances due to different kinds of illu-
mination.

§ 69. Reflected, incident, or direct light. — By this is meant light
reflected upon the object in some way and then irregularly reflected
from the object to the microscope. By this kind of light objects are
ordinarily seen by the unaided eye and the simple microscope (figs.

4-5). In histology, reflected light is
but little used; but in the study
of opaque objects, like whole in-
sects, etc., it is used a great deal.
For a simple microscope and low
powers of the compound microscope,
ordinary daylight that naturally
falls upon the object, or is re-
flected or condensed upon it with a
mirror, or a bull's eye condens-
ing lens, is sufficient. For high pow-
ers, special apparatus is necessary.
(See § 31).

§ 70. . Transmitted light. — By
this is meant light which passes
through an object from the opposite
side (figs. 20, 44). The details
of a photographic negative are in
many cases only seen or best seen
by transmitted light, while the
print made from it is best seen by
reflected light (figs. 19, 43).

Almost all objects studied in
animal and vegetable histology are
lighted by transmitted light, and
they are in some way rendered trans-
parent or semi-transparent. The light traversing and serving to illu-
minate the object in working with a compound microscope is usually
reflected from a plane or concave mirror, or from a mirror to a con-
denser, and thence transmitted to the object from below (fig. 18, 44).

FIG. 43. LOW-POWER OBJEC-
TIVE SHOWING WORKING DIS-
TANCE AND REFLECTED LIGHT.

Axis The principal optic axis
of the objective extended.

SI The glass slip on which the
object is mounted.

0 Object.

c Cover-glass over the object.

W The working distance be-
tween the cover and the objective.

Mirror The mirror is repre-
sented as above the stage and re-
flecting parallel beams upon the
object.

FC Front combination of the
objective.

BC Back combination of the
objective; it is composed of a
plano-concave of flint (F) and a
double convex lens of crown glass

CH. II]

THE BRIGHT-FIELD MICROSCOPE

§ 71. Axial or central light. — By this is meant light reaching the
object in such a way that it is symmetrically arranged around the
optic axis of the microscope, then the object will be equally illumi-
nated from all sides. If bundles of parallel rays are reflected upon
the object from the mirror, they must
be so disposed that the object will re-
ceive an equal quantity of light from
all sides. If the bundles of light are
made up of diverging or of converg-
ing cones, then the axes of the cones
should be coincident with or parallel
with and symmetrically arranged
around the optic axis of the micro-
scope.

§ 72. Oblique light. — By this is
meant light which reaches the object
with its axial beam oblique to the
optic axis of the microscope. With
oblique light the object cannot be il-
luminated equally from all sides, but
largely from one side, and consequently
the light is said to be unsymmetrical.

If no condenser is used, oblique
light is obtained by turning the
mirror so that parallel rays strike the
object obliquely to the optic axis of
the microscope (fig. 44c) or the axis
of the converging or diverging beam
from the concave mirror strikes the
optic axis obliquely.

If a condenser is used, oblique illu-
mination is produced by making the
diaphragm opening eccentric, or most

simply by putting the finger or other opaque body between the mirror
and the condenser to cut off part of the light (figs. 62, 135). The
result in all cases is that the object is lighted unsymmetrically.

FIG. 44. HIGH-POWER IM-
MERSION OBJECTIVE WITH CEN-
TRAL AND OBLIQUE TRANS-
MITTED LIGHT.

Axis The principal optic
axis.

Mirror This reflects the
light up through the object.

A B Central light.

C Oblique light.

Stage The microscope stage
in section.

0 The object.

7 Immersion liquid between
the objective and object.

FC The front lens of the
objective.

M C The middle combina-
tion.

B C The back combination.

54 THE BRIGHT-FIELD MICROSCOPE [CH. II

§ 73. Use of a diaphragm. — A diaphragm is an opaque disc with
an opening, and is placed somewhere between the object and the
source of light.

At the present time an iris diaphragm is almost universally em-
ployed. It, like the iris of the eye, can be expanded or contracted,
and thus gives a large range of openings to meet different conditions.

The object of a diaphragm is to cut off adventitious light and to
vary the aperture to suit the object and the objective.

§ 74. Size and position of the diaphragm with a mirror only. —
When no condenser is used in addition to the mirror, a diaphragm
opening about the size of the front lens of the objective may be
employed. Its position may be close to the object, in which case it
admits the greatest aperture of light, and cuts off the most adventi-
tious light. In this position it lights the smallest field, however.

If the diaphragm is far enough below the object, the field may all
be lighted, but the aperture will be smaller than when it is close to
the object, as one may see by removing the ocular and looking down
the tube into the back lens of a 16 mm. (lox) or 8 mm. (2ox) objec-
tive. On the other hand, while the aperture of the objective may
be filled even with a small diaphragm opening close to the object,
the field of view (§ 93, fig. 132) may be but partly lighted. In that
case the opening must be increased until the entire field is illumi-
nated. One must learn by practice how to get the best results,

ARTIFICIAL ILLUMINATION

§ 75. Artificial light. — While daylight is preferred by many for
all microscopic work, every one who must do much of that kind of
work, realizes very keenly its defects. It continually varies in
intensity and color from sunrise to sunset; and in most regions
where work is done it is frequently cloudy or stormy and sufficient
light is not obtainable. Then, too, it often happens that work
should be continued into the evening when no daylight is available.
Frequently, also, the worker must be in a room where suitable day-
light cannot be secured, no matter how favorable the day may be.
For all work it is advantageous to have a source that is uniform

CH. II] THE BRIGHT-FIELD MICROSCOPE 55

both in intensity and in color. This is especially necessary for
photography. All forms of artificial light have been used at some
time for microscopic work; and for a long time various means have
been taken to make the artificial light as nearly like daylight as
possible. This desire for artificial daylight is natural, for the eye
was developed for daylight, and all its standards of color and shading
have been worked out for that quality of light. In all of the
ordinary forms of artificial light, the relative intensity toward the
red end of the spectrum is much greater than with daylight, hence
color values with artificial light are distorted, and with most people
the excessive intensity of the red produces glare and a lack of
contrast, which is trying to the eyes.

§ 76. Artificial daylight. — For the production of artificial day-
light it is obvious from the curve (fig. 45) that there are two pos-
sible means: (i) The selection of two kinds of artificial light in which
the lack in one is made good by the excess in another, and by mixing
these in the right proportions the resulting light will have the same
relative intensity in different parts of the spectrum as is found in sun-
light. This is the " addative " method and has been quite success-
fully realized by combining a mercury arc light with its deficiency in
the red, but its richness in intensity in the blue end of the spectrum,
with a mazda incandescent lamp with its excessive red intensity.
If these two lights are enclosed in a glass globe, and the right
amount of each used, very good daylight is produced.

(2) As there is excessive intensity in the red part of the spectrum
it is evident that if this excess can be absorbed by a light filter of
some kind, then also the relative intensity of the light will be like
that of natural daylight. This is the " subtractive " method, and is
the method employed wherever a light filter or colored liquid,
colored gelatin, colored glass, or a combination is used. From time
immemorial various colored liquids like solutions of copper salts and
colored glasses have been used to whiten the artificial light.

During the last few years, however, the problem has been solved,
and now colored glass is made which gives to artificial light true
daylight qualities. As each artificial light has its own special curve
of intensity for the different parts of the spectrum, naturally a

THE BRIGHT-FIELD MICROSCOPE

[CH. II

special light filter must be worked out for each light source. Up to
the present, glass filters have been produced for the Welsbach gas

i r~n

12 Violet BUi0

Green Yellow Orange Red

.41 .43 .45 47 .49 .5t .63 .65 .57 .69 .61 -63 .65 .67 .69
Wave Length in Microns o>>

FIG. 45. CURVE OF ENERGY DISTRIBUTION IN SUNLIGHT; i\ THK MAZDA

C LAMP (TUNGSTEN AT 2800° ABSOLUTE); AND OF MAZDA LAMPLIGHT

FILTERED THROUGH DAYLIGHT GLASS 172 CD. (H. P. CAGE)

light, and for the incandescent, nitrogen-filled tungsten (mazda)
lamp. It may be said in passing that these glass filters whiten any
artificial light, but that true daylight color values are given only

CH. II] THE BRIGHT-FIELD MICROSCOPE 57

under the precise conditions for which the glass was worked out.
It is also gratifying to note that this successful solution o£ a long
vexing problem came only when the rigid training in physics and
chemistry and the facilities of a great manufacturing plant were
brought together.

§ 77. Daylight-lamp or lantern. — In the practical use of the
daylight glass filter, it was found that the light should be enclosed
in some kind of a lantern or lamp-house so that all the light de-
livered to the microscope might be of the daylight quality, and none
of the unfiltered light scattered about the workroom.

After much experimenting, a lantern having the general form of a
Swiss chalet was decided upon as it fulfilled all the requirements,
and besides by its extending roof excluded all light from entering the
eyes of the observer directly, one of the greatest causes of eye-
fatigue. The old opticians and astronomers knew and stated well
the conditions for the clearest vision, viz,; that no light should enter
the eyes except that which came from the object being studied.

It was found also that the best effect was secured when the 100-
watt lamp filament was opposite the middle of the window of day-
light glass (ms fig. 46),

§ 78. The daylight-glass filter. — Experience showed that the
windows in the lamp-house or lantern should be about 82 mm.
square in order to give sufficient area for lighting all the different
powers from the lowest to the highest. It also served to supply
light at the side of the microscope for drawing and note taking.

For the lower objectives, i.e., from the lowest up to the 4 mm.
(4ox), it is well to have one face of the glass filter ground with fine
carborundum or emery flour to diffuse the light so that the image
of the lamp filament will not show in the field. Formerly for these
powers it was recommended that both faces be ground, but since at
present all loo-watt, gas-filled lamp bulbs are inside .frosted, it is
necessary to grind only one face of the daylight filter to give the
desired diffusion. For objectives of 3 mm. (6ox) and less equivalent
focus and higher powers, it is better to have one of the daylight glass
windows smooth or polished like plate glass on both faces. If two
students are to use the same lantern at the same time, then it is

THE BRIGHT-FIELD MICROSCOPE

[CH. II

better to have both windows with one ground face. Even with the
highest powers the ground glass window gives light enough if the
lamp is brought close to the microscope. For powerful lamps to use
with the dark-field microscope, for seeing the blood-circulation and
for photography (figs. 78-82).

§ 78a. For a discussion of the requirements for the production of artificial
daylight, and the means so far employed, and the uses of artificial daylight, see:

Herbert K. Ives. Artificial Daylight. Journal of the Franklin Institute,
vol. 177, May, 1914, pp. 471-499. 19 figures.

Simon H. Gage. Artificial Daylight for the Microscope. Science, N. S.,
vol. 42, October, 1915, pp. 534-536. One curve.

M. Luckiesh. Artificial Daylight. Science, N. S., vol. 42, November, 1915,
pp. 764-765.

Henry Phelps Gage. "Daylite Glass," a color screen for producing daylight
artificially. The Sibley Journal of Engineering, Ithaca, N.Y., Vol. XXX, No. 8,
May, 1916. 4 quarto pages, 6 figures.

Simon H. Gage and Benjamin F. Kingsbury. Some apparatus for the micro-
scopical laboratory. Anatomical Record, Vol. X, No. 8, June, 1916, pp. 527-536.
7 figures showing the use of the daylight glass for microscopic work.

Anthony J. Brown. Some uses of artificial daylight in the psychological labo-
ratory. American Journal of Psychology. July, 1916, Vol. XXVII, pp. 427-429.

FIG. 46. CHALET MICROSCOPE LAMP IN SECTION.
(About one-fifth natural size.).

The metal part of the lantern or lamp-house is enameled white inside or pref-
erably painted with aluminium powder in a suitable lacquer. The outside may
be painted with any desired color of lacquer, or coated with black bakelite.

dg-dg Windows of daylight glass about 82 mm. square. One is ground on one
surface with very fine emery or carborundum, to diffuse the light, and the other is
left clear, or the glass may be polished on both faces.

CH. 11]

THE BRIGHT-FIELD MICROSCOPE

ms mazda C lamp bulb of 100 watts. The filament of the lamp should be op-
posite the middle of the daylight window.

5 The lamp socket with snap switch on the left, and the entering electric
cable.

v v v Ventilating spaces at the top and at the bottom. The lamp-house has
legs at each corner to elevate it and give free ventilation at the bottom. The
roof is supported at the two ends and has ventilating spaces over the two walls
containing the daylight filters, (figs. 53, 83, 198-199).

FIG. 47. NEW CHALET MICROSCOPE LAMP

The lamp-house is i J cm. lower than the one shown in fig. 46. This change was
made to bring the shorter, new form of loo-watt mazda lamps at the right level
with the window (W). E Plug to screw into the lamp socket of the supply wire.

EXPERIMENTS WITH SIMPLE AND WITH COMPOUND MICROCOPES

§ 79. Focusing a microscope. — Focusing is mutually arranging
an object and the microscope so that a clear image may be seen.

With a simple microscope either the object or the microscope or
both may be moved in order to see the image clearly, but with the
compound microscope the object more conveniently remains sta-
tionary on the stage, and the tube or body of the microscope is
raised or lowered (fig. 26).

In general, the higher the power of the whole microscope, whether
simple or compound, the nearer together must the object and the
magnifier be brought.

§ 80. Focusing a simple microscope. Use a reading glass, or any
form of simple microscope such as the tripod magnifier (figs. 15, 16).
Hold the magnifier over a printed page and look through the magni-
fier. The letters and words will appear as they do with the naked
eye, but larger (fig. 4).

60 THE BRIGHT-FIELD MICROSCOPE [Cn. II

In order to get the sharpest image it will be necessary to raise and
lower the magnifier until the best position is found. This mutual
arrangement of magnifier and object is called focusing, or getting
into focus.

§ 81. Size of the field. — With any given magnifier, the size of the
field, that is, the diameter of the area which can be seen at one time,
can be determined by using the ten-centimeter rule as object and
noting how many centimeters or millimeters can be seen at one time
without moving the magnifier or the measure sidewise. It will also
be found by trial that the greatest field can be seen when the eye is
at the level of the eyepoint as with the compound microscope
(§ 99)-

LIGHTING WITH THE SIMPLE MICROSCOPE

§ 82. Opaque objects. — For these the light strikes the surface
and is reflected, mostly in an irregular manner so that the object can
be seen almost equally well illuminated from any angle. Ordinarily
the daylight falling upon the object will sufficiently illuminate it,
also the light of a lamp.

Place a printed page in bright daylight or near a lamp where the
light can shine upon it and then look at it with the simple micro-
scope held in the hand, on the legs of the tripod (figs. 4, 15-17) or
held by a special stand. By varying the distance between the
microscope and the object one can soon find the best focus, and by
changing the position of the object, the best position for the light
available.

Of course if one wishes to discriminate colors precisely, daylight,
natural or artificial, must be available.

§ 83. Transparent or semi-transparent objects. — For these the
light should pass through the object. Use a lantern slide or printing
on very thin paper and hold it up toward the window or some
artificial light with one hand, and with the other hold the magnifier.
Look into the magnifier and move it toward and away from the
object till a clear image is seen. Here the light passes through the
object into the microscope and then to the eye instead of being
reflected from the surface as in looking at the page of a book.

CH. II]

THE BRIGHT-FIELD MICROSCOPE

EXPERIMENTS WITH THE COMPOUND MICROSCOPE
§ 84. Putting an objective in position and removing it. — Elevate

the tube of the microscope by means of the coarse adjustment (fig.

26) so that there may be

plenty of room between

its front or lower end and

the stage. Grasp the ob-
jective lightly near its

lower end with two ringers

of the left hand, and hold

it against the nut at the

lower end of the tube or

the revolving nose-piece

(figs. 48-50). With two

fingers of the right hand ^ ^ DQUBLE NosE.PIECE wmi THK

take hold of the milled OBJECTIVES IN PLACE.

ring near the back or

upper end of the objective and screw it into the tube of the microscope

or nose-piece. Reverse this operation for removing the objective.

By following this method
the danger of dropping the
objective will be avoided,
§ 85. Putting an ocular
I in position and removing
it. — Elevate the body of
the microscope with the
coarse adjustment so that
the objective will be 2 cm.
or more from the object,
grasp the ocular by the

milled ring next the eye-
FIG. 40. TRIPLE NOSE-PIECE WITHOUT , ° . 1^1

OBJECTIVES. lens (fig. 26) and the

Devices for quickly changing objectives of differ- coarse adjustment or the

ent powers date back nearly 150 years. (See tube of the microscope
Carpenter-Dallinger, 8th ed.. 1901, pp. 200-205.) , .

p fe and gently torce the ocular

THE BRIGHT-FIELD MICROSCOPE

[Cn. II

into position, In removing the ocular, reverse the operation. If
the above precautions are not taken, and the oculars fit snugly, there
is danger in inserting them of forcing the tube of the microscope
downward and the objective upon the object.

§ 86. Putting an object under the microscope. — This is so plac-
ing an object under the simple microscope, or on the stage of the
compound microscope, that it will be in the field of view when the

microscope is in focus
(§§ 93, 79, fig. 40).

With low powers, it is
not difficult to get an
object under the micro-
scope. The difficulty in-
creases, however, with the
power of the microscope
and the smallness of the
object. It is usually
necessary to move the
object in various direc-
tions while looking into
the microscope, in order
to get it into the field.

Time is usually saved by getting the object in the center of the
field with a low objective before putting the high objective in posi-
tion. This is greatly facilitated by using a nose-piece, or revolver
(figs. 48-50)-

Fie. 50. QUADRUPLE NOSE-PIECE WITH THE
FOUR OBJKCTIVKS IN PLACK.

FOCUSING EXPERIMENTS

§ 87. Focusing low objectives. — Place a mounted fly's wing
under the microscope; put the 16 mm, (rox) objective and the 5x
or 6x ocular in position. Select the. proper opening in the diaphragm
and light the object well with transmitted light (§ 70 V

Hold the head about the level of the stage, look toward the
window, and between the object and the front of the objective; with
the coarse adjustment lowei the tube until the objective is within

CH. II] THE BRIGHT-FIELD MICROSCOPE 63

about half a centimeter of the object. Then look into the microscope
and slowly elevate the tube with the coarse adjustment. The image
will appear dimly at first, but will become very distinct by raising
the tube still higher. If the tube is raised too high, the image will
become indistinct, and finally disappear. It will again appear if the
tube is lowered the proper distance.

When the microscope is well focused, try both the concave and the
plane mirrors in various positions and note the effect.

Pull out the draw- tube 4 to 6 cm., thus lengthening the body of
the microscope; it will be found necessary to lower the tube of the
microscope somewhat. (For reason, see fig. 151.)

§ 88. Pushing in the draw-tube. — To push in the draw -tube,
grasp the large milled ring of the ocular with one hand, and the
milled head of the coarse adjustment with the other, and gradually
push the draw-tube into the tube. If this were done without these
precautions the objective might be forced against the object and the
ocular thrown out by the compressed air.

§ 89. Focusing with high objectives. — Employ the same object
as before, elevate the tube of the microscope and, if no revolving
nose-piece is present, remove the 16 mm. (lox) objective as indi-
cated. Put a 4 mm. (4ox) or higher objective in place, and use
5x or 6x ocular.

Light well, and employ the proper opening in the diaphragm, etc.
(§ 74). Look between the front of the objective and the object as
before (§ 87), and lower the tube with the coarse adjustment till the
objective almost touches the cover-glass over the object. Look into
the microscope, and with the coarse adjustment, raise the tube very
slowly until the image begins to appear, then turn the milled head of
the fine adjustment (fig. 26), first one way and then the other, until
the image is sharply defined.

In practice it is found of great advantage to move the preparation
slightly while focusing. This enables one to determine the approach
to the focal point either from the shadow or the color, if the object is
colored. With high powers and scattered objects there might be no
object in the small field (§ 93, fig. 51 for size of field). By moving
the preparation an object will be moved across the field and its

64 THE BRIGHT-FIELD MICROSCOPE [CH. II

shadow gives one the hint that the objective is approaching the
focal point. (See also § 81.) If one lowers the tube only when
looking at the end of the objective as directed above, there will be
no danger of bringing the objective in contact with the object, as
may be done if one looks into the microscope and focuses down.

When the instrument is well focused, move the object around in
order to bring different parts into the field. It may be necessary to
refocus with the fine adjustment every time a different part is
brought into the field. In practical work one hand is kept on the
fine adjustment constantly, and the focus is continually varied.

§ 90. Focusing with scattered or transparent objects. — If the
objects in a preparation are few or much scattered, or if they are
unusually transparent it is sometimes difficult to find and focus
them. To overcome the difficulty one can use a low power and get
a specimen in the middle of the field. It is also advantageous in
making such preparations if fresh to make a delicate cross (X) in the
middle of the slide with India ink, or preferably a red glass pencil.
It is then easy to focus the highest powers at the right level, when
the scattered objects can be found by moving the slide. If a diffi-
cult preparation is permanently mounted, a delicate cross on the
middle of the cover glass will aid one in getting the objects in focus.

The above suggestions will greatly assist with glass micrometers,
fresh liquids like milk, blood, unstained bacteria, etc.

§ 91. Parfocal oculars and focusing. — On changing the oculars
from a higher to a lower or the reverse, it is necessary to refocus the
microscope. Formerly the change in focus was very marked in
changing from one power of ocular to another, but since Mr. Pen-
nock introduced parfocal oculars (1881) and their almost universal
adoption since, very little change in focus is necessary in passing
from power to power of ocular.

According to E. M. Nelson, such oculars were suggested by
Varley and constructed by Powell as early as 1839 (Jour. Roy.
Micr. Soc., 1908, p. 149).

§ 92. .Parfocal objectives. — These are groups of objectives, of
different power, so mounted that when screwed into the revolving
nose-piece of the microscope very little change in focusing is neces-

CH. II]

THE BRIGHT-FIELD MICROSCOPE

sary in passing from objective to objective. This arrangement of
objectives was a natural outgrowth from the parfocalization of the
oculars, the ocular remaining the same (§ 91).

In case the objectives are not nearly enough parfocal so that the
object is visible in turning from one objective to another, the de-
fect can be corrected easily by getting one of the objectives in exact
focus and then turning the others successively into place. If one
notes whether it is necessary to focus up, then it will be known that
the objective projects too far down toward the object; if, on the
other hand, one must focus down, then the objective is too high up.
To correct this lack of parfocalization use the objective which pro-
jects farthest toward the object
as standard. Focus it sharply
and then turn another in posi-
tion. Unscrew this slowly until
the image is also sharp. Now
wind a thread or string around
the lower end of the objec-
tive screw and then turn it
in place and slowly screw it
into the revolving nose-piece
until it is in focus. Proceed
with all until the entire number
are in focus at the same level.
With parfocal oculars and par-
focal objectives much time and
annoyance are saved, for one can
see the specimen in turning from
power to power, and it is neces-
sary to make only a small
focusing adjustment to get the

best image. Microscope manufacturers prepare thin washers that
ran be put on top of the objectives for parfocalizing them. The
washers are better than the string, but the string will answer if the
washers are not at hand. While it is relatively simple to parfocalize
different oculars, a group of objectives on a revolving nose-piece can

842

FIG. 51. FIELD WITH AND WITHOUT
OCULARS AS SHOWN BY THE PROJECTION
MICROSCOPE.

A The field of the 2 (gox), 4 (4ox),
8 (2ox), 1 6 (IQX) and 32 (4x) mm. ob-
jectives without an ocular.

B Field of the same objectives with a
5x ocular.

C Field of the same objectives with a
i ox ocular.

32 (4x), 16 (TOX), 8 (aox), 4 (4<«), 2
(gox). Equivalent focus of the different
objectives whose fields are shown.

66 THE BRIGHT-FIELD MICROSCOPE [Cn. II

be accurately parfocalized for only one ocular. The lox ocular is
generally selected. Fortunately when parfocal for the rox ocular,
the objectives will not be far from parfocal for the isx and the 6x,
the other oculars most used.

§ 93. Field or field of view of a microscope. — This is the area
visible through a microscope when it is in focus. When the field is
properly lighted and there is no object under the microscope, it ap-
pears as a disc of light. When examining an object, it appears within
the light circle, and by moving the object, if it is of sufficient size,
different parts are brought successively into the field of view.

In general, the greater the magnificatibn of the entire microscope,
whether the magnification is produced mainly by the objective, by
the ocular, by increasing the tube-length, or by a combination of all
three (§ 368), the smaller is the field.

The size of the field is also dependent, in part, without regard to
magnification, upon the size of the opening in the ocular diaphragm.
Some oculars, as the orthoscopic and periscopic, are so constructed
as to eliminate the ocular diaphragm, and in consequence, although
this is not the sole cause, the field is considerably increased.

§ 94. Method of determining the size of the field, and table with
different objectives and oculars. — Use a stage micrometer (fig. 148)
as object, and read off the number of spaces required to measure the
diameter of the light disc as seen in the microscope. Use first a low
objective 16 mm. (tox) and a low ocular (5x or 6x), then use a higher
ocular (tox or i5x). Do the same with the 4 (4ox) or 8 (2ox) mm.
objective arid the two oculars. Make a table giving the diameter of
the field in each case and compare with the accompanying table.
The tube-length (fig. 26) should be 160 mm. when making the
measurements. To see the effect of lengthening the tube, pull it out
as far as possible and note the effect on the size of the field. (The
longer the tube, the smaller the field).

FUNCTION OF AN OBJECTIVE

§ 96. Put a 50 -mm. (3-2x) objective on the microscope, or screw
off the front combination of a 16 mm. (lox) and put the back com-

CH. II]

THE BRIGHT-FIELD MICROSCOPE

bination on the microscope for a low objective. For object, use some
letters or numerals printed on thin paper and mounted in Canada
balsam (§ 330). Place on the stage so that they are erect to the
naked eye. Light as brilliantly as possible with transmitted light.

Differences in the magnification and the diameter of the field with the same objective
but with different oculars having the same designation.

Achromatic
objective
of 1 6 mm.
equivalent

5x Huygenian
(negative)
oculars

i ox Huygenian
(negative)
oculars

i ox Positive
oculars

i5x Posi-
tive
oculars

focus (e f )

I OX

Field

Mag.

Field

Mag.

Field

Mag.

Field

Mag.

16 mm. lox

i.Q5 mm.

6l.2X

1.48 mm.

97-5X

.Q5 mm.

g6x

o.QS

i68x

16 mm. lox

2.1 mm.

54X

.21 mm.

io5x

.67 mm.

IOOX

1.17

H7X

1 6 mm. i ox

2.3 mm.

51. 2x

.40 mm.

io5x

.70 mm.

g6x

1.20

i3ix

16 mm. lox

i. 98 mm.

6ox

•53 mm.

losx

.67 mm.

IOQX

I.I7

I39X

16 mm. rox

1.85 mm.

6ox

.45 mm.

iiSx

.52 mm.

IOQ. 2X

0.98

i68x

16 mm. lox

.64 mm.

g6x

.10 mm.

113. 2X

1-45

i fox

16 mm. lox

.27 mm

I02X

Differences in the diameter of the field and the magnification of different objectives
but with the same ocular.

Huyge-

16 mm. (e.f.)

8 mm (e.f.)

4 mm. (e.f.)

3 mm. (e f .)

(negative)

IQX objectives

2ox objectives

4ox objectives

6ox objectives

I OX

Field

Mag.

Field

Mag.

Field

Mag.

Field

Mag.

I OX

i. 60 mm.

96x

0.80 mm.

i96x

0.37 mm.

425*

0.27 mm.

58ox

I OX

1.67 mm.

9i.6x

0.85 mm.

i8sx

0.37 mm.

431*

0.26 mm.

S86x

I OX

i. 60 mm.

95.2X

0.76 mm.

203X

0.39 mm.

42OX

0.27 mm.

576x

I OX

1.65 mm.

94X

0.80 mm.

iQ5x

0.42 mm.

368x

I OX

1.64 mm.

96x

o 94 mm.

i68x

I OX

i. 53 mm.

102. 8x

0.74 mm.

2I2X

It U evident from the above tables that the magnification of the microscope
obtained by multiplying the designated magnification of the objective and of the
ocular would be only roughly correct. For the exact magnification, that for every
combination must be individually determined.

In place of an ocular put a screen of ground-glass, or a piece of
lens paper, over the upper end of the tube of the microscope.

Lower the tube of the microscope by means of the coarse adjust-
ment until the objective is within 2 to 3 cm. of the object on the
stage. Look at the screen on the top of the tube, holding the head
about as far from it as for ordinary reading, and slowly elevate the

68 THE BRIGHT-FIELD MICROSCOPE [Cn. II

tube by means of the coarse adjustment until the imagfe of the
letters appears on the screen.

The image can be seen more clearly if the object is in a strong
light and the screen in a moderate light, i.e., if the top of the micro-
scope is shaded.

The letters will appear as if printed on the ground-glass or paper,
but will be inverted.

If the objective is not raised sufficiently, and the head is held
too near the microscope, the objective will act as a simple microscope.
If the letters are erect, and appear to be down in the microscope
and not on the screen, hold the head farther from it, shade the
screen, and raise the tube of the microscope until the letters do
appear on the ground-glass.

95a. Ground-glass may be very easily prepared by placing some fine
emery or carborundum between two pieces of glass, wetting it with water, and
then rubbing the glasses together for a few minutes. If the glass becomes too
opaque, it may be rendered more translucent by rubbing some oil upon it.

§ 96. Aerial image. — After seeing the real image on the ground-
glass or paper, use the lens paper over about half of the opening of
the tube of the microscope. Hold the eye about 250 mm. from the
microscope as before and shade the top of the tube by holding the
hand between it and the light, or in some other way. The real
image can be seen as if in part on the paper and in part in the air.
Move the paper so that the image of a letter will be half on the
paper and half in the air. Another striking experiment is to have a
small hole in the paper placed over the center of the tube opening;
then if a printed word extends entirely across the diameter of the
tube, its central part may be seen in the air, the lateral parts on the
paper. The advantage of the paper over part of the opening is to
enable one to accommodate the eyes for the right distance. If the
paper is absent, the eyes adjust themselves for the light circle at the
back of the objective, and the aerial image appears low in the tube.
Furthermore, it is more difficult to see the aerial image in space than
to see the image on the ground-glass or paper, for the eye must be
held in the right position to receive the rays projected from the real
image, while the granular surface of the glass and the delicate fibers

CH. II] THE BRIGHT-FIELD MICROSCOPE 69

of the paper reflect" the rays irregularly, so that the image may be
seen at almost any angle, as if the letters were actually printed OP.
the paper or glass.

The function of an objective, as seen from these experiments,
is to form an enlarged, inverted, real image of an object, this ima£e
being formed on the opposite side of the objective from the object
(figs. 13, 18).

— - FUNCTION OF AN OCULAR

§ 97. Using the same objective as for § 95, get as clear an image
of the letters as possible on the lens paper or ground-glass screen.
Look at the image with a simple microscope (fig. 15), as if the image
were an object.

Observe that the image seen through the simple microscope is
merely an enlargement of the one on the screen, and that the letters
remain inverted. Remove the screen and observe the aerial image
with the tripod magnifier.

Put 5x ocular, i.e., an ocular of low magnification in posi-
tion (§ 85), Hold the eye about 10 to 20 mm. from the eyelens
and look into the microscope. The letters will appear as when the
simple microscope was used (see above); the image will become
more distinct by slightly raising the tube of the microscope with the
coarse adjustment.

The function of the ocular, as seen from the above, is that of a
simple microscope, viz., it magnifies the real image formed by the
objective as if that image were an object. Compare the image
formed by the ocular (figs. 2, 18) and that formed by a simple micro-
scope (figs, i, 6).

It should be borne in mind, however, that the rays from an object
as usually examined with a simple microscope extend from the object
in all directions, and no matter at what angle the simple microscope
is held, provided it is sufficiently near and points toward the object,
an image may be seen. The rays from a real image, however, are
continued in certain definite lines and not in all directions; hence, in
order to see this aerial image with an ocular or simple microscope, or

70 THE BRIGHT-FIELD MICROSCOPE [CH. II

in order to see the aerial image with the unaided eye, the simple
microscope, ocular, or eye must be in the path of the rays (figs. 1,2).

§ 98. The field lens of a Huygenian and other negative oculars
makes the real image smaller and consequently increases the size
of the field; it also makes the image brighter by contracting the
area of the real image (figs. 24, 25), Demonstrate this by screw-
ing off the field lens and using the eyelens alone as an ocular, refocus-
ing if necessary. Note that the image is bordered by a colored
haze.

When looking into the ocular with the field lens removed, the eye
should not be held so close to the ocular, as the eyepoint (fig. 24)
is considerably farther away than when the field lens is in place.

§ 99,, Eyepoint. — This is in the plane above the ocular where
the emerging rays cross (figs. 22-25). If the eye is placed at this
point it will receive the greatest number of rays from the microscope
and thus see the largest field. If the eye is too far from or too near
the ocular, part of the rays cannot enter the pupil of the eye and the
microscopic image is restricted.

Demonstrate the eyepoint by using a 16 mm. (lox) objective and
a 5x ocular. Light brightly and then focus the microscope on some
transparent specimen. Open the diaphragm widely so that the
entire aperture of the objective is filled with light (fig. 61). Shade
the ocular with the hand or a screen and hold above the eyelens a
piece of ground-glass or of the lens paper. By raising and lowering
the glass or paper one will find the level where the sharpest and
brightest light circle is located. The height varies with different
oculars.

The eyepoint is also known as the pupil of the lens; exit pupil;
Ramsden disc or circle; or Lagrange disc.

One can find the eyepoint of a simple microscope by placing it
on the top of the tube of the compound microscope after removing
the ocular. Then a piece of ground-glass or of lens-paper is held
over the simple microscope and moved up and down until the bright-
est point is found. This is the eyepoint, and if the eye is at that
level in looking into the simple microscope or magnifier, the largest
field can be seen.

CH. Il] THE BRIGHT-FIELD MICROSCOPE ft

§ 100. Erect and inverted images with the microscope. — By

glancing at fig. i, 6, it will be seen that with the simple microscope
the retinal image is inverted; that is, the arrow is turned end fox
end. In like manner the retinal image of any object seen with the
naked eye is also inverted (fig. 5).

On the other hand, with the compound microscope, the retinal
image is erect (figs. 2, 18); that is, the arrow points in the same
direction as the object. This happens because the eye does not see
the object directly, but the real image formed by the objective, and
this is inverted. From the crossing of the rays on entering the eye,
this inverted real image is reinverted, and thus gives an erect knage
on the retina. Now as objects or their images do not seem to be on
the retinal screen, but out in space in the direction of the light rays
entering the eye, it is very evident that if the light rays are traced
from the retinal image to the object or to a virtual image, this will
appear to be erect when the image on the retina is inverted as with
the simple microscope, and will appear inverted when the retinal
image is erect as with the compound microscope, because of the
crossing of the rays in passing the pupil- of the eye (figs, i, 2, 6, 18)
on their way to the retinal image, or on their way from the retinal
image to the apparent position of the object or the virtual image.

WORKING DISTANCE

§ 101. Working distance. — By this is meant the space between
the simple microscope and the object, or between the front lens of
the compound microscope and the object, when the microscope is
in focus. This working distance is always considerably less than
the equivalent focal length of the objective. For example, the front-
lens of a 4 mm. (4ox) objective would not be 4 millimeters from the
object when the microscope is in focus, but considerably less than
that distance, viz., less than half a millimeter. If now a cover-glass
of half a millimeter or more in thickness were used it would be impos-
sible to get the 4 mm. (4ox) objective near enough the object to get
it in focus.

§ 102. Free working distance. — (i) Where no cover-glass is used,

THE BRIGHT-FIELD MICROSCOPE

[CH. II

this is the distance between the front of the magnifier or the front
lens mount of the objective and the object (fig. 52 A).

(2) If a cover-glass is used, it is the distance between the upper
surface of the cover-glass and the magnifier or objective when the
microscope is in focus (figs. 52 B, 43). Strictly speaking, it is the dis-
tance between the objective front and the upper surface of a cover-
glass of the exact thickness for which the objective is corrected.

FIG. 52. WORKING DISTANCE AND THE COVER-GLASS.

Slide The glass slide upon which the object is mounted.

A Working distance with an uncovered object.

B Working distance when a cover-glass is used and the object is in contact
with the cover-glass. The object represented by the solid black oblong appears
to be elevated one- third the thickness of the cover to the level Obj., where it is
represented by dots.

The objective is elevated corresponding to the apparent elevation of the object.

C Working distance when a cover-glass is used and the objects are dis-
tributed in a stratum of Canada balsam.

It is evident from this figure why the focus must be different for objects at
different depths in the balsam.

As the working distance of an objective is practically always less
than its equivalent focus, one must take care to use cover-glasses
thin enough so that any suitable objective can be used for studying
the specimen. Furthermore, as microscopic specimens have con-
siderable thickness, the cover-glass should be thin enough so that the

CH, II] THE BRIGHT-FIELD MICROSCOPE 73

objective can be lowered sufficiently to enable one to bring the lower
strata of the specimen in focus without bringing the objective front
in contact with the upper surface of the cover-glass (fig. 52 C).

§ 103. Determination of working distance, no cover. — Some
manufacturers state this in the description of their objectives. The
information serves as a guide, for if a cover thicker than this working
distance is used, the objective cannot be put in focus. Occasionally
students and even experienced workers put unlabeled slides under
the microscope wrong side up. With low powers the specimen can
be focused through the thickness of the slide, but the high powers
cannot, because the slide thickness is greater than the working
distance. The working distance is always less than the equivalent
focus of the objective because the center of the lens combination is
some distance above the lower face of the front lens.

To determine the distance with low powers make a wooden wedge
10 cm. long which shall be exceedingly thin at one end and about
20 mm. thick at the other. Place a slide on the stage and some
dust or an ink or pencil mark on the slide. Do not use a cover-
glass. Use a 16 mm. (lox) objective and focus the dust or mark
carefully, and when the objective is in focus, push the wedge be-
tween the objective and slide until it touches the objective. Mark
the place of contact with a pencil and then measure the thickness of
the wedge with a rule opposite the point of contact. This thickness
will represent very closely the working distance. For measuring the
thickness of the wedge at the point of contact for the high objective,
use a steel scale ruled in \ mm. and the tripod magnifier to see the
divisions. Or one may use a cover-glass measurer (§ 518) for deter-
mining the thickness of the wedge.

For the higher powers, if one has a microscope in which the fine
adjustment is graduated, the working distance may be readily deter-
mined as follows:

Use the marked slide as above. Get the dust or mark in focus,
then lower the tube of the microscope until the front of the objective
just touches the slide. Note the position of the micrometer screw
and slowly focus up with the fine adjustment until the dust or mark
is again in focus. By noting the total and partial revolutions of the

74 THE BRIGHT-FIELD MICROSCOPE [CH. II

graduated fine adjustment the working distance will be known. For
example, suppose it required 5.5 revolutions of the micrometer screw
to raise the objective, from the surface of the slide where the object
is located to a point where the microscope is in focus, and the mi-
crometer screw raises the objective o.i mm. for each complete revo-
lution, then the total elevation will be o.i X 5.5 = 0.55 mm., that is,
the working distance in this case is 0.55 millimeter.

§ 104. Free working distance in covered objects. — Use a 4 mm.
(4ox) objective and the fly's wing or any covered object. Set the
fine adjustment head at zero (o). Lower the objective carefully
with the coarse adjustment until the objective just touches the
cover-glass. Now focus up with the fine adjustment until the object
is in sharp focus, noting the total and partial revolutions of the
screw to accomplish this. The distance the objective was raised is
the free space between the front of the objective and the cover-glass.
Suppose it required 3.2 revolutions of the fine adjustment to focus
the objective, then if each revolution represents o.i mm. the total
elevation is 3.2 x o.i = 0.32 mm. for the free working distance in
this case.

§ 106. Effect of the cover-glass on the working distance. — It is
obvious that if an object is covered with a layer of glass, the free
space between the front of the objective and the object will be
lessened, and if the layer of glass is considerably thicker than the
working distance of the objective, then it will be impossible to get
the object in focus. If the layer of glass is relatively thin, then it
will be possible to focus the microscope on the object, but from the
law of refraction it necessarily follows that the focus of the micro-
scope with and without a cover-glass will not be the same.

Now from the refraction of the rays in passing from one medium
to another of different refractive power, it follows that, when an
object is in or below a stratum of glass or water or other highly re-
fractive body, the object will appear as if raised (figs. 52 B, 64), the
amount of the apparent elevation depending on the refractive index
of the covering body, — the greater its refraction, the more the
apparent elevation. The general physical law is that, the eye being
in the air, the apparent depth of an object below the surface when

CH. II] THE BRIGHT-FIELD MICROSCOPE 75

viewed perpendicularly is the actual depth multiplied by the recipro-
cal of the index of refraction of the covering body. The index of
refraction of the cover-glass is 1.52 or approximately 1.50, and its
reciprocal is T*T> = §• That is, the apparent depth is only | its actual
depth, or in other words the object seems to be elevated f of the
actual depth.

Now if the object is apparently higher up, the microscope must
be raised an amount equal to the apparent elevation of the object.
This is illustrated in figs. 52 B-C. From this it follows that the free
working distance of the objective on a covered object is not lessened
the full thickness of the cover-glass, but only f of that thickness.

§ 106. Demonstration that the v, orking distance is lessened only §
the thickness of the cover-glass. — Use a clean, flat glass slide. Put
an ink or pencil mark on the upper face for object. Employ a 16
mm. (IQX) objective and a lox ocular. Focus the microscope on the
ink or pencil mark, then measure the free space between the slide
and the end of the objective \\ith the wooden wedge, as directed in
§ 103. This is the free working distance (\ 102) without a coverglass.

Cut a glass slide up into two or three pieces for cover-glasses.
Measure the thickness of one of the pieces with the cover-glass
measurer or in some other good way. Place this over the mark on
the slide which was in focus. If now one looks into the microscope,
the mark will not be in focus with the glass cover over it. Focus up
carefully until the mark is again in focus. Measure the space
between the top of the cover-glass and the objective as before. This
will represent the free working distance with this cover-glass.

Subtract the free working distance with this cover-glass from that
with no cover-glass and the difference will be the amount the free
working distance has been lessened by the addition of the cover.
This amount compared with the thickness of the cover-glass will
give the ratio of lessening of working distance by the addition of the
cover-glass.

In an actual case the results were as follows:

Free working distance without cover 4.62 mm.

u u " with cover 3.54 mm.

Lessening of the working distance by the cover-glass i .08 mm.

The actual thickness of the cover-glass was 1.62 mm.

76 THE BRIGHT-FIELD MICROSCOPE [Cn. II

That is, the lessening of the free working distance was not so great
as the thickness of the cover (1.62 mm,), but less; viz. 1.08 mm.;
that is, in the proportion of ~%~ = f of the actual thickness of the
cover-glass.

§ 107. Determining the thickness of the cover-glass with mounted
objects. — From what has been learned about the free working dis-
tance with covered objects, it is possible to determine the thickness
of the cover-glass over an object if the object is in contact with the
cover. If it is below, as shown in fig. 52 C, and the mounting me-
dium is Canada balsam with approximately the same refractive index
as glass, then it is possible to determine how great is the combined
thickness of the cover-glass and layer of Canada balsam over the
object.

Demonstrate the method as follows: (i) Where the object is in
contact with the lower surface of the cover-glass (fig. 52 B). Use a
4 mm. (4ox) objective and a cover-glass 7^ mm. thick. Make a
black ink mark on one side of the cover and a colored ink mark
directly opposite on the other side of the cover, or use glass pencils
of two colors. Set the graduations of the fine adjustment at zero
(o). Place the marked cover on a glass slide, and put under the
microscope. Focus with the coarse adjustment on the mark at the
upper surface of the cover. Then focus down with the fine adjust-
ment until the mark on the lower surface appears sharp. For veri-
fication, focus up until the upper mark is again sharp. The eleva-
tion will of course be the same as the lowering. If the total and
partial revolutions of the fine adjustment screw are noted, they will
show how much the objective was lowered to get the lower mark in
focus. In the case here given it was lowered i revolution. Now as
each revolution moves the objective up or down o.i mm., the objec-
tive was moved down o.i or -^ °f a millimeter. As this represents
| of the thickness of the cover from the effect of refraction, the
whole thickness must be o.io -*- f = 0.15 mm. For a cover of un-
known thickness with the object in contact with its under surface,
put an ink mark on the upper surface of the cover and proceed
exactly as above, focusing successively on the object and on the
ink spot.

CH. II J THE BRIGHT-FIELD MICROSCOPE 77

(2) Where the object is somewhere below the cover-glass (fig.
$2C). In this case the thickness of the cover-glass cannot be
determined, but one can determine very approximately the com-
bined thickness of the cover-glass and the mounting medium over
the object as follows: Put an ink or glass pencil mark on the upper
surface of the cover-glass. Focus the mark with the coarse adjust-
ment after setting the graduations of the fine adjustment at zero
(o). Then focus down with the fine adjustment until the object is
sharp. Note the number of revolutions and the partial revolution
of the fine adjustment drum. As this amount represents only f of
the actual thickness of the glass and mounting medium over the
object, divide the observed amount of movement by § and the quo-
tient will represent the total thickness over the object.

For example, in one case the microscope was focused on the ink
mark at the top of the cover, and then it was necessary to focus
down 13 revolutions of the fine adjustment screw to bring the object
in focus. That is, it was necessary to focus down 0.15 mm. As this
represents but § of the actual thickness of the cover-glass over the
object, the entire thickness must be (0.15 -s- f ) f or 0.225 mm. But as
the specimen was mounted in balsam which has nearly the refractive
power of glass, it represents the combined thickness of cover-glass
and balsam mounted object. Probably in this case the cover-glass
was 0.15 mm. and the object 0.075 mm-

LIGHTING EXPERIMENTS WITH THE COMPOUND MICROSCOPE

§ 108. Daylight with a mirror. — As the following experiments are
for mirror lighting only, remove the substage condenser if one is
present (see § 114, for condenser). Place a mounted fly's wing under
the microscope, put the 16 mm. (lox) or other low objective in posi-
tion, also a 5x ocular. With the coarse adjustment lower the tube
of the microscope to within about i.cm. of the object. Use an
-opening in the diaphragm about as large as the front lens of the
objective; then with the plane mirror try to reflect light up through
the diaphragm upon the object. One can tell when the field (§ 93)
is illuminated by looking at the object on the stage, but more satis-

78 THE BRIGHT-FIELD MICROSCOPE [Cn. II

factorily by looking into the microscope. It sometimes requires
considerable manipulation to light the field well. After using the
plane side of the mirror turn the concave side into position and light
the field with it. As the concave mirror condenses the light, the
field will look brighter with it than with the plane mirror. It is
especially desirable to remember that the excellence of lighting de-
pends in part on the position of the diaphragm (§73). If the
.greatest illumination is to be obtained from the concave mirror, its
Dosition must be such that its focus will be at the level of the ob-
ject. This distance can be determined very easily by finding the
.ocal point of the mirror in full sunlight.

\~Jfr 109. Use of the plane and of the concave mirror. — The Ifcrirror
should be freely movable, and have a plane and a concave face
(fig. 1 8). The concave face is used when a large amount of light is
needed, the plane face when a moderate amount is needed or when
it is necessary to have parallel rays or to know the direction of the
rays.

§ 110. Axial or central light (§71). — Place a preparation contain-
ing minute air bubbles under the microscope. The preparation may
be easily made by beating a drop of mucilage on the slide and cover-
ing it. (See § 334.) Use a 4 mm. (4ox) objective and a 5x ocular.
Focus the microscope and select a small bubble, one whose image
appears about i mm. in diameter, then arrange the plane mirror so
that the light spot in the bubble appears exactly in the center.
Without changing the position of the mirror in the least, replace the
air bubble preparation by one of Pleurosigma angulatum or some
other finely marked diatom. Study the appearance very care-
fully.

§ 111. Oblique light (§ 72). — Swing the mirror far to one side
so that the rays reaching the object may be very oblique to the
optic axis of the microscope. Study carefully the appearance of
the diatom with the oblique light. Compare the appearance with
that where central light is used. The effect of oblique light is not so
striking with histological preparations as with diatoms.

It should be especially noted in §§ iio-m, that one cannot de-
termine the exact direction of the rays by the position of the mirror.

CH. iij THE BRIGHT-FIELD MICROSCOPE 79

This is especially true for axial light (§ no). To be certain the
light is axial some such test as that given in § 334 should be applied.

EXPERIMENTS WITH ARTIFICIAL LIGHT AND A MIRROR

§ 112. Lighting with a kerosene lamp. — For this a lamp with a
flat wick from 3 to 5 cm. wide is best. It should be turned up well,
but not enough to smoke. The face of the flame should be turned
toward the microscope for low powers. For moderate powers the
flame should be made oblique, and for high powers the edge of the
flame should be used. This is because the thicker source of light
gtves a greater brilliancy. Use the fly's wing or any well-stained
preparation.

As the light is in diverging beams, it is best to use the concave
mirror to partly overcome the divergence. One must learn by ex-
perience and trial how far off to have the lamp. A distance of 15 to
20 cm. is usually satisfactory. There should be an opaque screen
between the lamp and the microscope to protect the eyes of the
observer and to screen the stage of the microscope (fig. 42).

This lamp illumination is brilliant, but the color values are quite
unlike those given by daylight.

§ 113. Lighting with artificial daylight. — For the source of light
use preferably a loo-watt nitrogen- filled mazda lamp enclosed in
a kind of lantern (figs. 46, 53). Have the lamp filament at about
the level of the center of the microscope mirror, and a frosted disc
of daylight glass, before an aperture in the lantern.

For object, use a fly's wing or any good, well-stained specimen.
It would be interesting to sit near a window, and to turn the mirror
in such a way as to bring in daylight a part of the time. In this way
one can get a good idea of the real similarity of the artificial and of
the natural daylight. If one also had an electric lamp without
any light filter one could pass in order from real daylight, through
the artificial daylight and then on to the unmodified artificial light.
Without seeing these in comparison, one is hardly able to appreciate
the likeness between the natural and artificial daylight and the great
unlikeness of unfiltered electric light and artificial daylight.

THE BRIGHT-FIELD MICROSCOPE

[CH. II

§ 114.vCondensers. — These are single lenses or lens systems to
aid in illuminating objects by either direct or transmitted light

FIG. 53. LABORATORY TABLE, STOOL, MICROSCOPE AND CHALET LAMP WITH

DAYLIGHT GLASS.
(About one-fifteenth natural size.)

CL Chalet microscope lamp with two windows of daylight glass on opposite
sides under the overhanging roof. The roof serves to protect the eyes (fig. 46).
M Laboratory microscope, slightly inclined.

It will be noted that the table rail is cut out in front to avoid interference with
the knees of the observer. A table drawer at the right can be pulled out without
moving. The revolving piano stool can be adjusted to any desired height.

(§§ 69-70). For direct or reflected light, such as is required for
opaque objects, condensers are usually simple, and are called " bull's-
eyes." They are mounted on a stand for holding them at different
heights and in any desired position (fig. 127).

Condensers for transmitted light (§ 70) are complex optical appli-
ances, sometimes almost as complex as objectives.

The student might fairly ask: Why be bothered with anything
more than a mirror for lighting translucent objects. A glance at

CH. II]

THE BRIGHT-FIELD MICROSCOPE

fig. 54 will show him that with a mirror only a narrow cone of light
can be sent to the object. He will find in actual work that for
powers of 8 (2ox), 4 (4ox),
and 2 (QOX) mm. the object
cannot be lighted with a suffi-
cient angle, or aperture, as it
is now called, to bring out the
details of structure that he
is trying to see and under-
stand.

If anything is certain in
vision, it is that the details
which can be made out clearly
depend upon the aperture of
the light from the object to the
eye. If that be true, then it
is essential that the object
be supplied with light at an

FIG. 54. DIAGRAM PROM BECK TO SHOW
THE APERTURE REFLECTED BY THE MI-
CROSCOPE MIRROR. IT is ONLY ABOUT
0.25 N.A.

The diagram represents the light origi-
nating from a white surface or from the
sky, and the small part which the micro-
scope mirror can receive and reflect,
aperture great enough to fur-
nish the required aperture of light for the eye.

With opaque objects like snow, white paper, etc., the reflections
are in all directions and if the light by which they are illuminated is
brilliant enough, any aperture up to i.oo N.A. will be satisfied if
there is air between the white surface and the microscopic objective.
If a greater aperture than i.oo N.A. is required, as for immersion
objectives, then the proper immersion liquid must be between the
object and the front of the objective.

It is sometimes stated that if one points the microscope toward the
sky out of doors, the aperture of any microscope objective will be
filled with light. This is true for dry objectives which never re-
quire an aperture greater than i.oo N.A. An immersion objective
with an aperture above i.oo N.A. would not be filled even from the
sky, as can be seen easily by trying the experiment (§124).

§ 115. Angular and numerical aperture in microscopy. — By angular
aperture is meant the angle in air formed by the border rays of the light
passing from the object into the front lens of the objective (fig. 116).

82 THE BRIGHT-FIELD MICROSCOPE [Cn. II

If the light entering and leaving a lens or lens system were always
from and into air, angular aperture would be entirely adequate; but
as the light in immersion instruments (objectives and condensers)
is from or into a medium with greater refractive index than air, the
cone of light is modified by the refractive action of the medium
which it traverses, hence the index of refraction of the medium in
which the light cone passes must be considered. Abbe devised an ex-
pression which meets the needs. It is called Numerical Aperture
(N.A.), and is found by multiplying the sine of the semi-angle of the
cone of light in the medium by the refractive index of the medium.
Stated mathematically it is: N.A. = n sin u. In which ;/ stands for
the refractive index, and sin u for the natural sine of half the angle
of the light in the medium.

The following generalizations can be made:

(a) With two media in contact, the sines of fhg a,ns1e<; °f ^e rays
of light in the two media are in inverse ratio to the index of refrac-
tion in the two media; consequently, the greater the difference in
refraction, the greater the difference of the anjle of the same light in
the two media. Knowing any three of the factors, the fourth is
readily found.

(b) The numerical aperture (N.A.) of the light passing from one
medium directly into another remains constant, no matter how great
the change in its angle.

(c) The numerical apeiturc (N.A.) of the light in any medium is
the sine of the semi-angle of the light multiplied by the index of
refraction of the medium (N.A. = n sin u). Knowing any two of the
factors, the third is readily determined.

By referring to fig. 116 and fig. 55, one can get a graphic view of
the significance of these terms.

In fig. 55 A, the angle of the light in the air is 180° and the sine of
half this angle is i.oo (sin 90° = i.oo). If now this 180° of light in air
passes directly into glass with an index of refraction of 1.52, its angle
will be reduced from 180° to 82°, but its numerical aperture (N.A.
= n sin u) is not changed, and is i.oo in both air and glass.

Suppose the light is passing from the glass into the air, as most
'requently happens with condensers, then the light cone of 82° will

CH. II]

THE BRIGHT-FIELD MICROSCOPE

expand into 180° or the whole hemisphere in the air.
numerical aperture (N.A.i) remains unchanged as before.

83
But the

FIG. 55. DIAGRAMS TO SHOW THE ANGLE OF LIGHT IN GLASS
REQUIRED TO FILL AN OVERLYING HEMISPHERE OF AIR,
WATER, GLYCERIN OR HOMOGENEOUS LIQUID WITH LIGHT.

The diagrams show that in each case the angle of light re-
quired in the glass represents a numerical aperture equal to
the refractive index (n) of the overlying medium. The dark
parts of the hemispheres in A, B and C represent the seg-
ments not lighted. In D the whole sphere is lighted. Any
light in excess of this aperture is reflected back into the con-
denser.

In fig. 55 D where there is glass below and homogeneous liquid
above, the angle of the light is 180° in both and the numerical aper-
ture is 1.52, agreeing with the refractive index.

§ 116. How to tell the part of the aperture filled with light. —
When an objective is focused upon any object one can tell the aper-
ture of the objective being used by taking out the ocular and looking

THE BRIGHT-FIELD MICROSCOPE

[CH. II

Table Showing the Angle of Light in Different Media for the same Numerical
Aperture with Dry, Water Immersion, and Homogeneous Immersion Objectives
or Condensers.

(From the Journal of the Royal Microscopical Society.)

Numerical
Aperture
(n sin u = N.A.)

Angle in
Air

(n » i)

Angle in
.Water

(n = 1.33)

Angle in
Homogeneous
Media

(n = 1.52)

1-52
1-33

1. 00

0.76
0.50
0.25

180°
g8°S6'
60°

1 80°
97o3I/,
44° 10^

180°

I22°6'

82°i7'
60°
38024'
i8°56'

Obj«ctlv»

down the tube. The bright spot seen is the back lens of the objec-
tive. If it is all lighted, then the entire aperture is filled. If there
is a bright spot in the middle and a dark rim around the edge, then
it is but partly filled. For experiment use a transparent object like

a stage micrometer or a very

B 07j«t,'vTe C Obj'lctlv0 thin, lightly colored section.

Use the 16 mm. (icx) objec-
tive. Focus the object. Then
take out the ocular and look
down the tube. Probably the
whole of the back lens will be
lighted. Close the iris di-
aphragm slowly and the

3ubtt«a»

FIG. 56. APERTURE OF THE SUBSTAGE
CONDENSER AND or THE OBJECTIVE.
(From Nelson, Jour. Roy. Micr. Soc.)
A The cone of light from the condenser margin of the back lens will
fills the aperture of the objective (B). uavp „ ,Joru r:m «rrmnri :* ac

D The cone of light of the condenser naVC a dark nm around lt as
only partly fills the aperture of the objec- the iris gets so small that the

is not filled. Turn

A and D the condenser and objective

are shown in section; in B and C, the back the 4 mm. (4Ox) objective in

lens of the objectives is shown in face view niarp flnri rf,npPf. f-v,- PYnPri

as when looking down upon it with the pla°e and repeat tne exPen-

ocular removed. ment. It is to be no ted that the

iris must be wider open for the

4 mm. Uox); and wider still for the 3 mm. (6ox) (fig. 56), One
can determine more easily and accurately the amount of aperture

CH. II]

THE BRIGHT-FIELD MICROSCOPE

filled in the objective, the centering of the condenser and the light-
ing if a central pinhole cap is placed over the top of the tube and
one looks down through the pinhole (fig. 58).

§ 117. Aperture; and centering the condenser by the eyepoint
— - As stated by A. E. Wright, the part of the aperture of an objec-
tive lighted in any given case is most easily and accurately deter-
mined in the focused microscope by examining the eyepoint with a
magnifier. One of the aplanats of 10 to 15 magnification is good for
this. The magnifier can be held in the hand, by a lens holder or by
Beck's lens holder for the eyepoint (figs. 24, 57). If the focused
microscope is brilliantly lighted, the eyepoint can be seen and its
position determined by the use of a piece of thin paper or by the
use of ground glass, § 99. If the magnifier is held above this point
and raised and lowered the eyepoint can be focused as if it were an
object. When in focus one will see an image of the back lens of the
objective and of the diaphragm opening. Open and close the iris and
change the focus of the magnifier if necessary to make the diaphragm
opening sharp. By closing the diaphragm the
back lens of the objective will be only partly
lighted. With low powers it is easy to open the
iris wide enough to light the entire back lens.
It will be seen in this experiment that the
higher the power the wider open must be the
iris to fill the back lens with light. This means
that the higher power has a larger aperture and
hence must be lighted by a wider aperture from
the condenser; and that necessitates a wider
opening to the iris. By looking at the eyepoint
with the magnifier one can tell exactly how much
of the back lens is lighted. That can be deter-
mined less certainly by taking out the ocular and
looking down the tube of the microscope, or by
using the pinhole cap (fig. 58).

As pointed out by Wright, p. 93, a study
of the eyepoint by means of the lens (fig. 57)
or pinhole cap (fig. 58) gives very definite information:

Focusing Glass

FIG. 57. COMPOUND
MICROSCOPE WITH
BECK'S ADJUSTABLE
ARRANGEMENT FOR
FOCUSING THE EYE-
POINT.

THE BRIGHT-FIELD MICROSCOPE

[Cn. II

(1) Whether the back lens of the objective is filled with light, or
how nearly filled. One can then judge whether the diaphragm is
opened the right amount.

(2) Whether the condenser axis is centered to the axis of the
objective. If it is not the opening of the diaphragm will be closer
to one side. If centered the diaphragm opening will be in the center
of the back lens of the objective.

(3) Dust or other opacities on the back lens will be brought out.

(4) The presence of air bubbles in the immersion liquid will ap-
pear.

§ 118. Experiments in centering. — Use a transparent or very
translucent specimen, and focus the microscope with any objective.
It is better to begin with an 8 mm. (2ox) or a 16 mm. (xox) objec-
tive.

Close the iris as much as possible, and then examine the eyepoint
with the magnifier. Or use the pinhole cap in place of the ocular

(fig. 58). The opening of the iris should
appear in the middle of the back lens of
the objective. Open the iris slowly while
looking into the magnifier, and note
whether as the iris opens, it disappears
around the edge of the back lens uniformly
or whether it remains in sight longer on
one side. If the small bright spot seems
to be in the center of the back lens, and
the iris disappears all around the back
lens at the same time, then the condenser
is centered. If the small opening is ec-
centric, and if the diaphragm does not
disappear all at once on opening the iris,
then the condenser and objective are not
centered, that is, are not on the same
optic axis.

If they are not centered, and the
substage fitting has centering screws (fig. 60) these should be
turned with the two hands while looking into the magnifier until

FIG. 58. PINHOLE CAP
TO Am IN CENTERING
AND DETERMINING APER-
TURE.

A Top view.

B Sectional view.

CH. II] THE BRIGHT-FIELD MICROSCOPE 87

the small opening seems central, and then until the iris disappears
equally all around the border of the back lens.

If there are no centering screws to the substage, as in student
microscopes and some others, one can ignore the fact if the eccentric-
ity is slight, or if the work is not too exacting. If the eccentricity is
great, it is best to send the microscope to the makers for centering,
unless the user has the knowledge and mechanical skill to make the
corrections himself.

With some microscopes the iris diaphragm is so placed that the
condenser projects a real image of it above the stage. If one closes
the iris and uses a 16 (icx) or 8 (2ox) mm. objective, this real image
of the iris can be seen in the field by focusing up from the position
where a specimen would be in focus. In some cases one might need
to focus down slightly. If the image of the iris opening is in the
middle of the field, the condenser is centered to the objective; if it
is not in the middle, one can center by using the centering screws
(fig. 60).

Sometimes also the iris can be made eccentric by means of a milled
wheel, therefore before trying the centering, one must be sure that
the iris diaphragm is centered, and not eccentric to the condenser.

§ 119. Experiment with the sky as light source. — Remove the body
of the microscope with its objectives in the nose-piece from the
microscope stand. Put some lens paper in the tube and insert the
ocular firmly. This is so it will not fall out when the tube is pointed
to the sky. Take along the magnifier to look at the eyepoint or
use its pinhole cap in place of an ocular (§117). Go out in the
open and point the microscope up at the sky. Use the objectives
in turn. Each of the dry ones will have its aperture completely
filled; but the immersion objective if of over i.oo N.A. will not be
filled. There will be a dark rim around the objective representing
the aperture above i.oo N.A.

§ 120. Aperture filled by a white opaque object. — Put a white
card or any thick piece of white paper on the stage of the micro-
scope. Put the microscope in a window where the sun can shine on
the paper, or use some brilliant artificial light and focus on the upper
surface of the paper under the objective. The daylight lamp (figs.

88 THE BRIGHT-FIELD MICROSCOPE [Cn. II

79 or 80) for dark-field work is excellent for this experiment. The
success of the experiment depends upon a bright light; how it is
obtained is not important.

Focus first the 16 mm. dox) on the top of the card. The back
lens will be full of light showing that the whole aperture is satisfied.
The same will be found for the 4 mm. Uox). Now turn the homo-
geneous immersion objective with an aperture of 1.25 to 1.40 in
place and focus it down almost to the paper. The back lens will
now have a dark rim around it.

To satisfy the aperture completely some homogeneous liquid must
be put between the paper and the front lens of the objective. Keep
the eye over the magnifier above the eyepoint or use a pinhole cap and
with a small brush or in some other way put some homogeneous
liquid on the paper at the edge of the objective. It will run under
by capillarity; and as it spreads over the face of the objective, the
dark rim will disappear. Prove this is true by focusing up until the
homogeneous immersion is broken. The dark rim will reappear.
Now focus down until the objective again becomes immersed and
the whole aperture will again be filled with light. In a word: These
experiments show that a numerical aperture not exceeding i.oo N.A,
can be supplied to the microscope by light in air.

§ 121. Aperture with transmitted light from a mirror. — With
transmitted light through thin white paper or ground glass or other
light scattering substance, the entire hemisphere will also be filled
with light by the mirror. This is easily demonstrated by placing a
piece of glass ground on both surfaces on the stage of the microscope,
and lighting it well with the plain mirror. If any dry objective is
focused on the upper granular surface of the glass, its aperture will
be found entirely filled with light. If now a homogeneous immersion
objective is focused on the granular surface, its aperture will be only
partly filled with light. Just as when pointing the objective toward
the open sky, the central part of the back lens will be lighted, but
there will fee a dark border all around it. This border may be dimly
lighted by the diffracted light, but it will not be anywhere near as
light as the central i.oo N.A. Now while looking at the image of the
back lens in the eyepoint (§117), if some homogeneous immersion

CH. II] THE BRIGHT-FIELD MICROSCOPE £9

liquid is put on the glass at the edge of the objective, it will gradu-
ally run in between the objective front and the glass and as it does
so, one can see the whole aperture becoming filled. If the objective
is focused up until the immersion liquid breaks, leaving air again
between the ground glass and the objective front, the aperture will
again be filled only to the i.oo N.A. as shown by the dark border.

If rather thick cedar oil is used for the immersion liquid, it is pos-
sible by focusing up carefully to have the oil column only partly un-
cover the front lens of the objective, then the part uncovered will
show the dark rim of unfilled aperture while the other part will be
completely filled. By careful focusing one can make the immersion
liquid flow back and forth over the front lens and see the change in
aperture from the covered to the uncovered portion. After such an
experiment one can never again doubt the efficiency of an immersion
fluid of greater refraction than air for increasing the aperture of the
light.

§ 122. Aperture with translucent objects. — If the object does
not scatter the light as with ground glass or thin paper, etc., then
the aperture of the light going to the object will pass on to the ob-
jective and will determine the amount of aperture the objective has
available for forming an image.

Aperture with translucent objects lighted by a mirror. — Use any
translucent or transparent object like a stage micrometer or a
thin histological section. Turn the condenser aside, and use the
mirror only for illumination. Use first a 16 mm. (lox) objective.
Focus the well lighted specimen and examine the back lens by the
pinhole cap after the ocular has been removed, or by looking at
the eyepoint with a magnifier (§117). With a 16 mm. (lox) of
N.A. 0.25, the entire aperture will be filled with light either from
the plane or from the concave mirror. With a 16 mm. (xox) ob-
jective of 0.30 N.A. the 5 centimeter mirror barely fills the aperture.
A mirror of 6 centimeters diameter fills the aperture completely.
As the usual microscope mirror is rarely over 5 centimeters in diam-
eter, it can be stated that with a mirror alone only about 0.25 N.A.
can be supplied.

§ 123. Aperture with translucent objects lighted by a substage

00 THE BRIGHT-FIELD MICROSCOPE [Cn. II

condenser. — Use a translucent object mounted in Canada balsam,
but put the condenser in position and close to the object. Light with
the plane mirror. First use the 16 mm. (lox) objective; open the
iris. Look down the tube or at the eyepoint. The aperture is
completely filled. Turn a 4 mm. (40*) objective into position, focus,
and then look down the tube or at the eyepoint with a magnifier.
Open and close the iris. By opening it widely the entire aperture
will be used as shown by the back lens full of light.

Now turn the homogeneous immersion into position, and without
adding any immersion liquid, focus the objective. Look at the back
lens and it will be found only partly filled with light, although the
iris is wide open. Add the homogeneous liquid so that there will be
immersion contact with the objective and specimen. Look at the
back lens again, and it will be found that the aperture is more com-
pletely filled, but there is still a dark rim around the outside just as
there was when looking at the sky (§ 119). This is in spite of the
fact that the immersion objective is in homogeneous immersion con-
tact with the object or its cover-glass. In § 120 when the homo-
geneous liquid made immersion contact, the aperture was completely
filled with light; why is it not in this experiment? With §§ 120-
121 the object itself scattered the light and filled the whole hemi-
sphere above, consequently when the homogeneous liquid was added,
the full aperture could be satisfied. Tn this case the specimen does
not scatter the light to any extent, and the objective receives only
the aperture of light that went from the condenser through the
object to the objective.

But as the substage condenser has an aperture of 1.20 to 1.40
N.A., why does it not supply the adequate aperture to the object
so that the objective aperture will be completely satisfied? The
law of optics by which no aperture greater than i.oo N.A. can
pass from a denser medium to air prevails here, for there is air be-
tween the top of the condenser and the under surface of the glass
slip bearing the specimen, hence the object cannot be lighted by such
an arrangement with a cone with an aperture greater than i.oo N.A.
The way the difficulty is overcome is shown in the next section.

§ 124. Immersion substage condenser. — If the substage con-

CH. II] THE BRIGHT-FIELD MICROSCOPE 91

denser has air between its upper face and the under surface of the
glass slip on which the specimen is mounted, the condenser, no
matter what its possible aperture, can send into the air only an
aperture of i.ooN.A. (fig. 55 A). If the condenser is to illuminate
the object with an aperture greater than one, then there must be a
medium of higher refractive index than air between the top of the
condenser and the under surface of the glass slip.

This statement is very easily verified by the following experiment.
Use a homogeneous immersion objective, and some rather transpar-
ent object, or take a clean glass slip and with a fine pen make a line
in the middle with India ink. This is to give something to focus on.
Use the homogeneous immersion fluid on the objective as usual and
focus the ink line, then move the slip so that the black line will not
obscure the field. Open the iris diaphragm to its full extent so that
the condenser will have a chance to work at full aperture. Light
well with the plane mirror. Examine the eyepoint with a magnifier
(§ 117) or use the pinhole cap (fig. 58) and note that there is a
dark rim around the margin of the back lens of the objective just as
there was when the microscope was pointed toward the sky or used
with the ground glass. That is, as only a numerical aperture of
i. oo could be supplied by the condenser with air between it and the
glass slip, only a numerical aperture of i.oo of the objective is
filled. Without changing the objective in any way, lower the
condenser and put on its upper face a good drop of the immersion
liquid, and then slowly run it up until the oil on its upper face comes
in contact with the lower face of the slip. Now look at the back
lens of the objective and note that the aperture of the objective is
filled. To make still more striking the demonstration, while looking
at the back lens of the objective, lower the condenser very slightly.
This will break the immersion contact on one side. Lower till
about half of the aperture shows immersion contact. On the side
where the immersion is lost the dark rim will show, and on the side
still immersed it will be absent (§§ 120-121).

If then one is to use the full aperture of any objective with an
aperture greater than one i.oo N.A., both the objective and the con-
denser must be immersed in some fluid with a higher refractive index

92 THE BRIGHT-FIELD MICROSCOPE [Cn. II

than air. In the experiment the homogeneous liquid was of much
higher index (1.52).

§ 125. Correlation of objective and condenser aperture. — It is
evident from the experiment in § 124 that if the objective is to be
used at its full aperture the condenser on the microscope must be of
sufficient aperture to satisfy the objective. Strange as it may appear,
however, many microscopes are regularly supplied with objectives
of 1.25 to 1.30 N.A., and much is made of the fact, but the con-
denser supplied on the microscope has at the outside not over 1.20
N.A. and not one in a hundred who uses such a microscope ever
makes immersion contact with the condenser and glass slip, and
therefore never uses more than an aperture of i.oo N.A. The few
that would like to utilize the high aperture, of which so much is said
in the microscope catalogues of all manufacturers, cannot do so
with the low apertured condenser regularly supplied.

Fortunately it is now becoming the fashion of users of the micro-
scope to know something of the optics of their most efficient servant
and the manufacturers are helping to spread the knowledge needed,
and to supply most excellent condensers to meet the most exacting
requirements. It is to the credit of the British microscopists and
manufacturers that they have always been in the lead in such mat-
ters. — (See Brewster, Carpenter-Dallinger, Nelson, Coles, and Con-
rad Beck, etc.)

§ 126. Experiment with a 1.20 N.A. condenser (fig. 59) and an
objective with 1.25 to 1.40 N.A. — Use a transparent specimen in
Canada balsam like that used in § 125. Connect condenser and
glass slip with homogeneous liquid (§ 125). Use the homogeneous
immersion objective with as high an aperture as is available, and
employ the correct homogeneous liquid. Focus sharply. Now look
at the back lens of the objective and open and close the iris diaphragm
so that the opening shows unmistakably. Now open the iris to its full
extent. There will still be a dark rim around the outside. That is,
a condenser with an aperture of 1.20 cannot supply an aperture of
1,25 to 1.40 N.A. even though it is in immersion contact with the
under side of the slip.

If one has available a substage condenser of 1.40 N.A. and it is in

CH. II]

THE BRIGHT-FIELD MICROSCOPE

WITH

immersion contact with the glass slip, the full aperture of the objec-
tive can be filled. For the methods of determining the numerical
aperture (N.A.) of objectives and condensers
see § 261 et. sq.

§ 127. Diffracted light and aperture. — With
many objects when illuminated by transmitted
light there is considerable, and with all objects a
certain amount of diffraction of the light going
through them; consequently there is a greater or
less amount of diffracted light in addition to
the original cone. This additional diffracted
light may be so great in amount that the whole
aperture of the objective of 1.30 to 1.40 N.A. may
be filled, although the condenser may have an CONDENSER
aperture of only 1.20 N.A. In most cases the vc

diffracted light is very weak as compared with Iris diaphragm.
that directly from the condenser and object, hence
the aperture supplied by the condenser will be very bright in com-
parison with the aperture filled by the diffracted light, and the
zone at the edge of the objective appears as a gray rim around the
edge. If there were no diffracted light, it would look black, be-
yond the aperture supplied by the condenser.

Much has been said in recent years about the supreme importance
of diffracted light in microscopy. As diffracted light forms an im-
portant part of all light, no one can doubt that it is of importance.
It seems to the writer, however, that this constituent has been by
some over-emphasized and given undue prominence in microscopic
vision.

§ 128. Optical corrections of the substage condenser. — Funda-
mentally the substage condenser has for its purpose the illumination
of the object. One of the pictures showing the use of a condenser for
the microscope is given by Descartes (fig. 312) and is a plano-convex
lens with the plane side toward the source of light.

In the early period of uncorrected lenses, and also at the present
time with the popular Abbe condenser, which is neither achromatic
nor aplanatic, the main purpose seems to be to supply an abundance

94 THE BRIGHT-FIELD MICROSCOPE [Cn. II

of light. If one bears in mind that no matter how carefully corrected
the objective may be, if the light received by it from the condenser
is full of chromatic and spherical aberration, it is not fair to expect
the objective to make a perfect picture. Over 100 years ago Sir
David Brewster appreciated the difficulties, and stated with great
clearness what seemed to him the means for obtaining correct micro-
scopic images. He says of illumination in general: " The art of
illuminating microscopic objects is not of less importance than that
of preparing them for observation." " The eye should be protected
from all extraneous light, and should not receive any of the light
which proceeds from the illuminating center, excepting that portion
of it which is transmitted through or reflected from the object."
So likewise the value and character of the substage condenser was
thoroughly understood and pointed out by him as follows: " I
have no hesitation in saying that the apparatus for illumination
requires to be as perfect as the apparatus for vision, and on this
account I would recommend that the illuminating lens should be
perfectly free of chromatic and spherical aberration, and the greatest
care be taken to exclude all extraneous light both from the object
and from the eye of the observer." (See Sir David Brewster 's treatise
on the Microscope, 1837, pp. 136, 138, 146, and the Edinburgh
Journal of Science, new series, No. n (1831) p. 83.)

While the simple and relatively inexpensive substage condensers
of the Abbe type (fig. 59) serve fairly well for student and general
work it is evident that for the most exacting work achromatic-apla-
natic condensers are required. If it is true, as all agree, that for a
perfect image of an object no light should reach the eye except from
the object, then it is readily seen that an achromatic-aplanatic con-
denser must be used for it is only by such a condenser that the
object can be so lighted that no light reaches the eye except from
the object. Condensers with both chromatic and spherical aberra-
tion cannot direct their entire cone of light upon the object and
hence much light must reach the eye which does not come from the
object. In so far as that is the case, the image will be imperfect.

The clearness of the images with the best dark-field condensers
gives abundant evidence for this contention. With the highest

CH. II]

THE BRIGHT-FIELD MICROSCOPE

Subttago
Condenser

powers, if perfection is sought for, the object must be mounted on
glass of cover-glass thickness, and a homogeneous immersion objec-
tive used for condenser as well
as for forming the microscopic
image, and both are in homoge-
neous contact below and above
the object respectively. Or a
condenser of objective perfection csl
must be used. Special homoge-
neous immersion condensers are
demanded by some workers, and
are willingly produced by the
opticians. However, the com-
mon practice of using the same
substage condenser, dry or im- ^ 6o ACHROMATIC.APLANATIC>
mersed, as occasion requires, is CENTERING, SUBSTAGE CONDENSER.
justified from the fact that the (From the Catalogue of the Spencer
working distance of the sub- CA coarse Adjustment for the con-
stage condenser is exceedingly denser.

, , , ., , . ", FA Fine adjustment for the con-

short, and* hence its spherical denscr>

and chromatic corrections are OS', CS Centering screws for center-
v i -LI cc *. A u 4.1- ing the condenser.

very slightly affected whether klr Handle for working the iris dia.

used dry or immersed. Of phragm.

.- , .Obi Milled head for working the

course, if an aperture greater oblique-light iris.

than i. oo N.A. is desired the

condenser must be immersed. (§§ 124-125.)

g __8a. The writer is indebted to Mr. H. N. Ott of the Spencer Lens
Company for help in this matter. Mr. Ott says: "If the object were in optical
contact with the objective, that is, the working distance reduced to zero, and
the condenser in like manner in optical contact, there would be no difference
whether the objective or the condenser were immersed or not. The working
distance of the objective, small as it is, is relatively great as compared with the
working distance of the condenser. The working distance of the condenser ap-
proaches so nearly to zero that there is no great difference in its chromatic
and spherical corrections whether it is immersed or not. Hence all forms of
condensers, the Abbe type and the aplanatic-achromatic type, may be used
either immersed or not as required. It is of course impossible to get an aper-
ture greater than i.oo N.A. unless the condenser is immersed."

§ 129. Condensers for student microscopes. — These are usually

THE BRIGHT-FIELD MICROSCOPE

[CH. II

of the non-achromatic or Abbe form (fig. 61); and the construction
of the modern microscope is sufficiently accurate so that they are
nearly enough centered for all practical purposes
(Beck). Sometimes they get so badly decentered
that they should be either sent to the makers, or
should be put right by some one with mechanical
ability connected with the laboratory.

§ 130. Mirror and light for the condenser. —
It is best to use light with parallel rays. The
rays of daylight are practically parallel; it is
best, therefore, to employ the plane mirror for
all but the lowest powers. If low powers are
CONDENSER WITH used, the whole field is not illuminated with the
PARALLEL LIGHT. plane mirror when the condenser is close to the
obJect5 furthermore, the image of the window
frame, objects outside the building, as trees, etc.,
would appear with unpleasant distinctness in the field of the
microscope. To overcome these defects one can lower the con-
denser and thus light the object with a diverging cone of light, or
use the concave mirror and attain the same end when the condenser
is close to the object (fig. 57).

§ 131. Lighting the entire field with a condenser. — With the
condenser there are two conditions that must be fulfilled; the proper
aperture must be used, and the whole field must be lighted. As seen
in § 124 the diaphragm of the condenser regulates the aperture of the
illuminating cone but does not affect the size of the lighted field
unless it is far below the condenser. The size of field that is lighted
by a condenser can be modified in two ways:

(1) Suppose that the image of the source of light is focused on the
object, the size of that image will determine the size of field which
is illuminated in a given case. If the illuminated field is not so large
as the objective field, then the source of light is too small, or too far
away. In that case, use a larger source or bring the source closer to
the microscope.

(2) By lowering the condenser or using the concave mirror a
much larger object can be fully lighted, as it is in a diverging cone

CH. II]

THE BRIGHT-FIELD MICROSCOPE

of light above the focal point of the condenser where the light is
spread over a greater area (fig. 61).

For quite low objectives, 35 (4.5x) to 60 mm. (2.6x) focus, it is
better to remove the condenser
and use the mirror only. The
whole field can be illuminated
easily and sufficiently in this
way.

§ 132. Substage condensers
with removable top. — In most
of the good modern, sub stage
condensers the upper or top ele-
ment is removable, and the ele-
ment next the mirror used alone.
With this lower element a suffi-
ciently large field can be lighted
to satisfy nearly all require-
ments. Indeed the lower ele-
ment is so well corrected that
for most purposes objectives as
high as the 4 mm. (4ox) are
well lighted, provided the con-
denser is placed at the right dis-
tance below the stage. One can
easily determine that by trial.
As the lower element is of much
longer focus than the entire
condenser, it must be lowered.

FIG. 62.

OBLIQUE LIGHT WITH \
CONDENSER.
(From Chamot).

The iris diaphragm is opened com-
pletely and the light from one side is
blocked out by inserting the finger; this
gives unsymmetrical light and all of it is
oblique to the optic axis.

For the dark-field element to

go with these separable condensers see § 181.

§ 133. Axial and oblique light with the condenser. — To demon-
strate the effect of the methods of illumination when a condenser is
used, take any striking preparation like a diatom (Pleurosigma
angulatum, for example); employ a 4 mm. (4ox) objective. Being
sure that the condenser is centered, fill the aperture of the objective
about f full of light (§ 124). Study the preparation with the central

98 THE BRIGHT-FIELD MICROSCOPE [Cn. II

light and note the appearance of the markings. Cover a part of the
diaphragm opening by putting the finger or some other opaque
object between it and the mirror (fig. 62). Note that the markings
come out more strongly. Hold the finger in position and open the
diaphragm widely and see if the markings can still be made out.
Now remove the finger so that the object is lighted by the full
aperture of central light. Probably the markings will not appear at
all. Put the finger back in position to give oblique light and the
markings will again be seen. Remove the finger and slowly close up
the diaphragm, When the proper aperture is reached the markings
will again appear.

For histological preparations the oblique light is not a help in
bringing out details of structure. There the end is reached by using
the proper aperture, regulating the source of light, and by differen-
tial staining.

§ 134. Lateral swaying. — Frequently in studying an object,
especially with a high power, the image will appear to sway from
side to side in focusing up or down. A glass stage micrometer or
fly's wing is an excellent object. Make the light central or axial,
focus up and down, and notice that the lines simply disappear or
grow dim. Now make the light oblique, either by making the
diaphragm opening eccentric or, if simply a mirror is used, by
swinging the mirror sidewise. On focusing up and down, the lines
will sway from side to side. What is the direction of apparent move-
ment in focusing down with reference to the illuminating ray?
What in focusing up? If one understands the experiment it may
sometimes save a great deal of confusion.

§ 136. Critical illumination. — This expression strictly used means
that the image of the source of illumination is projected upon the
object by the condenser (fig. 63). The object then appears in the
image of the light source. If the image of the light source is to be
used with the chalet lamp, then the daylight glass filter must be
polished on both sides and the bulb be of transparent, not frosted
glass. The filament is not large enough to cover the field except
when using high objectives.

CH. II]

THE BRIGHT-FIELD MICROSCOPE

Exc

FIG. 63. FIKI n OF THK MICROSCOPE
SHOWING THE LIGHT IN THE CENTER AND
TO ONE SIDE.

C, Fl The light is in the center and
illuminates the object.

Exc, Fl The light is at one side of the
center and does not illuminate the object.
(The field is not fully lighted, as a low
powe.r is used to center the object and
the light.)

Used less strictly it means any very exact method of illumination
which will give the clearest image in any given case. Many good
workers get a sharp image of the source of light upon the object and
then focus the condenser down
just enough to throw the
image out of focus. If one
uses ground daylight glass or
ground ordinary glass over
the window of the chalet
lamp for example, the ground
glass becomes the source of
light and if that is focused on
the specimen, the granulation
of the surface takes away from
the sharpness. This is en-
tirely obviated by throwing
the image of the ground glass
just out of focus. A lamp
flame in like manner, or the coils of an incandescent lamp have
irregularities that injure the microscopic image if the image of the
light source is sharply focused on the object. Unless, then, the
light source is entirely homogeneous, it is better to have its image
out of focus on the object.

§ 136. Aperture of the condenser with critical illumination. —
The most general rule followed — the Nelsonian rule — is to open
the iris diaphragm of the condenser till about three-fourths of the
aperture of the objective is filled with light. For moderate powers
one can tell easily by taking out the ocular and looking down the
tube of the microscope at the back lens of the objective. About
three-fourths of the lens should be lighted. One can judge by open-
ing and closing the iris until it is judged that three-fourths the
diameter is bright, and one-fourth dark. For high powers one can
tell more accurately by looking with a magnifier or the pinhole cap
(§ 1 18, fig. 58) (figs. 57-58, § 1 17) at the eyepoint above the ocular when
the microscope is in focus. In the actual study the diaphragm is
frequently closed more or less and opened to get the best effect in

100 THE BRIGHT-FIELD MICROSCOPE [Cn. II

any given case. One has to keep in mind always, however, that
the amount of fine detail that it is possible to see depends upon the
numerical aperture by which it is studied. As will appear in the
next section the amount of aperture which is usable in a given case
depends partly on the size of the source of light, and the character
of that light.

§ 137. The source and character of the light. — The statements
of three British workers are so to the point in this connection that
they are quoted: — Sir David Brewster, 1831-1837. " The gieatest
care (should) be taken to exclude all extraneous light both from the
object and from the eye of the observer." Wright, " Principles of
Microscopy, " p. 219: " The necessity for the regulation of the
source of illumination will appear when we consider the optical
conditions which obtain where an extended radiant field such as is
furnished by the sky or a broad lamp tlame is employed as a source
of light. With such an extended source, its image will be larger
than the field of any objective. .

From the radiant points included within this illuminated area
beams will pass into the aperture of the objective. Those from the
center of the field — always assuming that their numerical aperture
does not exceed the numerical aperture of the objective — will pass
through the aperture unmutilated. It will be different with respect
to the beams which proceed from the periphery of the field. These,
taking the aperture obliquely, will, unless in the case where their
numerical aperture is much less than that of the objective, be cut
down in an unsymmetrical manner by the margin of the objective,
exactly in the same way as would be the case if transmitted through
an elliptical, or, in the extreme case, through a slit aperture.

" It follows that while the radiant points in the center of the field
will be represented in the image by circular antipoints whose dimen-
sions will be determined by the full numerical aperture of the objec-
tive, the radiant points on the periphery of the field will be represented
in the image by elliptical or linear antipoints whose long axes will in
each case be disposed radially to the aperture, overlapping the anti-
points in the center of the field in such a manner as to fog the image."

Conrad Beck, Journal of the Royal Microscopical Society, 1922,

CH. II] THE BRIGHT-FIELD MICROSCOPE 101

pp. 399-405, and in his book " The Microscope," Part II, 1924, p.
105: " For the correct delineation of a microscopic object seen with
transmitted light, no light should reach the eye that has not passed
through the object." It is well also to keep in mind what was found
out by the older opticians, viz., that for the discrimination of the
fine details of an object one is far more successful when working in a
dark or a dimly lighted room so that the light from the object under
the microscope or seen through any optical instrument is the only
light entering the eye. The astronomers also found long ago that no
successful study could be made of the faint nebulae on a bright
moonlight night.

—-§ 138. Brightness for the best visual acuity. — For the best
visual acuity with the naked eye it is believed that light objects
should be illuminated by i~ to 3-foot (10- to 3o-meter) candles. Dark
objects must have a proportionally increased illumination.

As the microscope gives an apparent increase in the area of the
object looked at, i.e., in proportion to the square of the magnifying
power, it follows that if the same brilliancy is to be maintained the
light must be increased in the same proportion. That is, if a square
centimeter of an opaque object had a given brilliancy when seen by
the naked eye, when magnified 10 diameters giving an area of 100
square centimeters, the illumination must be increased 100 times if
the brightness remains constant.

For translucent objects lighted by transmitted light, the matter
is complicated because microscopic objectives vary in aperture from
the lower to the higher powers regularly, and with the same powers
depending on their construction, also on the aperture of the cone of
light sent to the object by the mirror or through the condenser. As
the brilliancy varies directly as the square of the numerical aperture,
but inversely as the square of the magnifying power, both the aper-
ture and the magnifying power must be considered when estimating
the intensity of the light required to maintain a constant favorable
brilliancy of the object for the discrimination of its fine details.

Conrad Beck in his recent work on " The Microscope," Part II,
p. 104, taking into account the aperture, the magnifying power and
the light losses in the microscope, gives the following table of de-

102 THE BRIGHT-FIELD MICROSCOPE [Cn. II

sirable foot candles for different powers. Candle meters in round
numbers have been added in the third column.

Mag. power Foot candles Candle meters

50 16 172

100 20 255

300 So 538

600 80 86 1

1000 250 2691

2OOO 1050 H3O2

Diffuse daylight can rarely supply more than 100 foot candles
(1076 candle meters) and hence is not sufficiently brilliant for powers
above 500 to 600 diameters. Besides it is exceedingly variable in
intensity during the different hours of daylight, and in different
seasons of the year. For these reasons many workers discard day-
light altogether for exacting work, and utilize some artificial source
like one of the different daylight lamps (figs. 46, 80), the " tungsarc"
or the " pointlite " lamp. Even the petroleum or kerosene lamp has
its enthusiastic advocates where color values are not involved.
From personal observation the yellow petroleum light does not
permit so clear an appearance of fine details as the daylight lamp.
Furthermore it is much more tiring to the eye with most observers
who have made comparative tests.

For some special work it has been found desirable to use color
screens or even pure spectral colors. In general the shorter the wav^
length of the light used, the finer the detail that can be made out.
For example, with rulings and diatoms, finer lines and finer markings
can be seen with blue light than with red.

In recent times there has been a revival of the use of polarized
light for the study of microscopic objects, and much is hoped from
that source (§216). It is anticipated that the ultra-violet micro-
scope will add even greater information (§ 303).

§ 139. Glare and fog in the microscope. — Even when one has a
good microscope, the image may not be sharp and clear, but in-
distinct and hazy. This may be due to mist on some of the lenses.
For example, on a cool morning, mist may collect on the ocular from
the breath of the observer, or from the moisture emanating from the
eye. No clear image can be seen through mist. Again, the dry

CH. II] THE BRIGHT-FIELD MICROSCOPE 10^

objective may have been smeared with immersion liquid or with
Canada balsam or other mounting substance. No clear image can
be seen through dirty glass surfaces.

Although the microscope lenses may all be clean and of excellent
quality there may also be glare and fog from the illumination. It
is well also to keep in mind that the image may be greatly interfered
with or even spoiled by the glare of an air bubble in the mounting
medium near the object, or in the immersion iluid for the objective
or the condenser. See Sir A. E. Wright, pp. 219-222.

§ 140. Experiments for glare and fog. — Use a stage micrometer
or some other very transparent specimen under a cover-glass in air.
Put the microscope facing a window. Use a 16 mm. (lox) objective
and a rather high eyepiece (lox to 25x). Have the condenser up
close to the glass slip carrying the object. Put an ink spot or a faint
mark with a glass pencil near the object so that it will be possible to
focus easily. With the iris of the condenser nearly closed, focus the
specimen. Then while looking into the microscope gradually open
the iris and note the gradual dimming of the image. When the iris
is wide open it will look as if a veil or mist were over the image, or
the image may be wholly obliterated. This occurs because there is so
much light around and near the object that gets into the microscope
with the light from the object itself. One can prove that it is the light
which is not from the object in this* way: (i) Use a piece of dark,
thick paper and make a hole about 10 millimeters in diameter in it.
Hold this opening between the window and the mirror so that the
light reaching the mirror comes only through the 10 millimeter
opening. The outline of the object will again appear. It may be
necessary to use a screen with even a smaller hole. Restricting the
source of light cuts off the adventitious light which did not come
from the object. (2) The second method is to eliminate the more
oblique rays of the cone of light by closing the condenser iris to a
greater or less extent. Remove the screen with the 10 mm. aperture
and let the full light of the window strike the mirror. Then gradu-
ally close the iris of the condenser. The image will appear. One
can make it appear or disappear by opening and closing the iris.
In case the specimen is not so delicate as the lines of a stage microm-

104 THE BRIGHT-FIELD MICROSCOPE [CH. II

eter ruled by a diamond, the specimen may not disappear entirely,
but it will become much fainter when flooded with light by the full
aperture, as many of the oblique rays enter the microscope that did
not come from the object. In practice one really makes use of both
the iris diaphragm and the regulation of the source of light for
rendering the image distinct. However, if the finest details are to be
seen the aperture must be retained. It is not to be forgotten, too,
that an aperture above i.oo N.A. cannot be obtained except by
immersing the condenser (§ 124).

Fortunately objects mounted in Canada balsam, or other medium
with the refractive index of glass, are not so subject to the disturbing
effects of glare as when mounted in air or in media of low refractive
index, hence one can use the full aperture of the objective most
satisfactorily with balsam mounts.

As stated in § 136 Mr. Nelson makes the generalization that the
best results are obtained in critical work by using about three-fourths
the aperture of the objective. Conrad Beck contends that, with
the proper source of light, one can use the full aperture; he also
emphasizes the statement that to avoid the glare from the surround-
ing parts of the specimen while one should use the whole aperture of
an objective, the iris of the condenser should be opened only enough
just to fill the aperture.

The whole matter may be understood if one keeps in mind the
fundamental principle that for the clearest vision only the light from
the object should enter the microscope and form the image in the
eye. In the first method there is the defect that the whole field may
not be illuminated although the whole aperture of the objective may
be filled. The second method restricts the aperture of the objective
and hence in so far limits the resolving power of the microscope.
By a judicious selection of the size of the source of light, and the
correct intensity, and by regulating the aperture in each special case
one can get the best results. This ability comes, like other good
things, only by much practice even after the principles are under-
stood.

§ 141. Microscopic objectives for research. — For the modern
researcher there are three types of objectives to choose from: (a)

CH. II] THE BRIGHT-FIELD MICROSCOPE 105

Achromatic objectives with lenses made wholly of glass. — With these
the spherical aberration is corrected for one spectral color, the one
selected being in the brightest part of the visible spectrum in the
yellow green. For chromatic correlation, two colors only can be
brought to one focus. While this statement might lead one to think
that such objectives were very inferior optical instruments, in prac-
tice this is found not to be the case for the corrections being made in
the part of the visible spectrum brightest to the eye, the defects in
the outer, less brilliant parts (red and blue ends of the spectrum),
while they exist, are overpowered, in the words of the older opti-
cians, by the more brilliant part of the spectrum and hence do not
obtrude themselves unduly.

(b) Fluor tie or semi-apoclromatic objectives. — This second group
now coming largely into use for the more exacting work has added or
substituted for one of the glass lenses in the combination, a lens of
the natural mineral fluorite. This with its moderate refractive index
and very small dispersion, makes it possible to construct objectives
with more perfect corrections than the achromats. They are very
good for photography.

(c) A pochromatic objectives. — These were so named by Abbe.
Their characteristic is that spherical correction can be made with
two colors, and chromatic correction for three colors of the visible
spectrum. As it was next to impossible to make the full corrections
in the higher power objectives, a special series of oculars were made
to go with the apochromats and finish the corrections. These are
for that reason called compensation oculars.

Besides the use of fluorite in apochromats and fluorite ob-
jectives, all modern objectives and other optical apparatus make
use of. new forms of glass, which is now made in America as well as
in Europe. With these new forms of glass, and their range of re-
fractive and dispersive power, it is now possible to render all optical
instruments more perfect, even without the use of fluorite. For the
most perfect results, however, fluorite must be used.

It is strongly advised that every one who is to use the microscope
for research should become thoroughly familiar with experiments
such as were devised by Abbe, and made possible with his test-

106 THE BRIGHT-FIELD MICROSCOPE [CH. II

plate. Without these experiments it would be almost impossible to
believe that thickness of cover and length of the tube of the micro-
scope could make such a difference in the clearness of the image seen
with high objectives.

With this test-plate, too, one can get a most striking proof of the
higher corrections of the apochromatic and fluorite objectives over
even the best achromatic objectives. After such a clear demonstra-
tion as the test-plate affords, the researcher can never again feel that
it is a waste of time to make sure that the optical apparatus is ad-
justed to enable it to give its best effects.

§ 142. Condensers for research. — From what has been said in
this chapter it must be evident that to get the full benefit of the
best research objectives one must use with them a well corrected
condenser with sufficient aperture; and furthermore it must be borne
in mind that no condenser can pass on to the object an aperture
greater than i.oo N.A. unless the slide is connected with the con-
denser by means of a medium of sufficient refractive index (fig. 55).

§ 143. The Abbe Test-plate and the method of its use. — This
test-plate is intended for the examination of objectives with reference
to their corrections for spherical and chromatic aberration and for
estimating the thickness of the cover-glasses for which the spherical
aberration is best corrected.

" The test-plate consists of a series of cover-glasses ranging in thick-
ness from 0.09 mm. to 0.24 mm., silvered on the under surface and
cemented side by side on a slide. The thickness of each is written
on the silver film. Groups of parallel lines are cut through the film
and these are so coarsely ruled that they are easily resolved by the
lowest powers, yet from the extreme thinness of the silver they form
a very delicate test for objectives of even the highest power and
widest aperture. Full directions accompany the test-plate.

§ 144. Oculars to use. — The Huygenian oculars with magnifica-
tion of 2x to IQX answer very well for the achromatic and fluorite
objectives of all powers, but for the apochromatic objectives oculars
should be so constructed that they compensate for defects in the
objectives. These so-called compensating oculars are also good for
the high power modern achromatic and fluorite objectives, when high

CH. II] THE BRIGHT-FIELD MICROSCOPE 107

power oculars are to be employed. This is especially true, for dark-
field microscopy, as is shown in Chapter III.

Besides the compensating oculars strictly so called, a great and
successful effort has been made in the last few years to give the
ocular a greater optical perfection by the use of combinations of
lenses instead of the simple lenses of the Huygenian form. These
go by various trade names as indicated in §43a.
~*$ 145. Oculars and spectacles. — As the eye of the observer is
the last link in the optical chain forming a microscope, the perfec-
tion of the magnified image depends in part at least upon the per-
fection of the eye. As the seeing brain must gets its data from the
retinal image, no argument is needed to show that this retinal image
is of supreme importance. Indeed, the whole purpose of the entire
microscope is to aid in making a perfect retinal image.

At present nearly every researcher must wear spectacles to correct
dioptric eye defects such as astigmatism, etc. Fortunately the area
of the eyepoint of the microscope is so small that only a small part
of the cornea of the eye is involved, and the iris of the eye serves to
cut out border rays that would make confusion. Nevertheless a
defective optical part of the eye cannot give a perfect retinal image,
and if the spectacles serve to make the retinal image more perfect it
follows that the spectacles should be worn in exacting microscopic
work. The difficulty in keeping the spectacles on in microscopic
observation is that the eyepoint of most oculars is so close to the
eyelens that one cannot get the eye close enough to the ocular if
spectacles are worn. This is especially true if the curved toric
glasses are used.

To make it possible to wear spectacles for microscopic observation,
Swift of London has produced an ocular — " telaugic " (airy 17, eye;
rrjAe, far) — of two combinations having a high eyepoint. With
such an ocular the spectacles can be worn without interfering in any
way, and with the advantage of vhe correction to eye defects which
the glasses give. Twenty-six (26) oculars of all types and of five
different makers were examined for the distance of the eyepoint
from the eyelens of the ocular, that is, the height of the eyepoint.
For measuring the height of the eyepoint a Beck swing-out lens

To8 THE BRIGHT-FIELD MICROSCOPE [Cn. II

holder (fig. 57) was used. In place of the magnifier, a piece of
ground glass less than i mm. thick was put on the holder. The
microscope was focused on a very transparent object and brilliantly
lighted with a daylight lamp (fig, 199).

With the ground glass over the ocular it was very easy to find and
to focus the eyepoint. The distance from the upper surface of the
eyelens to the ground glass was then measured with a millimeter
scale. To make sure that the eyepoint was sharply focused and
that the measurement of the height was accurate, a low-power
magnifier was used. It was found that the eyepoint was practically
constant in height with objectives from 20 (8x) to 2 (QOX) mm.
equivalent focus, consequently the low power was used throughout.

The following is a summary of the findings:

5 different 5x oculars Height of eyepoint 10-14 mm.

6 different oculars, 6x, 6.45X, yx and 7,$x 7-10 mm.

9 different oculars, TOX 6-20 mm.

4 different oculars, 1 5x 4 5-18 mm.

i Compensation ocular of 2ox 12 5 mm.

i Periplane ocular of 25X 2 oo mm.

i Telaugic ocular of lox 20 oo mm.

From this summary it will be seen that there is at present great
variety in height of eyepoint even for the same magnification, there-
fore it seems quite possible to construct a series of oculars for users
of spectacles. See " telaugic " oculars, § 41.

§ 146. Centering of the ocular. — From the size of the lenses of
oculars, an exact correspondence of the optic axis of the ocular with
that of the objective and the condenser is not so essential as with
the condenser and objective. The tubes of microscopes are so
mechanically perfect that when the ocular is in place its axis will be
sufficiently near the center.

§ 147. Changing objectives and centering. — With student micro-
scopes and those for most purposes are now so well made that
one need not worry overmuch about the centering in passing from
one objective to another by means of a revolving nose-piece. If one
wishes to test a microscope for centering of the condenser with the
different objectives the directions given in § 118 will serve as a guide.
Of course it is very easy for any one to see whether an object in the

CH. II] THE BRIGHT-FIELD MICROSCOPE 109

center of the field with one objective is also in the center of the field
when the other objectives are swung into position.

§ 148. Exact centering impracticable. — A perfect microscope
would have the condenser centered to each objective, and at the
same time an object in the center of the field with one objective
would be in the center for all the others on the revolving nose-piece
or with any other form of objective changer. While the approxima-
tion is good, no microscope has yet been tested which shows the
perfect centering of both condenser and field; and microscopes of the
principal makers have been critically examined for this information.

On the whole the revolving nose-pieces have been found as ac-
curate as the more expensive objective changers; and the revolving
nose-piece is very much more convenient.

In exacting research and with the dark-field microscope the ques-
tion of centering is a very vital one. For research, the condenser
should not only be of the best quality (§ 128), but it should be sup-
plied with a centering device (fig. 60) by which its axis may be put
in line with the axis of the objective bein^ usei. From rigid tests
with many microscopes of many different makes the writer is forced
to the conclusion that if successive objectives are to be centered,
none of the objective changers or revolving nose-pieces are suffi-
ciently accurate. The different objectives must be screwed directly
into the lower end of the body tube of the microscope. Even then
there will be slight differences, for so exacting are the requirements
that no two objectives have yet been found by me that put in place
successively will be exactly centered. The difference is usually very
slight, much less than with a revolving nose-piece, but still the
centering is not perfect. For the most perfect correlation the sub-
stage condenser should be centered for each objective.

ADJUSTABLE, WATER AND HOMOGENEOUS IMMERSION OBJECTIVES

Experiments

§ 149. Adjustment for objectives. — As stated above (§ 29) the
aberration produced by the cover-glass (fig. 64) is compensated for
by giving the combinations in the objective a different relative posi-

THE BRIGHT-FIELD MICROSCOPE

[CH. II

Balsam

tion than they would have if the objective were to be used on uncov-
ered objects. Although this relative position cannot be changed in

unad jus table objectives, one
can secure the best results
of which the objective is
capable by selecting covers
of the thickness for which
the objective was corrected.
Adjustment may be made
also by increasing the tube-
length for covers thinner
than the standard and by
shortening the tube-length
for covers thicker than the
standard.

In learning to adjust ob-
jectives, it is best for the
student to choose some ob-
ject, like Pleurosigma whose
structure is well agreed
upon, and then to practise
lighting it, shading the

stage and adjusting the objective, until the proper appearance is
obtained. The adjustment is made by turning a ring or collar which
acts on a screw and increases or diminishes the distance between the
systems of lenses, usually the front and the back systems (fig. 44).
§ 150. Directions for adjustment. — (i) The thicker the cover-
glass, the closer together are the systems brought by turning the
adjusting collar from the zero mark and conversely; (2) the thinner
the cover-glass, the further must the systems be separated, i.e., the
adjusting collar is turned nearer the zero or the mark " uncovered."
This also increases the magnification of the objective (§ 368).

The following specific directions for making the cover-glass adjust-
ment are given by Mr. Wenham (Carpenter, yth Ed., p. 166):
" Select any dark speck or opaque portion of the object, and bring
the outline into perfect focus; then lay the finger on the milled-head

FIG. 64. ABERRATION PRODUCED BY THE

CoVER-dLASS.

Axis The extension of the principal optic
axis.

Cover The cover-glass.

z, 2, 3 Three rays originating from the
object mounted in balsam.

r, r, r Points of refraction as the three
rays emerge from the upper surface of the
cover into the air.

0 Object from which the rays originate.

j, 2, 3 The three levels from which the rays
seem to originate when traced backward from
their points of emergence. This gives the
effect of spherical aberration (Ch. IV).

CH. II] THE BRIGHT-FIELD MICROSCOPE in

of the fine motion and move it briskly backwards and forwards in
both directions from the first position. Observe the expansion of the
dark outline of the object, both when within and when without the
focus. If the greater expansion or coma is when the object is with-
out the focus, or farthest from the objective [i.e., in focusing up],
the lenses must be placed further asunder, or toward the mark un-
covered [the adjusting collar is turned toward the zero mark, as the
cover-glass is too thin for the present adjustment]. If the greater
expansion is when the object is within the focus, or nearest the
objective [i.e., in focusing do\*n], the lenses must be brought closer
together, or toward the mark covered [i.e., the adjusting collar
should be turned away from the zero mark, the cover-glass being
too thick for the present adjustment]." In mcst objectives the
collar is graduated arbitrarily, the zero (o) mark representing the
position for uncovered objects. Other objectives have the collar
graduated to correspond to the various thickness of cover-glasses for
which the objective may be adjusted. This seems to be an admirable
plan; then if cne knows the thickness of the cover-glass on the
preparation (§ 518) the adjusting collar rray be set at a corresponding
mark, and one \ull feel confident that the adjustment will be
approximately correct. It is then only necessary for the observer to
make the slight adjustment to compensate for the mounting medium or
any variation from the standard length of the tube of the microscope.
In adjusting for variations of the length of the tube from the stand-
ard it should be remembered that: (i) If the tube of the microscope
is longer than the standard for which the objective was cor-
rected, the effect is approximately the same as thickening the. cover-
glass, and therefore the systems of the objective rrust be brought
closer together, i.e., the adjusting collar must be turned away from
the zero mark. (2) If the tube is shorter than the standard for
which the objective is corrected, the effect is approximately the
same as diminishing the thickness of the cover-glass, and the systems
must therefore be separated (fig. 44), i.e., turned toward the zero
mark.

In using the tube-length for cover correction shorten the tube for
too thick covers, and lengthen the tube for too thin covers.

H2 THE BRIGHT-FIELD MICROSCOPE [CH. II

Furthermore, whatever the interpretation by different opticians
of what should be included in tube-length, and the exact length in
millimeters, its importance is very great, for each objective gives the
most perfect image of which it is capable with the tube-length for
which it is corrected, and the more perfect the objective the greater
the ill effects on the image of varying the tube-length from the
standard. The plan of designating exactly what is meant by tube-
length and engraving on each objective the tube-length for which it
is corrected, is to be commended, for it is manifestly difficult for
each worker with the microscope to find out for himself for what
tube-length each of his objectives was corrected (see Ch. IV).

§ 151. Water immersion objectives. — Put a water immersion
objective in position (§ 84) and the fly's wing for object under the
microscope. Place a drop of distilled water on the cover-glass, and
with the coarse adjustment lower the tube till the objective dips into
the water, then light the field well and turn the fine adjustment one
way and another till the image is clear. Water immersions are ex-
ceedingly convenient in studying. the circulation of the blood, and for
many other purposes where aqueous liquids are likely to get on the
cover-glass. If the objective is adjustable, follow the directions
given in § 150.

When one is through using a water immersion objective, remove
it from the microscope and with some lens paper wipe all the water
from the front lens. Unless this is done dust collects and sooner or
later the front lens will be clouded. It is better to use distilled water
to avoid the gritty substances that are likely to be present in natural
water, as these gritty particles might scratch the front lens.

> REFRACTION AND COLOR IMAGES

§ 162. Refraction images are those mostly seen in studying micro-
scopic objects. — They are the appearances produced by the re-
fraction of the light on entering and on leaving an object. They
therefore depend (a) upon the form of the object, (6) upon the rela-
tive refractive powers of object and mounting medium. With such
images the diaphragm should not be too large (§§121-122).

CH. II] THE BRIGHT-FIELD MICROSCOPE 113

If the color and refractive index of the object were exactly like the
mounting medium, it could not be seen. In most cases both refrac-
tive index and color differ somewhat; there is then a combination of
color and refraction images which is a great advantage. This com-
bination is generally taken advantage of in histology. The air
bubble in § 333 is an example of a purely refractive image.

A purely refractive image like that given by an air bubble or a fat
globule gives a dark border for central transmitted light, and a light
border on a black field with very oblique light, such as is given by
the mirror turned far to one side or by a central stop when the con-
denser is used (§§ 138, 340). In both cases the object is in outline.
As pointed out by Wright (p. 5, 41) the visibility of the object
shown in outline depends on the width of the outline and not on the
diameter of the whole object. If the width of the outline is too
narrow to include the necessary visual angle of i minute (§ 360) the
whole object fades into the background and is no longer visible. On
the other hand, if the object is colored, then it is visible so long as
its entire diameter gives a visual angle of i minute or more.

One can see from the above what a tremendous advantage it is in
studying the finest details of structure to have them brilliantly
colored.

HOMOGENEOUS IMMERSION OBJECTIVES

Ex^erimenis

As stated above (§ 23), these are objectives (fig. 44!) in which a
liquid of the same refractive index as the front lens of the objective
is placed between the front lens and the cover-glass.

§ 163. Refraction images. — Put a homogeneous immersion ob-
jective in position; employ a condenser. Use some histological
specimen like a muscular fiber as object; make the diaphragm open-
ing about 9 mm. in diameter, add a drop of the homogeneous immer-
sion liquid, and focus as directed in § 89. The object will be clearly
seen in all its details by the unequal refraction of the light traversing
it. The difference in color between it and the surrounding medium
will also increase the sharpness of the outline. If an air bubble

114 THE BRIGHT-FIELD MICROSCOPE [Cn. II

preparation (§ 334) were used, one would get pure refraction
images.

§ 154. Color images. — Use some stained bacteria as Bacillus
tuberculosis for object. Put a drop of the immersion liquid on the
cover-glass or on the front lens of the homogeneous objective. Re-
move the diaphragms from the illuminator or in case the iris dia-
phragm is used, open it to its greatest extent. Focus the objective
down so that the immersion fluid is in contact with both the front
lens and the cover-glass; then with the fine adjustment get the
bacteria in focus. They will stand out as clearly defined colored
objects on a bright field.

If one closes the diaphragm until one-half or three-quarters of
the aperture of the objective is used, the image will be a combined
color and refraction image.

§ 155. Shading the stage of the microscope and the eyes of the
observer. — As emphasized before, the clearest possible image of an
object can be obtained when the only light reaching the eye comes
from the object. With opaque objects and with the dark-field
microscope this is literally true. With the bright-field microscope
where the light is transmitted through and around the object it is
necessary to exclude any other light than that which is transmitted
by shading the stage of the microscope so that the image will not
be blurred by light upon the object from various angles above the
stage. This shading is easily accomplished by means of a screen
(fig. 42) if daylight is used, or by having a shield as shown with the
chalet microscope lamp (fig. 46). Both the screen and the shield in
the daylight lamp also keep the light from the eyes of the observer.

§ 156. Removing homogeneous immersion liquid from glass
surfaces. — Homogeneous immersion objectives, condensers and
other glass surfaces covered with cedar oil or other homogeneous
liquid are cleaned as follows: — The main part of the liquid is re-
moved by a clean piece of gauze, then a piece of gauze or lens paper
is wet with xylene or chloroform and the glass surface wiped. Im-
mediately afterward a fresh piece of the lens paper is used to wipe
away the last traces of the solvent. This leaves the glass surfaces
clean and ready for the next experiment.

CH. II] THE BRIGHT-FIELD MICROSCOPE 115

EXPERIMENTS WITH BINOCULAR MICROSCOPES

§ 157. Arranging the microscope for binocular vision. — Until one
has had some experience with binocular microscopes it is not easy
to tell whether one is seeing with one eye or with both. In order to
see with both eyes it is necessary that each eye should receive the
beam of light from its own ocular at*the same time, and this can
occur only when the oculars are spread the right amount to bring
the eyepoints the same distance apart as the pupils of the eyes of
the observer, and the eyes are at the correct level.

Hold the head close to the oculars and look into the microscope.
Focus as usual and the image will be satisfactory. Now to tell
whether the image is seen with one eye or with both, hold the head
still and shut the eyes alternately. If only one eye is being used no
image at all will be seen when that eye is closed, but when the other
is closed there will be no change in the appearance (§ 158).

If it is found that only one eye is being usei, change the spread of
the oculars by grasping the prism holder or drums or the tubes above
these with the two hands and increase and diminish the distance
between the tubes until both eyes are receiving the light, and there
is an image in each eye. When this occurs and one once gets the
stereoscopic effect there will never be any doubt in the future
whether the vision is monocular or binocular.

§ 168. — In some makes of binocular microscopes (the Spencer
Lens Co.'s, for example), there is a little shutter just above the
objectives which can be turned to either side, covering the back of
the corresponding objective. If the image is still apparent which-
ever objective is covered then, of course, both eyes are seeing the
image, but if the image is wholly obliterated when the shutter is on
one side, that is the only side giving an image, and the tubes must
be changed in position to get the correct pupillary distance of the
eyepoints.

EXPERIMENTS WITH DOUBLE-OBJECTIVE BINOCULAR MICROSCOPES

§ 159. Opaque and transparent objects. — Place the binocular

microscope (fig. 27) near a window where there is an abundance of

Il6 THE BRIGHT-FIELD MICROSCOPE [Cn. II

light or if artificial light must be used, employ the dark-field lamp
(figs. 79 or 80) or a bull's-eye condenser (fig. 127) to concentrate
the light upon the object. At first use low power objectives and
oculars. As it is somewhat easier to get the stereoscopic effect with
opaque objects, use a black background like a piece of black velvet.
Put a flower or some folded white gauze, a bunch of keys or other
familiar object under the mic?oscope and look at the object. Focus
sharply. Make sure that both eyes see the image as directed above

(§§ 157, 158).

After trying various opaque objects, and becoming familiar with
the necessary adjustments, use a large transparent object like a
preparation with the blood vessels injected. The different levels of
the blood vessels will stand out with amazing distinctness.

The double-objective binoculars are excellent for studying the
circulation of the blood and all injected preparations. For dissection
the microscope is mounted on an arm which may be swung into
position.

§ 160. There are three precautions to keep in mind: The oculars
must be the right distance apart for the observer's pupillary sepa-
ration; (2) the two oculars must be of the same power; and
(3) finally the observer must make sure that the image is in focus
for both eyes. In all the best modern binoculars of all kinds ad-
justments are provided for this purpose.

If the special focusing device for eye differences is at the left as in
fig. 27, then one closes the left eye and focuses the microscope for the
right eye as sharply as possible. The right eye is then closed and
the image examined with the left eye. If it is equally sharp with
the left eye, the. microscope is properly adjusted for both eyes, and
will give a good binocular image. If the image should not be sharp
for the left eye, then without changing the focus of the microscope,
one turns up and down with the focusing device on the left objec-
tive^ until the image is sharp to the left eye. Make sure that it is
also sharp for the right eye. If it should not be, one must repeat the
entire operation. In this way one can have a perfect image in each eye.

Correct movement of the specimen or instruments under an erecting microscope. —
For one who has become thoroughly trainee1 in using the ordinary inverting com-

CH. II] THE BRIGHT-FIELD MICROSCOPE 117

pound microscope it is very difficult to make the proper motions to move the
specimen, or to move the dissecting instruments correctly under an erecting com-
pound microscope. This illustrates the power of training. The beginner with
the inverting microscope finds it hard to move his hands in the opposite way from
what his eyes dictate, but when the correlation between the appearance and the
motion necessary has become fixed, it is equally difficult to move the hands in
the direction which the eyes indicate, although it is known that this is now cor-
rect. This difficulty is soon overcome by practice.

Under the simple microscope, however, in which there is no reversal or in-
version, the eyes and hand work together automatically as with the naked eye.

EXPERIMENTS WITH SINGLE OBJECTIVE BINOCULARS

§ 161. Experiments with low powers. — Arrange the binocular
microscope so that it stands squarely before you, otherwise it will
not be easy to hold the head so that the eyes are directly over the
eyepoints of the two oculars.

As it is simpler, use first an opaque object like some loosely woven
gauze or other white cloth, a light colored insect or other opaque
object with very definite features which are at different levels.
Light well by having the microscope before the window or by the
use of a bull's-eye lens or best of 'all by the use of the dark-field
microscope lamp. Use a low objective, one not higher than 16 mm.
(IQX) and low oculars x5 or x6, and make sure that the oculars are
of the same power. With the eyes in the correct position and the
object well lighted there should be no difficulty in getting the
stereoscopic effect. It is well also to close one eye and get the ap-
pearance, and then the other, or to use a monocular microscope and
compare the appearance of the object with monocular and with
binocular vision. For many who have had considerable experience,
the image looks just as stereoscopic with one eye as with both.

For transmitted light, use some translucent object like a section
in which the blood vessels have been injected. These are thick and
when well lighted by the mirror or the mirror and condenser show
the stereoscopic effect very strikingly. Try all powers, including the
homogeneous immersion. A fly's wing mounted in balsam is good
for all powers.

§ 162. Unlikeness of the two eyes. — If the two eyes are markedly
unlike, true binocular vision is impossible. If the difference is not

n8 THE BRIGHT-FIELD MICROSCOPE [Cft. II

great, correction can be made with spectacles, or with the special
focusing adjustment on one side.

With the mon-objective binoculars the correcting device for un-
likeness of focus of the two eyes is usually on the left tube below the
ocular. It may be on the right as in fig, 35. To make sure that the
two eyes have sharp images, proceed as described for the double-
objective binocular (§ 160), only in this case the correcting device is
on the tube of the microscope and not on the objective.

§ 163. Experiment with unlike oculars. — It occasionally happens
that oculars of different powers get into the two tubes of the bin-
ocular. It produces great confusion as one can see by an experi-
ment. Use some well known object with like oculars and get the
image as perfect as possible, then put a higher or lower ocular in one
of the tubes. Get both images sharp as directed in § 162. If both
eyes are then opened there will not be a good single image of the two
differently magnified images, although separately both are good.

§164. Change from binocular to monocular observation. — The
method first adopted was the removal of the binocular arrangement
and the substitution of a monocular tube. It takes only a moment
to make the change (fig. 32). The latest device is to slide the
binocular arrangement side wise. When this is done the prism of one
tube is swung aside and this gives one tube in the main axis of the
microscope to serve in place of the single tube formerly employed.

TESTING THE MICROSCOPE

§ 165. Testing the microscope. ~— To be of real value this must
be accomplished by a person with both theoretical and practical
knowledge, and also with an unprejudiced mind. Such persons are
not common, and when found do not show overanxiety to pass
judgment. From the writer's experience it seems safe to say that
the inexperienced can do no better than to state clearly what he
wishes to do with a microscope and then trust to the judgment of
one of the optical companies. The makers of microscopes and
objectives guard with jealous care the excellence of both the mechan-
ical and optical part of their work, and send out only instruments

CH. II] THE BRIGHT-FIELD MICROSCOPE 119

that have been carefully tested and found to conform to the stand-
ard. This would be done as a matter of business prudence on their
part, but it is believed by the writer that microscope makers are
artists first and take an artist's pride in their work; they therefore
have a stimulus to excellence greater than business prudence alone
could give.

What has just been said does not by any means imply that the
purchaser of a microscope should blindly accept anything which is
offered him. It simply means that if one has no knowledge of a
microscope one can hardly pass expert judgment upon it.

§ 166. Mechanical parts. — All of the parts should be firm, and
not too easily shaken. Bearings should work smoothly. The mirror
should remain in any position in which it is placed (fig. 26).

§ 167. Focusing adjustments. ~— -The coarse or rapid adjustment
should be by rack and pinion and work so smoothly that even the high-
est power can be easily focused with it by an experienced observer.

This coarse adjustment is liable to work too hard or too easily. If
it works too hard, the bearings of the pinion are too tight or the
gliding surfaces are sticky and not properly lubricated. If the
bearings are too tight, loosen the screws very slightly; if the bear-
ings are not lubricated or the surfaces are covered with sticky oil,
wet a cloth with a good lubricating oil and rub the gliding surfaces
well. This will clean them and lubricate them at the same time.

If the tube runs down too easily the bearings of the pinion are too
loose and the screws should be tightened a little.

§ 168. The fine adjustment is m'ore difficult to deal with. —
From the nature of its purpose, unless it is approximately perfect,
it would be better off the microscope entirely. It has been much
improved recently.

It should work smoothly and be so balanced that one cannot tell
by the feeling when using it whether the screw is going up or down.
Then there should be absolutely no motion except in the direction of
the optic axis; otherwise the image will appear to sway even with
central light. Compare the appearance when using the coarse and
when using the fine adjustment. There should be no swaying of the
image with either if the light is central (§§ 133-134)-

120 THE BRIGHT-FIELD MICROSCOPE [Cn. II

§ 169. Testing the optical parts. — As stated in the beginning,
this can be done satisfactorily only by an expert judge. It would be
of very great advantage to the student if he could have the help of
such Q. person. In no case is a microscope to be condemned by an
inexperienced person. If the beginner will bear in mind that his
failures are due mostly to his own lack of knowledge and lack
of skill, and will truly endeavor to learn and apply the principles
laid down in this and in the standard works referred to, he will learn
after a while to estimate at their true value all the parts of his
microscope.

If one can compare a new or unfamiliar microscope with one with
which there is entire familiarity, a very good estimate can be made.
The first principle is to use some microscope with which one is familiar
and to use microscopic preparations of which one knows the structure;
then a fair judgment can be made of the excellence of the per-
formance of the new instrument. If there seems to be any defect in
the image, make sure

(1) that the lighting is good;

(2) that the proper aperture of the objective is being used and
that the condenser is centered; (§§ 123, 118).

(3) that the stage is shaded;

(4) that the tube-length of the microscope is that for which the
objectives were corrected.

(5) that the preparation is clean and gives a good image with the
microscope with which one is familiar. If all the precautions have
been taken and still a good im£ge cannot be obtained one should get
some more expert friend or the makers to show wherein the trouble
lies.

COLLATERAL READING

The same as for Chapter I. Consult the catalogues of the Micro-
scope Manufacturers, and the small guides they send out with their
microscopes.

CHAPTER HI
THE DARK-FIELD MICROSCOPE AND ITS APPLICATION

§ 170. Comparison of bright-field and dark-field microscopy. —

In most work with the microscope the entire field of view is lighted
and the objects to be studied appear as colored pictures or as
shadows — in extreme cases, as silhouettes — on a white ground.
As the field is always light, this has come to be known as Bright-
Field Microscopy.

FIG. 65. FIG. 66.

Bright- and dark-field photo-micrographs of the same objects (starch grains).

In contrast with this is Dark-Field Microscopy in which the field
is dark, and the objects appear as if they themselves emitted the
light by which they are seen.

The study of objects in a bright-field probably comprises 95 per
cent of all microscopic work, and is almost universally applicable.
On the other hand dark-field microscopy has a more limited appli-
cability, and yet from the increased visibility given to many objects
it is coming to be appreciated more and more.

121

122

THE DARK-FIELD MICROSCOPE

[Cn. Ill

§ 171. Definition of dark-field microscopy. — In its comprehensive
sense, Dark-Field Microscopy is the study of objects by the light
which the objects themselves turn into the microscope.

THROMBQCYTES
ERYTHROCYTES / L LEUCOCYTES

BRIGHT
• FIELD

DARK *
• FIELD

MTCBRIN •>
^CHYLOMICRONS'

FIG. 67. FKESH BLOOD.
Half the field is with dark-ground and half with bright-ground illumination,

There are two principal cases: (A) The objects which are truly
self-luminous like phosphorescent animals and plants; burning or
incandescent objects, and fluorescent objects. (B) The objects

CH. Ill] THE DARK-FIELD MICROSCOPE 123

which emit no light themselves, but which deflect the light reaching
them from some outside source into the microscope.

These two groups are well represented in astronomy. If one looks
into the sky on a cloudless night, the fixed stars show by the light
which they themselves emit, but the moon and the planets appear
by the light from the sun which they reflect to the earth, the sun
itself being wholly invisible at the time. As there is relatively very
little light coming from the intervening space between the stars and
planets, all appear to be self-luminous objects in a dark field. This
reference to the sky at night will serve to bring out two other points
with great clearness: (i) The enhanced visibility. Everybody
knows that there are as irany stars in the sky in the daytime as at
night, but they are blotted out, so to speak, by the flood of direct
light from the sun in the daytime, ^hile at night when these direct
rays are absent and no light comes from the background the stars
and the planets show again by the relatively feeble light which they
send to the earth.

(2) The other point is that in dark-field microscopy the objects
must be scattered, not covering the whole field (fig. 66).

§ 172. Light in the workroom. — As brought out in the previous
section in referring to the stars, they appear brighter* in a dark
clear night, so with dark-field microscopy. If one works at night
or in a dark room the effects are more satisfactory for two reasons:
(i) The scattered light of daylight or lamp light does not enter the
eye and thus lessen the effect of the dark-field appearances in the
microscope.

(2) In a darkened room the eyes of the observer are adapted for
relatively dim light and therefore the details of structure in the
dark-field microscope are apparently emphasized, just as when one
goes into a relatively dark room from a brightly lighted one. At
first almost nothing can be seen, but when the eyes become adapted
to the dim light, much can be seen.

§ 173. Dark-field and ultra-microscopy. — In both of these the
objects seem to be self-luminous in a dark*field, and no light reaches
the eye directly from an outside source, but only as sent to the eye
from the objects under observation.

124 THE DARK-FIELD MICROSCOPE [Cn. Ill

The terms simply represent two steps, and merge into each other.

Dark-Field Microscopy deals mainly with relatively large objects,
o.2ju or more in diameter, that is, those which come within the re-
solving power of the microscope.

Ultra-Microscopy deals principally with objects so small that they
do not show as objects with details, but one infers their presence by
the points of light which they turn into the microscope. This can be
made clear by an easily tried naked-eye observation. Suppose one
is in a dark room, and a minute beam of brilliant light like sunlight
or arc light is directed into the room. Unless one is in the path of
this beam of light, it will remain invisible; but if there are vapor or
dust particles present, they will deflect some of the light toward the
eye and will appear as shining points. The character of the particles
cannot be made out, but the points of light they reflect indicate their
presence. As Tyndall used this method in determining whether a
room was free from dust in his experiments in spontaneous genera-
tion, the appearance of the shining dust particles is sometimes called
the " Tyndall effect."

The two forms are said to merge, because in studying objects like
saliva, etcv with the microscope designed especially for dark-field
work, some of the objects seen will show details, but some are so
small that they show simply as points of light usually in the form of
so-called diffraction discs. The larger objects in the saliva come in
the province of dark-field microscopy, and the smallest ones, of
ultra-microscopy, and in this case the instrument used might with
equal propriety be called a dark-field or r,n ultra-microscope.

§ 174. Visibility and resolution with the dark-field microscope. —
Visibility refers only to the possibility of seeing that some object is
present; resolution to the possibility of seeing details so that one can
judge not only that an object is present but also see some of the
structure and relations of the object. These two terms come over
from the ancient science of astronomy where the questions were
whether what seemed a single bright point in the sky was a single
star or a double or triple star or a star cluster; and whether the
surface of the planet Mars was uniform or had markings and whether
the planet Venus was always a round disc of light or had phases

CH. Ill] THE DARK-MELD MICROSCOPE 12$

like the moon. The telescope, which increased the visual angle under
which the different things could be seen, added a certain amount of
resolution, and details were made visible which are invisible to the
naked eye.

It is contended by some workers that the whole purpose of the
dark-field microscope is to make objects visible, just as the stars
and planets are visible on a clear, dark night. Other persons are
equally emphatic that the dark-field microscope not only makes
objects visible, but it also is a powerful aid in resolution, bringing
out details of structure not even visible with the bright-field micro-
scope. The matter is admirably stated by Beck, and has the writer's
emphatic endorsement: With the dark-field microscope when skill-
fully used, " There is no glare or flooding and the whole aperture of
the object-glass is evenly filled with light so as to give the maximum
resolution. There is no foundation for the statement that has been
made that this form of illumination does not give the full resolving
power of the object-glass in use. Anything that can be resolved by
transmitted illumination can be resolved by dark-ground .illumina-
tion, and in general with much greater brilliancy, because of the
increased contrast between different parts of the structure,"

§ 175. Naked-eye demonstration of dark-field effects. — Use
some black velvet and scatter upon it some minute pieces of white
paper, also a small piece of black velvet. Place in a well lighted
window or light well by a bull's-eye (fig. 68) or the dark-field lamp
(fig. 80).

The paper reflects the light to the eyes; the small piece of velvet
is very obscure. For comparison with bright-field appearances, use
a piece of white paper and put on it small pieces of white paper and
a piece of the black velvet. These experiments bring out clearly
the advantages of contrast as well as the light and dark back-
ground.

§ 176. Dark-field effects with light above the stage. — Use a
16 mm. (IQX) or lower objective, and a low ocular. Place some black
velvet on the stage of the microscope, and upon it a glass slip on
which are some grains of flour or starch or other white powder.
Place the microscope near a well lighted window or let the light from

126

THE DARK-FIELD MICROSCOPE

. lit

FIG. 68. LAMP WITH PLANO-CONVEX
LENS ABOVE THE STAGE.

a lamp fall upon the top of the slip (fig. 68). In the microscope the
particles will appear brilliantly white on a dark background. In all

work with the microscope,
whether with bright- or dark-
field illumination, one would do
well to remember this experi-
ment, for it illustrates how
light from above the stage may
enter the microscope. Such
scattered, irregular light often
spoils the sharpness of the
image with transmitted light.

This is why it is best to have no light strike the object from
above the stage when the lighting is from below the stage, whether
for bright- or for dark-field illumination.

§ 177. Dark-field effects with light from below the stage. — As
commonly understood, dark-
field illumination refers espe-
cially to light from below the
stage. The light may be
directed upon the object from
some source as in fig. 68, or it
may be reflected from a mirror.
For the higher powers it is nec-
essary also to use a condenser
to light the object with a suffi-
cient aperture.

Use the same microscope and

the same specimen as in § 176. Swing the mirror far to one side
and direct the light very obliquely upon the glass slip supporting the
white granules; or use a bull's-eye to direct the light very obliquely
upon the object. The purpose is to have the light all so oblique
that none of it can get directly into the objective. The only light
that should pass into the objective is that reflected from the white
granules. If light were to pass directly into the objective the back-
ground would be light.

FIG.

69. LAMP AND BULL'S-EYE
LENS BELOW THE STAGE.

CH. Ill] THE DARK-FIELD MICROSCOPE 127

DARK-FIELD ILLUMINATION BY THE* AID OF CONDENSERS

Just as with the bright-field microscope the mirror alone does not
give light of high enough aperture for the modern objectives used and
a condenser must be employed, so with the dark-field microscope a
condenser must be used to give rays of sufficient obliquity. With the
bright-field microscope the light illuminating the object is in the form
of a solid cone (figs. 61, 287-290), but with the dark-field microscope,
where only oblique rays must light the object, the central part of the
solid cone must be blocked out somewhere in its course and only the
oblique rays forming a hollow cone of light must be permitted to
illuminate the object. If the objects in the field are thus illuminated
by rays so oblique that none of them can enter the objective directly,
the objects will appear bright by the light which they deflect into the
microscope and the background or field will be dark ; that is, the ob-
jects will appear to be self-luminous in a black field, as with the moon
and the planets in the sky at night.

The light reaching the condenser from the mirror is mostly in the
form of a solid beam of parallel rays, and the stop to eliminate the
central part of the beam may be below all the elements of the con-
denser as in figures 70, 84, 279, or it may be between the elements of the
condenser as in figures 71, 77, 280-282. In all cases the central part is
blocked out and only the outer shell or hollow cone composed of the
most oblique rays is permitted to pass on and illuminate the object.
This is shown in the illustrations of all the dark-field condensers
(figs. 71, 74, 77, 84, 126, 279, 280-281) except figure 70.

§ 178. Spot lens and refracting condensers for dark-field effects. —
Besides the early devices for dark-field shown in figures 68-69, a con~
densing lens with a black central patch cemented to the middle was
much used. Since 1854 at least, the achromatic and non-achromatic
bright-field condensers have been found to give excellent black back-
grounds if central stops are used. (See Ch. XIV.)

In general, these refracting condensers are entirely satisfactory for
use with low objectives, that is, those of 8 (2ox) to 16 mm. (lox) equiv-
alent focus and still lower ones. (See also §§ 717-723.) They have the
further advantage of lighting a large field. By using care in lighting, by

128

THE DARK-FIELD MICROSCOPE

[CH. Ill

selection of slips of the correct thickness and by the use of brilliant
light, dark-field work can be done satisfactorily with objectives of
8 mm. (2ox) and 4 mm. (e.f.) Uox), provided the numerical aperture
does not exceed 0.60 to 0.66. Homogeneous immersion objectives of
2 (oox) to 3 mm. (e.f.) (6ox) and indeed dry objectives of any power
and aperture can be used provided there is inserted a reducing dia-
phragm to bring the aperture slightly below 0.65. For the higher
powers one must use a strong light and it is also of great advantage
to use homogeneous immersion liquid between the condenser and
glass slip bearing the object, for then the most oblique rays can pass
into the slip and illuminate the object up to the refractive index of
its mounting medium. For the 16 mm. (lox) and lower objectives
immersion contact of the condenser and slip is unnecessary unless
especial brilliancy is needed.

§ 179. Thickness of slips to use with refracting condensers. —
The object should be in the focus of the condenser (figs. 70, 77),
hence the slip used should be of the thickness to put the objects
upon it at the level of the focus. This is, of course, more important
with the higher powers than with the lower ones. The ordinary
Abbe condenser does not have a sharp focus
on account of its aberrations, consequently
one need not be so particular about selecting
the slips. With the achromatic condensers,
however, there is a sharp focus and, as a rule,
it is quite near the surface of the top of the
condenser, hence thin slips should be used.
Fortunately many microscope makers give the
equivalent focus of their condensers and also
the working distance, that is, the distance be-
tween the top of the condenser and its focus.
This working distance shows the thickness of
slip to use, and if the slip is of that thick-
ness, it will bring the focus of the condenser
at the upper surface of the slip where the ob-
ject is situated, and thus insure the most brilliant illumination.
If the makers do not state the equivalent focus and working dis-

FIG. 70. REFRACT-
ING CONDENSER WITH
CENTRAL STOP (C-S)
FOR DARK-FIELD IL-
LUMINATION.

CH. Ill] THE DARK-FIELD MICROSCOPE 129

tance, one can find it by holding the condenser with its lower end
toward the sun. The sun's rays are practically parallel and hence
will be brought to a focus above the upper end of the condenser. If
one measures the distance between the focal point and the top of
the condenser it will show the working distance. A piece of ground
glass is a good object to use to show where the focal point of the
condenser is situated. This working distance indicates the thick-
ness of the slide to use.

§ 180. Size of the central stop required. — Evidently the central
stop must be of a size to exclude all rays which could pass directly
into the microscope objective, and allow those to pass which were of
an aperture greater than that of the objective in use. The size can
be easily determined in any given case as follows: The iris, dia-
phragm is opened widely, and the light reflected up through the
condenser. The objective should be focused on a transparent speci-
men; the ocular is removed and one looks directly down the tube of
the microscope. The back lens of the objective will be brilliantly
lighted. Now slowly close the iris, and soon its edge will be seen
all around the bright area. Close the iris until sure that it is clearly
seen. Then slowly open it until its opening is just at the edge of the
back lens of the objective. The use of the eyepoint magnifier or the
pinhole cap is convenient in this connection (figs. 57-58). This
opening of the iris which just fills the aperture of the objective indi-
cates how large a central stop is necessary to exclude all this light.
Turn the microscope over on its side and, with fine pointed dividers,
measure the diameter of the iris opening. Make a central stop of
the size of the iris opening or slightly larger out of thick paper like a
visiting card. As shown in figure 70 there must be three or four
arms left to support the central stop. Blacken the paper with black
ink, and put it in the holder under the condenser. If now a suitable
object is put on a slip and the objective focused, there should be a
dark-field, and the objects present should shine as if by their own
light. If the field looks gray instead of black it is because the
central stop is too small or is not centered, or the white particles
used on the slip are far too numerous and do not leave enough blank
space.

130 THE DARK-FIELD MICROSCOPE [Cn. Ill

One can determine what is at fault thus: The ocular is removed.
If the central stop is too small the back lens of the objective will
show a ring of light around the outside. If the central stop is not
centered there will be a meniscus of light on one side. If the ob-
jects are too numerous the whole field will be bright. To verify
these statements one can use a specimen with flour or starch all over
the slide.

For the meniscus of light when the central stop is decentered,
purposely pull the ring holding the stop slightly to one side and the
meniscus will appear in the back lens. To show the ring of light due
to a too small size of the stop, the easiest way is to use a higher
objective, say one of 3 (6ox) or 4 mm. (40%), in place of the 16 mm.
(lox) objective. While it is necessary to eliminate all the light which
could enter the objective directly, the thicker the ring of light which
remains to illuminate the objects, the more brilliantly self-luminous
will they appear, therefore one uses only the stop necessary for a
given objective. If one makes central stops for the different objec-
tives as described above, it will be greatly emphasized that the
objectives differ in aperture; in general the higher the power, the
greater the aperture, and consequently the larger must be the central
stop, and the thinner the ring of light left to illuminate the object.
As one needs more light for high powers instead of less than for low
powers, the deficiency of light caused by the large central stop
must be made good by using a more brilliant source of light for the
high powers.

-> § 181. Dark-field element for refracting condensers. — Recently
there has been devised a dark-field element for the improved Abbe
:ondensers. The upper lens of the condenser is removed and in its
3lace is a lens with the lower end ground away, and blackened
directly or by means of a special diaphragm which can be placed
very close to the lens. By this arrangement only the very oblique
rays at the edge of the solid cone can enter the sides of the upper
element, and dark-field illumination results as the rays in the hollow
cone which illuminates the object are all too oblique to enter the
objective directly (fig. 126).

These dark-field elements do not give so perfect dark-field illumi

CH. Ill]

THE DARK-FIELD MICROSCOPE

13*

nation as do the regular paraboloid or reflecting condensers, but they
do have the advantage that they are relatively cheap, and serve to
light rather a large field. As with the regular dark-field condensers,

rti

FIG. 71.

REFRACTING CONDENSER WITH UPPER DARK-FIELD
ELEMENT (5) IN PLACE.

(For full explanation, see fig. 126.)

it is better to have the slide in immersion contact with the con-
denser.

§ 182. Light for dark-field work with refracting condensers. —
For objectives of 8 mm. (2ox), 16 mm. (lox), and lower powers,
ordinary daylight or lamplight answers fairly well, and the Chalet
microscope lamp answers for the 8 mm. (zox) objective. If the 4
mm. (4ox) of 0.66 N.A., or the immersion objective reduced to 0.80 or
0.85 N.A. is to be used, then the dark-field microscope lamp gives
more satisfactory results. For many objects it is advantageous to
have a piece of finely ground glass in the path of the light. For all

132

THE DARK-FIELD MICROSCOPE

[Cn. Ill

powers the light through the piece of daylight glass without any
condenser (fig. 82) is good for many purposes. As for the reflecting
condensers, it is well to keep in mind that too great intensity of light
tends to make the background gray from the stray light which gets
into the field.

§ 183. Objectives and oculars for dark-field work with refracting
condensers. — With the dark-field element in place one can use all
powers, including an oil immersion, but the lower powers are the more
satisfactory. For the 4 mm. and the oil immersion it is advantageous
to have their iris diaphragms closed somewhat more than when using
the cardioid or paraboloid condensers.

With the ordinary refracting condenser of 1.20 to 1.40 N.A., fairly
good dark-field effects can be produced by using the proper dark-field
stop (fig. 282) if the objectives of 8 mm. and higher powers have iris
diaphragms that can reduce the apertures sufficiently. They light a
large field and permit change from dark- to light-
field without disturbing the specimen (§ 718).

REFLECTING DARK-FIELD CONDENSERS
As was first pointed out by Wenham, 1850-
1856, refracting condensers are not so well
adapted for obtaining the best ring of light for
dark-field work as a reflecting condenser, on
account of the difficulty in getting rid of the
spherical and chromatic aberration in the re-
fracted bundles of such great aperture. He first
(1850) used a silvered paraboloid and later (1856)
one of solid glass as is now used. Within the last
10 or 15 years there have also been worked
out reflecting condensers on the cardioid princi-
ple (Fig. 77). The purpose of all forms is to
give a ring o{ light which shall be of great
aperture, and be as free as possible from chro-
matic and spherical aberration, and hence will
form a sharp focus of the hollow cone upon
the level where the objects are situated.

FIG. 72. HIGH-
POWER OBJECTIVE
WITH APERTURE RE-
DUCING DIAPHRAGM
FOR DARK-GROUND
ILLUMINATION.
(From Chamot.)
D Funnel-shaped
reducing diaphragm
screwed into the
lower end of the
"boot" (opposite #).

CH. Ill]

THE DARK -FIELD MICROSCOPE

133

In the reflecting as in the refracting condensers the central part of
the light beam from the source is blocked out by a central opaque
stop and only a ring of light enters the condenser (figs. 70, 75, 77,

84).

While the purpose of the reflecting condenser is to produce a hol-
low cone of light of great aperture for illuminating the object, it is
seen at once that the law of refraction will prevent the light from
passing from the condenser to the object unless the glass slip bearing
the object is in immersion contact with the top of the condenser.

Air

Glyo

Gl.M

' FIG. 73. HEMISPHERES OF GLASS TO SHOW THE RE-
QUIRED ANGLE OF THE CONE OF LIGHT IN THE GLASS TO FILL
THE OVERLYING HEMISPHERE WITH LIGHT.

The diagrams show that in each case the cone of light in
the glass must have an aperture equal to the refractive index
of the overlying medium: For air, N A i.oo; for water,
N A 1.33; for glycerol, N A 1.47; for homogeneous liquid,
N A 1.52. Any aperture of the light cone in the glass in
excess of the refractive index of the medium above is beyond
the critical angle and is therefore reflected back into the con-
denser.

134

THE DARK-FIELD MICROSCOPE

[Cn. Ill

If the objective is traversed by the light, then the aperture will be
limited by the refractive index of the object. In like manner any
medium between the objective and the object limits the aperture
depending on the refractive index of the medium.

Figure 73 shows the maxi-
mum aperture that can pass
from the condenser to the ob-
ject where there is (i) air, (2)
water, (3) glycerin or (4) ho-
mogeneous immersion between
the top of the condenser and
the glass slip. If there is
homogeneous contact then the
only limit up to 1.52 N.A. is
the mounting medium of the
object itself.

§ 184. Numerical aperture
of reflecting dark-field con-
densers. — This must be
greater than the objective with
which it is to be used, and the
central part of the cone of
light up to the full aperture of
the objective must be stopped
out, leaving a hollow cone of
light all of whose rays are at a

FIG. 74. BECK'S FOCUSING DARK-FIELD
CONDENSER.

SB Solid beam of light from the
mirror to the stop, which permits only the
border rays to pass on to form the hollow
cone of light. greater aperture than that of

/, 2 (cd in § 181) The lower movable \ . \
and the upper fixed elements of the con- the objective,
denser. The closer the elements the
thicker can be the slide, the farther sepa-
rated the thinner.

SI, c Slide and cover-glass in immer-
sion contact below with the condenser and
above with the objective.

By means of a screw the
upper and the lower elements
can be separated or approxi-
mated to change the position
of the focus of the hollow cone
and thus to make it possible to use slides of different thickness. The
closer the elements the thicker can be the slide, the farther apart the
elements the thinner must be the slide (Beck, p. 132),

OH. Ill] THE DARK-FIELD MICROSCOPE 135

In practice it has been found safer to reduce the aperture of the
objective to 0.80 or 0.85 N.A., then one can get a dark-field with any
good dark-field condenser. (The writer has not found the reducing
diaphragms furnished by the makers to exceed 0.90, and some were
even as low as 0.60 N.A.) See further under § 186.

§ 185. Homogeneous objectives for dark-field microscopy. —
From the uncertainty in the use of these reducing diaphragms, it was
urged in the former edition of this book that opticians prepare
homogeneous objectives especially for dark-field work. As a result
of tests of several of the standard reflecting dark-field condensers
of both the paraboloid and the cardioid form, it was found that an
objective aperture of 0.80 would give a dark-field, hence this aper-
ture is mentioned as safe for all the standard makes, although in some
cases an aperture as great as 0.90 could be safely used.

In 1921, at my earnest personal solicitation, the Bausch & Lomb
Optical Company, and the Spencer Lens Company did construct
homogeneous immersion objectives with an aperture of 0.80 to
0.85 N.A. for dark-field work. These have proved thoroughly satis-
factory for the dark-field work and also for most of the work in
histology and pathology where the substage condenser was used dry
and therefore could not supply an aperture greater than i.oo to the
object and to the oil immersion objective. (See discussion of the need
for a homogeneous immersion condenser, §§ 124-125.)

In England and on the Continent homogeneous immersion objec-
tives of about 3 mm. (6ox) focus have been produced for some time.
Some of these have an aperture below i.oo N.A. for dark-field work.
In the special micro-catalogue of Zeiss No. 306, received in 1922,
there was found an announcement of two apochromatic, homoge-
neous immersion objectives especially designed for dark-field work.
They are designated X (60) (f. 3 mm.) and W (120) (f. 1.5 mm.).
Each has an aperture of 0.85, and both are excellent for dark-field
use and also for bright-field work.

§ 186. Dark-field condensers for high apertures. — During the
last two years there have been developed in England by the veteran
optician, Edward Nelson, and by the Messrs. Beck, dark-field con-
densers which permit the use of homogeneous immersion objectives

I36

THE DARK-FIELD MICROSCOPE

. Ill

up to 1.25 to 1.40 N.A. The type of Nelson's condenser is known as
a Cassegrain reflecting condenser and is produced by Watson & Sons

(fig- 75).

The high apertured condenser by Beck is known as a " Special
Focusing Dark Ground Illuminator " and is said to give an illuminat-
ing aperture from 1.32 to 1.45 N.A. It is described in Beck's last
catalogue and in Conrad Beck's book, " The Microscope," Part II,
1924, pp. 128-129. In general it is like fig. 74 which gives an aper-
ture from i .00 to 1.40. The special focusing condenser used with
slips of 0.5 mm. thickness gives an aperture of 1.32 to 1.45, and
objectives of an aperture of 1.25 can be used successfully with it.

While the Nelson Cassegrain reilecting condenser permits the use
of high apertures, even up to 1.40 according to Dr. F. J. Brislee,
President of the Liverpool Microscopical Society, in Watson's Micro-
scopic Record, Sept. 1924, pp. 4-7, it must be remembered that such
high apertures require a mounting medium of high refractive index,
for the aperture of light finally entering the objective is limited by
the medium of lowest refractive index between the objective and the

object. It is also evident that

objects like blood-corpuscles
which are normally in a medium
of about n I) 1.33, cannot be
viewed by an aperture greater
than the mounting medium.
Furthermore, the very great
obliquity of the high-apertured
light is a severe test of the cor-
rections of the condenser, and is
likely to cut down the intensity
of the illumination.

§ 187. Combined dark-field
and bright-field condensers. —
Every one who uses the dark-
field microscope for serious
work often feels the need of bright-field observation upon the
identical object to make it possible to arrive at a just interpreta-

FIG. 75. NELSON'S CASSEGRAIN
DARK-FTELD CONDENSER FOR IMMER-
SION OBJECTIVES OF APERTURE ABOVE
1.00 N A.
,(From Watson's Microscope Bulletin.)

c-s Central stop, these are of differ-
ent size depending on the aperture of the
objective to be used.

s-s Silvered border of the upper,
reflecting element.

CH. Ill] THE DARK-FIELD MICROSCOPE 137

tion of a given appearance. To meet the insistent demand three
Continental opticians have designed and manufactured three
different types of combined condensers, and they all enable one to
see the same structure by a slight change in the accessories of the
condenser, but no change in the specimen under observation.

In the form of Leitz, by the change in position of a central stop
and an iris diaphragm either bright- or dark-field illumination can
be used, and when the iris is open and the central stop turned
aside, both kinds of illumination can be used at the same time. This
condenser is better for dark- than for bright-field illumination, and
like the following, not so good for either as condensers especially
designed for one purpose.

The form of Reichert is a supers tage condenser in which is present,
besides the upper element of the dark-field, a disk with central stops
of different sizes, a ground glass for mild, low power bright-field
lighting, and a very convex lens serving the purpose of an Abbe
condenser. These different elements are brought into the optic axis
by rotating the disk. This is a convenient and effective instrument,
and when once centered, is satisfactory (§ 189).

The form of Zeiss is a modification of his paraboloid condenser by
the addition of elements below the paraboloid proper and changed in
position by means of two levers. One of the levers turns the central
stop in position for dark-field work, and to one side for bright-field
work. The other, shorter lever actuates an element which adjusts
the condenser for slips of different thickness between i.oo and
2.00 mm. This makes it possible to examine successfully prepara-
tions which were not especially mounted for dark-field work. On
the whole this condenser is easiest to work with and gives satis-
factory results with a wide variety of thickness of slip, and different
magnifications.

All of these condensers require immersion contact of the slip and
the top of the condenser. For low powers water immersion is suffi-
cient. For high powers the homogeneous immersion contact is best,
and it gives more brilliant pictures for all powers because of the
additional aperture that it makes possible.

§ 188. Focusing dark-field condensers. — With dark-field con '

138 THE DARK-FIELD MICROSCOPE [CH. Ill

densers the thickness of the glass slip is determined by the fixed focal
distance and working distance of the condenser, and unless the slip
is of the corresponding thickness the object will be above or below
the apex of the cone of light and therefore not in the most favorable
position to bring out its details of structure (fig. 77).

Beck Limited of London and Zeiss of Jena have designed dark-
field condensers in which the focal-point and consequently the work-
ing distance can be made to vary, and hence make possible the
successful use of slips of different thickness. This is convenient
when examining objects not originally mounted for dark-field work.

With the Beck form, by means of a lever below, the elements of
the condenser (fig. 74C, d) can be separated or approximated for
slips 0.5 to 1.85 mm. As evident from the diagram, the nearer
d is to c the thicker the slip, and the farther d and c the thinner the
slip. Furthermore \uth this form, by using a very thin slip (0.5
mm.), it is possible to increase the aperture of the condenser and
hence to irake it pcssille to use higher apertured homogeneous
irrrrersion objectives and thereby gain the advantage in resolution
rrade possible by the added aperture. With the form figured, Beck
says that with the adjustment made for a half millimeter slip and
specimens mounted upon it, an aperture as high as 1.20 in the objec-
tive may be used. See also § 186. The Zeiss form is focused for
different thickness of slips (i.oo to 2.00 mm.) by drawing a short
lever to the right for thin and to the left for thick slips. According
to leaflet No. 365, this displaces an element within the condenser
without changing the position of the upper lens. Therefore, as with
the Beck form, the immersion contact with the under surface of the
slip bearing the specimen is undisturbed.

§ 189. Superstage dark-field condensers. — These are dark-field
condensers to put on the stage instead of in place of the substage
illuminator. They are made with the same exactness as the sub-
stage form, indeed, in some the optical part is exactly the same
as for the substage condenser. In its use the bright-field sub-
stage condenser is turned aside and the light reflected by the mirror
directly into the condenser on the stage. The only special difficulty
in its use is that in moving the preparation to find a desirable field

CH. Ill] THE DARK-FIELD MICROSCOPE 139

one is likely to get the condenser out of center, as it is held in place
only with the stage clips, or the mechanical stage.

Reichert has overcome this difficulty by attaching arms with
pegs to fit into the holes in the stage designed for the spring clips.
The sliding joint of the two arms connected with the condenser and
the pegs is clamped when the condenser is centered, then it is almost
as fixed as the substage condenser, and the preparation can be
moved freely without much danger of decentering the stage.

§ 190. Immersion contact of condenser and glass slip for high
apertures. — As shown in § 1 24 for the bright-field condenser, and
as indicated in the diagrams (fig. 73) showing the aperture of light
that is required to fill the hemisphere above the condenser with light,
if a high aperture is required, it can only be obtained by making
immersion contact between the upper face of the condenser and the
lower face of the glass slip bearing the object to be studied.

As will be seen by the different figures in this group the numerical
aperture that can illuminate the object is limited by the refractive
index of the medium in contact with the upper face of the con-
denser.

A general and complete statement is that the aperture of light
which can be concentrated upon an object is limited by the medium
of lowest refractive index between the condenser and the object,
hence the advice to use homogeneous immersion.

As in the reflecting condensers, practically no light can escape
from the condenser which has an aperture greater than i.oo, objects
mounted in air require immersion contact of the mounting slip and
the top of the condenser, although this would be unnecessary with
refracting condensers (§178). If in air or water, it would seem that
water immersion of the slip and condenser would suffice; if the
object were in glycerin, then glycerin immersion, and if in Canada
balsam or other medium of the refraction of glass, then only would
the homogeneous immersion seem to be necessary (fig. 73).

It is advocated, however, that in all cases the homogeneous im-
mersion be made with the condenser for two reasons: (i) If there
is homogeneous contact between condenser and slip, not so much
light will be lost by reflection from the lower face of the slip. H not

140

THE DARK-FIELD MICROSCOPE

[Cn. Ill

immersed, this loss is very great from the obliquity of the light from
the condenser to the slip. (2) It is advantageous to use the homo-
geneous immersion contact because some of the objects mounted in
air rest directly on the glass slip, as is also the case with any fluid
mounting medium. Being in contact with the glass slip the light
passes directly from the glass into it up to the aperture of the index
of refraction of the object; hence the object in optical contact with
the glass slip receives a greater aperture of light than the surround-
ing medium could, and is therefore more brilliantly illuminated, as
the illumination is as the square of the aperture. One can see the
importance of this consideration by a very simple experiment:
Clean the top of the condenser thoroughly, then illuminate as
strongly as possible. The top of the condenser will remain relatively
black. Scatter a few grains of Hour or powdered starch upon the
face of the condenser and those that are in optical contact with
the condenser will be very white from the light passing from the
condenser to them, and being reflected by them (fig. y6B).

§ 191. Slips and cover-glasses for dark-field work. — As most
dark-field condensers have a fixed focal distance, and the object
must be placed in the focus to be properly illuminated, it follows
that one must select glass slips of the thickness to bring the objects

mounted upon them at the level
of the focal point. Slips 0.05
less and 0.05 more than the
standard are permissible with
most dark-field condensers. The
makers mark upon the con-
denser mounting the thickness
of slip to use, hence one can
select those of the right thick-
ness by the use of micrometer
calipers (figs, 219, 220). Dealers
in microscopical supplies will
also furnish slips of the required
thickness. As these measured
are often used over and over it is better to have them of a

FIG. 76. TWICE ENLARGED UPPER
FACE or A FULLY-LIGHTED, PARABOLOID,
DARK-FIELD CONDENSER TO SHOW THE
SMALL CENTERING CIRCLE.

A Glass surface perfectly clean.

B Starch granules in optical contact
with the glass.

CH. Ill] THE DARK-FIELD MICROSCOPE 141

permanent glass. Those of a slight greenish tinge now made in
America are much to be preferred over the white, unstable glass so
common in the market.

Cover-glasses should be about 0.15 to 0.18 mm. in thickness.
That is, they should not be thicker than the working distance of the
highest objectives to be used.

§ 192. Cleaning slips and covers for dark-field work. — From
much personal experience the writer urges all who are to undertake
serious work with the dark-field microscope to use the method of
Stitt for cleaning the slips and covers. This method is given in full
in §§ 512, 515.

§ 193., Test preparations for the dark-field. — As the most perfect
effects are obtained with some difficulty, it is advised that prepara-
tions be made with slips and covers of known thickness by which the
performance of the dark-field condenser can be tested.

Suppose the thickness of slip to be used with a given condenser
is 1.30 mm. Select a slip of exactly 1.30 mm. and a cover-glass of
0.15 mm. in thickness. Clean well. In the middle of the slip write
the thickness with thin white ink. A delicate brush is good for this.
Some parts should appear white to the naked eye and others must be
very faint. After the ink is dry, add a drop of Canada balsam and
put on the cover-glass and press it down well. The particles of
which the white ink is composed serve well for deflecting the light.

When ready to undertake the study of some preparation, if this
standard is used to get the optimum lighting, one can feel confident
that the conditions are favorable for the object to be investigated.
This may seem like too much trouble, but no trouble is too great to
enable one to get the best possible results if the work is worth doing
at all.

§ 194. Determining the thickness of slip for the best results with
a dark-field condenser. — The writer has not always found the
thickness of glass slip recommended by the makers of the apparatus
the most perfect for that particular condenser. Indeed, sometimes
the results obtained by using the recommended thickness of slips
were very imperfect.

With dark-field condensers of the cardioid form which give a sharp

142 THE DARK-FIELD MICROSCOPE [Cii. Ill

focus, the proper thickness of slide to use for the best results can be
readily found by the use of slips of various thickness ground on
one face.

FIG. 77. BISPHERIC DARK-FIELD CONDENSER.

SB Solid beam of light from the mirror (M) to the central stop, which cuts
out all but the border rays.

I, 2 Lower and upper elements of the condenser. There is reflection from the
lower element only. Compare with the cardioid form (fig. 84), where there is
reflection from both elements.

HC Hollow cone of light.

F Slide of the thickness to bring the object in the focus of the hollow cone.

A Slide too thick, bringing the object above the focus. There will be a dark
central spot in the field.

B Slide too thin. This will locate the object below the focus in the hollow cone
and give a dark spot in the field.

To determine the thickness the smooth glass surface is connected
with the top of the condenser by homogeneous liquid. The micro-
scope is brilliantly lighted by an arc light or by one of the lamps
used for dark-field observation (fig. 80). Of course sunlight can also

CH. Ill] THE DARK-FIELD MICROSCOPE 143

be used. Employ a low objective and low ocular. Focus the spot of
light on the top of the glass slip. As it is much too thick, there will
appear a ring of light with a black center (fig, 77). If one uses
thinner slips, the bright spot on the top of the slip will grow smaller
and smaller and the central black spot disappear. The slip showing
the smallest bright spot is the one of correct thickness, for it brings
the focus of the hollow cone of light from the condenser at the sur-
face of the slip where the object to be examined is situated. If one
uses a slip much thinner than required, there will also be a ring of
light with a dark center (fig. 77). For measuring the thickness of the
slips a micrometer caliper is used. For grinding the surface, a piece
of plate glass or other smooth, flat glass is used for the grinding sur-
face. On this is placed some very fine carborundum or emery flour,
and a small amount of water added. The glass slip is then put down
on the grinding powder and rubbed around on the plate glass. After
a little experience one can grind one face of a glass slip in 20 to 30
seconds. After being ground the surface is well washed with water
and wiped dry. For use, the unground surface of the slip is con-
nected with the top of the condenser by homogeneous liquid. The
ground surface will then be up and it serves to show the ring or spot
of light from the condenser exactly as the ground glass in a photo-
graphic camera shows the image. A ground glass slip serves also to
aid in centering the condenser (§ 202).

In practice one must be sure that the glass slip is pressed down in
close contact with the top of the condenser. If there is a consider-
able stratum of the homogeneous liquid between the slip and the
condenser the thickness of the slip is thus virtually increased in
thickness and one would conclude that a thinner slip was needed,
which would be wrong. If one works in a warm room and uses an
immersion liquid which is not too thick, it is easier to make proper
contact between the slide and the top of the condenser. This is
taken advantage of when by necessity or mistake an object has been
mounted on too thin a slip. By using plenty of the immersion
liquid between slip and condenser the condenser can be lowered
sufficiently to bring the focus at the level of the object on the upper
surface of the thin slip.

144 THE DARK-FIELD MICROSCOPE [Cn. Ill

jLld

GHTING AND LAMPS FOR DARK-FlELD MICROSCOPY

§ 195. A glance at figures 70, 74 and 84 will give a clear notion of
how little of the light passing through the condenser is deflected by
the object into the microscope, consequently the source of light must
be of great brilliancy or there will not be enough to give sufficient
light to render the minute details of the objects visible, when high
powers are used. This visibility of minute details involves three
things: (i) The aperture of the objectives; (2) The aperture of the
illuminating pencil; (3) The intensity of the light.

The most powerful light is full sunlight. Following this is the
direct current arc, the alternating current arc and then the glowing
filament of the gas- filled or mazda lamps, and the " pointolite, or
tungsarc " lamps.

The reflecting condensers are designed for parallel beams, con-
sequently the direct sunlight can be reflected into the condenser
with the plane mirror of the microscope. If the arc lamp, a mazda
lamp, or any other artificial source is used, a parallelizing system
must be employed. The sirrplest and one of the most efficient is a
planoconvex lens of about 60 to 80 mm. focus with the plane side
next the light and the convex side toward the microscope mirror
(fig. 81), i.e., in position of least aberration. This is placed at about
its principal focal distance from the source whether that be arc
lamp, mazda lamp, or any other source and the issuing beam will
be of approximately parallel rays. These can then be reflected up
into the dark-field condenser with the plane mirror.

In dark-field work it is wise to recall that it is somewhat like
studying the planets and the moon, that is, one is observing objects
which are seen by light deflected by them, hence it is better to work
in a dark or dimly lighted room so that the comparatively mild
light from the object shall not be rendered obscure or misty by an
admixture of scattered light reaching the eye at the same time.

§ 196. Direct sunlight. — The early workers, Wenham, Shad-
boldt, Stephanson, Edmunds, Quekett, Carpenter, etc., advocated
the use of full sunlight, and that is the best light for the most diffi-
cult demonstrations. For continuous* observation one must use a

CH. Ill] THE DARK-FIELD MICROSCOPE 145

heliostat to keep the light in position. Unfortunately, also, clouds
are likely to obscure the sun, and night is sure to come before the
study is completed. Hence artificial sources of light are utilized.
For many purposes sunlight is too brilliant, but that difficulty is
easily overcome by the use of one or more pieces of ground glass
between the sun and the mirror.

§ 197. The arc lamp. — Next to the sun the arc lamp gives the
most brilliant light. Small forms have been devised using 4 to 6
amperes of current (fig. 78). The direct current arc is most satis-
factory for there is but one brilliant crater supplying the light. The
alternating arc has two equally brilliant craters, and the double light
is not easy to utilize.

FIG. 78. SMALL ARC LAMP FOR DARK-FIELD
ILLUMINATION AND FOR PHOTO-MICROGRAPHY.

A Support for the lamp; H C, V C, The
horizontal and vertical carbons. With direct
current the upper carbon is made positive and the
lower one negative.

Ch The hood and sleeve to cover the crater and
contain the parallelizing lens.

R The resistance or rheostat.

W 2, 3, 4 Wiring for the arc lamp.

So, K S p Socket with its key-switch and sepa-
rable plug below.

146

THE DARK-FIELD MICROSCOPE

[CH. Ill

Like sunlight, the arc is brighter than necessary for much of the
work with the dark-field microscope. It can be softened to the
desired brightness by pieces of ground glass between the lamp and
the microscope.

§ 198. 6- Volt headlight lamps. — Next to the arc light, and far
more satisfactory to use is a 6-volt headlight lamp. This has a very
small, closely coiled filament or a band filament giving a source not
much larger than the crater of the arc lamp and hence closely
approximates a point source of light. The brilliancy is also very
great as the filament is at about 2800° absolute. Thfe two sizes that
have been found most useful are the 7 2- watt and the io8-watt
bulbs.

FIG. 79. ADJUSTABLE 6-voLT, 1 08- WATT LAMP
FOR BRIGHT-FIELD AND FOR DARK-FIELD ILLU-
MINATION.

1 Coiled filament, 6-volt lamp.

2 Mogul base.

3 Connection for the iio-volt circuit.

4 Step-down transformer, i.e. no to 6 volts.
5-5 Mistakeless connection for the lamp.

6 Mogul socket in the lamp-house.

7 Lamp-house.

8 Tube with condenser.

9 Screen carrier to attach to the condenser
tube.

jo-ii Adjusting screws for tipping the lamp,
and raising and lowering it.

(Modified from the Catalogue of the Bausch &
Lomb Optical Co.)

CH. Ill]

THE DARK-FIELD MICROSCOPE

147

In use these lamps require some means of reducing the voltage
from 1 10 or 220 to 6 volts. A rheostat is sometimes used, but this is
exceedingly wasteful. If the current is alternating, a step-down
transformer accomplishes the reduction with practically no loss.
As the wattage is the voltage into the amperage, it follows that the

FIG. 80. ADJUSTABLE, 6-vot/r, IO&-WATT RESEARCH LAMP.

(Modified from the Bausch & Lomb Optical Co.'s Catalogue.)

1 Daylight-glass screen.

2 Frame for holding water cell.

j Handle for opening and closing the iris diaphragm.

4 Handle for focusing the condenser.

5 Lamp-house.

7 Ribbon-filament of the upper cylindrical lamp.

8-9 Coil-filament and cylindrical lamp.

w Water cell of a glass tube with heat absorbing faces
(Dr. H. P. Gage).

ii -12 Set screws for the inclination and elevation ad-
justments.

13 Adjusting screws for centering the lamp.

14-15 Mistakeless connection for the lamp cable.

16-17 Step-down transformer (no to 6 volts), and con-
nection for the no-volt circuit.

transformer in reducing the voltage from no to 6, must raise the
amperage a corresponding amount. Then the ys-watt lamp with a
6- volt current requires — or 12 amperes, and the io8-watt lamp ^
or 1 8 amperes of current.

148 THE DARK-FIELD MICROSCOPE [Cn. Ill

The heating of the lamp filament depends upon the amperage.
It is also to be remembered that the greater the amperage, the larger
must be the wire conducting the current. Hence the wire from the
transformer to and from the lamp must be of much larger size than
the wires to and from the transformer to the no- volt circuit of the
ordinary lighting system.

FIG. 81. DIAGRAM TO SHOW THE CONSTITUENT ELEMENTS OF THE 6-VoLT,
DARK-FIELD LAMP AND TRANSFORMER.

Supply wires from the no volt-circuit to the primary (P) side of the trans-
former with its many coils.

Transformer to step the voltage down from ITO to 6.
P Primary side of the transformer with many coils.

5 Secondary side of the transformer with few coils.

6 Volts The number of volts in the wires to the lamp but as the voltage is
stepped down the amperage is proportionally increased to hold the wattage con-
stant.

M C Mistakeless connection. A connection which prevents joining the lamp
wires with a no-volt circuit.

Lamp wires These must be heavy to carry the high amperage.
D G Polished daylight glass, and L parallelizing lens.

Ordinary cable used on desk lamps, etc., is plenty large enough to
carry the current to the transformer, but from the transformer to
and from the lamp the conductor should be much larger. Heater
cable has been found good, especially if a double cable is used as

CH. Ill] THE DARK-FIELD MICROSCOPE 149

shown in the diagram, (fig. 81.) To avoid mistakes in connecting the
lamp and transformer there should be a connection wholly different
from that connecting the transformer to the ordinary lighting circuit.
If the 6- volt lamp is connected directly with the i lo-volt lighting cir-
cuit, the lamp will burn out almost instantly.

The transformers for use with the dark-field microscope lamp
should be substantial and designed for continuous use. Furthermore,
in introducing them into the circuit between the lighting system and
the 6-volt lamp, one must be sure to connect the 6-volt side or wires
with the lamp and the no- volt side with the lighting circuit. If
the transformer is reversed the voltage would be stepped up a corre-
sponding amount and the fuses in the lighting circuit burned out.

FIG. 82. ORIGINAL 6-Voi/r LAMP FOR DARK-FIELD AND BRIGHT-FIELD

ILLUMINATION.

In this lamp the daylight window for bright-field illumination is on the side.

The transformers are clearly marked on the two ends either by
the voltage, or as they are often used for ringing door bells the end
for the circuit is marked " line," and that for the bell is marked
" bell." The " bell " wires are the ones to connect with the lamp.
If one looks at the wires, it will be found that those on the 6-volt
side are much heavier than those on the no- volt side, for they must
carry 12 to 1 8 amperes, while the wires on the lighting circuit side
(no- volt side) have to carry less than i ampere. The transformers

150 THE DARK-FIELD MICROSCOPE [CH. Ill

sent out with the 6-volt lamps are usually so wired and their con-
nectors so arranged that there is no chance of mistake.

§ 199. Chalet microscope lamp (fig. 83). — The Chalet Micro-
scope lamp, while not designed for dark-field work, answers fairly
well for the lower powers, i.e., up to objectives of 8 mm. (2ox) used
either with a refracting or a reflecting condenser. If it is to be used
for the highest powers, it is better to remove one of the daylight
glasses. In all uses of this lamp where there is no bull's-eye con-
denser the lamp should be brought up close to the microscope. If a
bull's-eye lens is used with the lamp the distance may well be 20 to

30 centimeters. If the lamp is
used without the daylight glass
in the window, that is, with the
lamp bulb direct, one often gets
better results with the concave
mirror, as that tends to make
the rays from the lamp more
nearly parallel. If the ground
daylight glass is used or if the
naked lamp is used with a

bull's-eye lens, the plane mirror
FIG. 83. ORIGINAL CHALET DAY- • _¥.r.,_ ~{r0/>f •, ~
LIGHT MICROSCOPE LAMP WITH Two 1S more enecuve.
WINDOWS. § 200. Dark-field condensers

with small attached lamps. — In

order to meet the needs of those who can have very limited space,
and therefore require minimum bulk of apparatus, substage condenser
lamps of small size are connected directly under the condenser.
These give fair results, but are not satisfactory as a laboratory
instrument, for the light is not brilliant enough to meet the varied
demands made in a laboratory, and for research on difficult subjects.
§ 201. Blackness of the dark-field and intensity of the light
source. — A perfect dark-field condenser would give a perfectly
black field with any source of light. Tests made on ten different
forms with the arc lamp and uranium glass have shown that in all
forms a certain amount of the light from the source does pass into
the microscope without being directed by the objects in the field of

CH. Ill]

THE DARK-FIELD MICROSCOPE

view. This tends to render the background grayish, instead of

leaving it perfectly black as it would be if absolutely no light entered

the microscope except that deflected to it by the object. The

amount of this adventitious light increases with the brilliancy of the

illumination, even when the

condenser is perfectly centered,

the correct thickness of slip used

and the lamp and microscope

mirror in the most favorable

position.

With all forms of dark-field
condensers the background may
be rendered darker by lessening
the intensity of the light either
by using a weaker light or by
putting in the path of the beam
from the lamp one or more
sheets of ground glass. These
ground glass sheets are conven-
iently held in wooden blocks,
then they can be placed any-
where in the beam of light.
The closer to the microscope

mirrnr HIP rhnrp brilliant the FlG' 84- CARDIOID REFLECTING DARK-

mirror, the more brilliant tne FlELD CoNDKNSBR>

light. If the unmodified ground (slightly modified from thc wr c^ta.

glass subdues the light too logue of the Bausch and Lomb Optical

much, the ground surface may Coj$ansyjid beam from the mirror (M).

be oiled, and most of the oil Only the border rays of great aperture

pass on to form the hollow cone (HC).

i, 2 Lower and upper elements of the
condenser. In the cardioid form both
elements reflect. Compare with the bi-
spheric forms (figs. 74, 77) where the re-
flection is only from the lower element.

rubbed off with a clean cloth.
With the paraboloid condens-
ers (fig. 84) the field can also be
made darker by closing the iris

more or less. This is because in

closing the iris the outside part of the ring of light entering the con-
denser is blocked. A glance at the figure will show that in closing
the iris the outside rays that are excluded are those which, after the

152 THE DARK-FIELD MICROSCOPE [Cn. II J

single internal reflection by the condenser, become the inside rays
at the least aperture. The rays of greatest aperture are left to
illuminate the object under the microscope. With the cardioid
forms of condenser with two internal reflections, closing the iris
would exclude the most oblique rays. This would darken the field,
but at the expense of the most favorable part of the light for dark-
field illumination.

FIG. 85. FACE AND SECTIONAL VIEWS OF THE Focus OF THE HOLLOW CONE OF
LIGHT FROM DARK-FIKLD CONDENSERS.

A Sectional view of an optically perfect dark-field condenser in which the sun
is represented as focused nearly to a point. No such condenser exists.

B Sectional view of a possible condenser focus. It is drawn out somewhat
and spreads laterally. The variation in the thickness of slide which might prop-
erly be used is shown by the two parallel lines enclosing the elongated focus.

C Sectional view with a still more elongated focus. The parallel lines show
that the variation in thickness of slide permissible is correspondingly increased.

The apparent size of the sun's image is shown on the axis above in each case.
It is least sharp in C, and represents fairly the paraboloid condenser where the
different zones have different foci, and hence permit of considerable latitude in
thickness of glass slip, and give a large, lighted field.

c d The white line above the letters A B C is at the level of the top of the
condenser.

a b The vertical elongation of the focal point. It is very marked in C.

It is to be remembered that any device for darkening the field —
except of course the correct arrangement of the apparatus and lamp
— makes the object less brilliant at the same time that it darkens
the field; and it is the intensity of the light that determines the
smallness of the object which can be seer. This is easily demon-
strated by using a preparation like fresh blood with very fine ele-
ments (fibrin filaments, chylo-microns, etc.), (fig. 89). With a
brilliant light all the elements can be clearly made out. Insert one
or more sheets of ground glass in the path of the beam from the

CH, III] THE DARK-FIELD MICROSCOPE 153

lamp and the smallest elements seem to disappear. That is, -as
shown in fig. 67, minute details may be swamped by the excessive
surrounding light, and they may also be obliterated if the light from
them is so faint that not enough reaches the observer's eye to make
them visible.

EXPERIMENTS WITH DARK-FIELD CONDENSERS

§ 202. Centering a substage, dark-field condenser. — While
centering the condenser and objective is important with a bright-
field condenser, it is far more important if one is to realize anything
like the perfect images that are possible with a dark-field condenser.
To render centering certain and easy, most makers put a small ring
in the middle of the upper face of the dark-field condenser (fig. 76).
The ordinary bright-field condenser is removed from the substage,
and the dark-field inserted in its place. It must be possible to raise
the condenser so that it is at the level of the upper face of the stage,
otherwise it cannot be brought close to the glass slip. When it is in
position, the iris diaphragm of the substage is 'opened widely, and
also that of the condenser if one is present. For centering it is
advantageous to use a low ocular, and a low objective, say one of
50 mm. (3x), then the end of the condenser can be seen as a whole
as well as the little ring. The condenser is lighted as strongly as
possible and the upper face brought into focus by the coarse adjust-
ment. If well lighted and clean, the centering ring will appear as a
shining circle for it allows some of the light to escape from the con-
denser and scatters it in all directions. Now with the special center-
ing screws of the condenser or with the substage centering screws,
change the position of the condenser until it is exactly in the middle
of the field of the microscope. It should then be in a favorable
position for all powers, although as the different objectives may not
have their centers in exactly the same line, some further slight ad-
justments may be necessary.

§ 203. Special methods of finding the centering circle of the
condenser. — Wipe the end of the condenser with clean gauze, using
also xylene if necessary to remove any cedar or other immersion

154 THE DARK-FIELD MICROSCOPE [CH. Ill

oil. Raise and tilt the lamp used for dark-field work till the beam
crosses the condenser face at right angles. The little centering ring
usually appears with much brilliancy. Often when the ring does not
show with the light coming from below it will appear if the end of
the condenser is made thoroughly clean.

Another excellent method of centering the condenser whether
there is a small centering circle or not is to employ a slip of the
proper thickness with one ground face (§ 179). The unground face
is put in immersion contact with the condenser and the light di-
rected up through the condenser by the mirror. One can see with
the naked eye when there is a bright spot on the ground surface of
the slip, Look into the microscope and tilt the mirror in various
directions until the smallest, brightest spot is in the middle of the
condenser face. The condenser and this spot of light are then put
exactly in the middle of the field of the microscope by means of the
centering screws. By this method both condenser and light are
centered at the same time. See the following section.

§ 204. Centering the light for the dark-field microscope. — If
an arc lamp, the 6-volt headlight lamp or tungsarc is used it should
be about 25 centimeters from the microscope, and the lamp-con-
denser should be in a position to give practically parallel light. This
can be brought about by focusing the arc crater or the lamp filament
on a white wall 5 to 10 meters from the lamp. One can work at
night for this adjustment if the room cannot be darkened. When
the position is once determined, a mark should be made on the lamp
so that it can be adjusted at any time. If the chalet lamp or one
having no condenser is used, the lamp should be as close as possible
to the microscope.

Arrange lamp and microscope so that the light strikes the middle
of the mirror. Use a ground slip of the proper thickness for the
particular condenser. Connect the unground face to the condenser
with homogeneous liquid, turn the mirror until the light passes up
through the condenser and forms a spot of light on the ground face
of the slip. Look into the microscope and focus the spot of light.
Turn the mirror until the smallest and brightest round spot is ob-
tained. This should be in the exact middle of the upper face of the

CH. Ill] THE DARK-FIELD MICROSCOPE 155

condenser. One can also see whether the condenser is accurately
centered to the objective (§ 202).

If a suitable preparation like fresh blood (§§ 211-212) is substi-
tuted for the ground glass and a homogeneous objective used in place
of the very low power, the elements of the blood should appear with
great brilliancy. Sometimes a slight change in the mirror will in-
crease the brilliancy.

§ 205. Focusing the dark-field microscope with immersion ob-
jectives. — The object on a slip of proper thickness is put in immer-
sion contact with the condenser and a drop of immersion liquid is
put on the cover-glass in the middle of the preparation. The mirror
is arranged so that the light shines in the drop of immersion liquid
on the cover. The objective is then focused down until it touches the
immersion liquid. When this happens, a flash of light will be seen if
one works in a dimly lighted room and looks toward the lower end
of the objective. When one is sure that the objective is in the im-
mersion liquid, look into the microscope. There will be a diffuse
bright area or field. This will be the appearance whether the ob-
jective is above or below the focus of the object. To make sure that
it is not below, focus up slightly. If nothing appears focus down
slowly. As the objective approaches the focus of the object the
field will be very bright all over. As one continues to focus down
the field will become gray and in many cases appear like a bank of
clouds; focusing still farther down the field will become darker and
darker and finally the field will be dark with the bright objects
appearing as if shining by their own light in blank space.

§ 206. Indicator to aid in focusing. — The above assumes that
there are enough particles so that there will be some in every field.
In case a liquid is being examined where there are few particles,
and therefore the possibility of blank fields, it is a good plan to
make a faint x with a red glass pencil on the middle of the slip
before the preparation is added. One can then focus on the red
cross and feel sure that the objective is at the right focus to give an
image of any particles which may be in the preparation.

§ 207. Objects suitable for the dark-field microscope. — Fresh
preparations: Any of the body fluids, — saliva, milk, blood, chyle,

156 THE DARK-FIELD MICROSCOPE [Cn. Ill

lymph, pleural, pericardial, peritoneal, and all other normal body
fluids, secretions and excretions; all pathological fluids and the
body fluids in pathological conditions of the body; isolated elements
of the tissues of the body.

In biology, use all the body fluids of plants or animals and their
isolated tissue elements and for minute organisms — microbes — the
entire organism (bacteria, protozoa, etc.). For the most satisfactory
results the elements must be scattered so that there will be blank
space between them. If they are so numerous or so close together
that the whole field is filled with light from them, the benefit of
contrast is lost. Ordinary thick microscopical sections are therefore
not suitable. Very thin sections, stained and unstained, may be used
for the difference of refractive index is sufficient to give differentia-
tion, and if the differential stains used are fluorescent, this will help
in the differentiation.

Dr. Chamot has pointed out that the dark-field is of great help in
the study of foods, fibers, crystallization phenomena, sub-microscopic
particles and colloids. He adds further (pp. 35-37): " This method
is invaluable for demonstrating the presence of very minute bodies or
those whose index of refraction is so nearly the same as that of the
mounting medium in which they occur as to cause them to escape
detection when illuminated by transmitted light," that is, the or-
dinary light used for bright-field microscopy.

§ 208. Mounting fluid preparations. — Take saliva for a trial
specimen. The tongue is rubbed around on the gums and cheeks and
the saliva brought to the lips. Some of this saliva is transferred by a
clean toothpick to the middle of a suitable glass slip. A cover-glass
is put over it and pressed down moderately. If any liquid is pressed
out, it is wiped away with a piece of gauze. Then with a fine brush
the cover-glass is sealed by painting a ring around the edge of the
cover. For the sealing one can use some thick oil like castor oil, or
thick automobile oil or shellac.

With watery preparations like saliva the cover-glass is likely to be
dragged around in moving the slip for studying different parts, be-
cause of the viscosity of the immersion liquid. The shellac cement
dries quickly and anchors the slip firmly.

CH. Ill] THE DARK-FIELD MICROSCOPE 157

Some workers prefer the immersion liquid made of heavy white
petroleum mixed with alpha-bromo-naphthalene because it is not so
sticky as cedar oil.

In case very few particles are present in the liquid to be studied,
remember to make the faint red cross on the slip to aid in focusing
(§ 206).

§ 209. Preparations to show spirochsetes. — (a) Those of the
mouth, Spirochala bucalis: Especially around the base of the
teeth next to the gums in most adult human beings, there are spiral
micro-organisms which show the cork-screw form and movements of
that group of organisms with great clearness. Those from the mouth
are large enough and numerous enough to make them easy of ob-
servation and thus to gain some adequate notion of the character-
istic appearance and movements of spiral organisms (Treponema,

etc.).

For making the preparation use a fresh toothpick and collect some
of the material around the base of the teeth and put it on the middle
of a suitable glass slip. Add a drop of saliva, and put on a cover-
glass and seal the cover (§ 208).

Focus as described in § 205, and in the field will be found salivary
corpuscles, and large epithelial scales from the lining epithelium of
the mouth, minute particles, bacteria, motile and stationary, and
the cork-screw-like buccal spirochaetes. It is of great help to get
thoroughly familiar with the appearance and movements of these
apparently harmless spirochaetes in preparation for the detection of
the spirochaetes of Vincent's angina and those of syphilis.

(b) Spirochceia pallida; Treponema -pallidum of syphilis. — This
spiral micro-organism discovered by Schaudinn in 1905 in the lesions
of syphilis is rather difficult of demonstration by any method, but
most easily shown by the dark-field microscope in fresh preparations.
The use of the dark-field microscope for such demonstrations has had
great influence in bringing the possibilities of the dark-field to the
attention of the medical profession, and has stimulated the micro-
scope makers to make convenient and efficient lamps and condensers
for general use. The biologist has thus put at his service a powerful
aid in pure research. Wenham, Edwards and many others years

158 THE DARK-FIELD MICROSCOPE [Cn. Ill

before had shown how clearly living microbes could be demonstrated
by its help, but it was the practical application that brought the
dark-field microscope into general use.

The method of making an examination for diagnostic purposes
here given is compiled from Stitt, Thro, and from personal observa-
tions in a clinic held in the office of Dr. M. A. Dumond.

Slips and covers are first carefully cleaned and placed in a con-
venient position. Then the suspected lesion is rubbed with a match
stick or a toothpick around which has been wrapped some cotton or
a strip of gauze. The rubbing is continued till the covering is re-
moved and the lymph commences to exude. Stitt recommends that
the lesion be first washed with alcohol and dried with cotton or
gauze. In a few minutes, (3-5), a clear lymph will exude. A drop
of normal saline solution is placed in the middle of a slip, and with a
pipette or a platinum loop some of the serum is transferred to the
salt solution and mixed with it. Then the cover-glass is added.
Or the cover-glass is touched to the exuded serum and then put over
on the slip with the salt solution. The edge of the cover-glass can
be sealed with oil or preferably with shellac so that it will be
anchored firmly and not be displaced when moving the slip around,
due to the viscosity of the immersion liquid between it and the

FIG. 86. SMALL TRAY WITH CLEANED SLIPS AND COVER-GLASSES.
The covers are set up in grooves in a cork so that they can be easily grasped

by the edges.

objective. The organisms are sometimes few, requiring much search-
ing, hence the microscope should have a mechanical stage to aid in a
systematic search. If present, and the specimen is examined soon,
the active movements of the spirochaetes will help in their detection.

CH. Ill] THE DARK-FIELD MICROSCOPE 159

After becoming familiar with the appearance of the organism they
are easily detected when motionless. In the preparation there will
be many minute particles undergoing the Brownian movement.
There may also be present blood corpuscles, white and red. The
spirochaetes are so characteristic in form and movement that there
should be no confusion.

§ 210. Infusoria and other micro-organisms in ditch-water in-
fusions. — A world of interesting forms can be easily obtained for
study with the dark-field microscope with all powers by getting some
water from a long established ditch or pond, and adding to it some
of the grass along the edge of the water. If this mixture is kept in a
warm room the organisms will multiply with amazing rapidity. If
some of the scum, scrapings from the plants or mud on the bottom
is placed on the center of a glass slip it can be studied with the 16
mm. (IQX) and lower objectives without any cover-glass. If the
Abbe condenser is used with a central stop (fig. 70) below the entire
condenser the field lighted will be sufficient for the 16 mm. (lox)
objective; if the higher powers are needed then a dark-field element
(§ 181) or a paraboloid dark-field condenser works well. The
lighted field will be of sufficient size for the lower powers. For both
forms it is best to have the slide in immersion contact with the
condenser, although with the dark-field element one can get fairly
good results without immersing the slide. For the highest powers
the reflecting, cardioid dark-field condensers are most satisfactory
because of the sharp focus of the hollow cone of light. These must
always be in immersion contact with the slide. For all objectives
above the 16 mm. (lox) the specimens must be covered with a cover-
glass; it is well to seal the cover glass so that it will not move, and
so the liquid will not evaporate. A study of such a preparation will
give a wonderful insight into the form and activity of these lowly
creatures, and reveal a beauty of design that will always be re-
membered.

§ 211. Fresh blood and the dark-field microscope. — Perfectly
fresh blood is one of the best objects to study by this method. As
pointed out by Dr. Edmunds nearly 50 years ago, blood with the
dark-field illumination appears like a new object so many things are

i6o

THE DARK-FIELD MICROSCOPE

[CH. Ill

seen with the greatest distinctness that are wholly invisible or merely
glimpsed when examined by the bright-field method.

(1) Carefully cleaned slips and
cover-glasses of the right thick-
ness are placed where they can be
easily grasped (fig. 86).

(2) For obtaining the blood the
part to be punctured is washed with
soap and water and then cleaned
well with a piece of gauze well
wet with 95% alcohol to which
has been added 2 grams of mer-
curic chlorid to the liter (two-
tenths of i %). A needle or Dr.
Moore's haemospast is cleaned by
the alcohol also and then the
puncture is made. The ball of

FIG. 87. LARGE SLIDE TRAY the middle finger of the left hand
WITH SUPPORTS FOR THK GLASS has been found a favorable place

SLIPS SO THAT THE IMMERSION , , ,, U1 , rp. • r

LIQUID ON THE UNDER SURFACE to get the blood- Tie a Piece of

SHALL NOT TOUCH THE TRAY. gauze around the base of the

A Face view of the slide tray. finger and then squeeze it and
B Sectional view. ° M

In all experiments the prepara- a drop of blood will exude. The

drop should be fairly large. Now
grasp the cover-glass by the edges
with the thumb and index of the
right hand and touch the cover

to the top of the drop. Enough blood will adhere to the cover.
Put it on the middle of the cleaned slip and the blood will
spread out. It should be%pressed down moderately with curved
forceps or by a finger covered with gauze, and the blood running out
at the edge of the cover should be wiped away or it will run back by
capillary attraction. Some parts of the preparation should appear
almost transparent to the eye. If it looks red all over, the blood
layer will be too thick and not leave enough blank spaces. Seal the
cover-glass with heavy oil. The fibrin network will adhere to both

points may be cleared up by re-
examining earlier preparations.

CH. Ill] THE DARK-FIELD MICROSCOPE 161

slip and cover and hold the cover in place. No diluting substance
is added to the blood.

FIG. 88. BLOCK WITH BOTTLES OF REAGENTS AND WITH THE MOORE
ELEMOPAST FOR FRESH BLOOD STUDIES.

§ 212. Appearance of blood under the dark-field microscope. —

(a) The erythrocytes will appear like dark discs with bright rims
owing to their convex borders.

(b) The leucocytes appear as real white corpuscles owing to the
granules within them which turn the light into the microscope. If
the room is moderately warm — 20° C or more — the leucocytes,
some of them, will undergo the amoeboid movement, and the picture
they present will be a revelation to those who never saw it or saw it
only with the bright-field microscope. From the clearness with
which everything can be seen the minutest change can be followed,
and also the most delicate pseudopod detected. Another striking
feature will be noticed in the moving ones, that is, the vigorous
Brownian movement of the granules jn the part of the leucocyte
with the amoeboid movement. In those showing no amoeboid move-
ment there is usually no sign of the Brownian movement of the
granules; also, if a part of the leucocyte is not undergoing amoeboid
movement, the particles in it are usually motionless.

(c) The fibrin network will be seen like a delicate cobweb between
the corpuscles. In different parts of the specimen one can find all

1 62 THE DARK-FIELD MICROSCOPE [Cn. Ill

the appearances of the fibrin shown in textbooks on the blood (fig.

FIG. 89. Two FIELDS OF FRESH BLOOD TO SHOW THE FEW CHYLOMICRONS
OR FAT PARTICLES IN FASTING, AND THEIR ABUNDANCE AFTER A FLTLL MEAL
WITH PLENTY OF FAT.

(From the American Journal of Anatomy, Sept. 1924.)
A 3 Microscopic field in fasting with 3 chylomicrons in the counting net.
B gi Microscope field 4 hours after a full meal.
The counting net encloses 91 chylomicrons.

The numerals along the curve show the number of chylomicrons during each
hour of the digestive cycle of 10 hours.

(d) Chylomicrons appear everywhere like bright points in the
empty spaces between the corpuscles. They are in every active
Brownian or pedetic movement. These chylomicrons will probably
be the most unusual part to those studying blood with the dark-field
for the first time. The term Chylomicron is from two Greek words,
xOX6s (chylos), juice or chyle, and p,ucpov (micron), any small thing.
In modern metrology it signifies the millionth of a meter (§ 380). 1

CH. Ill] THE DARK-FIELD MICROSCOPE 163

have introduced this word to show the origin of these bodies from
the chyle, and to indicate their average size. In 1840-1842, Gulliver
called these minute granules the " molecular base of the chyle " and
showed that they were identical in the thoracic-duct and in the blood
vessels of the same animal. He gave their average size as 1/36,000
to 1/24,000 of an inch (i/z to o.5/z). They have been called by others
free granules or granulations, elementary particles, haemokonia,
blood dust, etc. (fig. 89).

(e) A very striking view of the fibrin network may be obtained by
irrigating a thick unsealed blood preparation. If a drop of normal
salt solution is placed on one edge of the cover-glass and a piece of
blotting paper on the other the liquid is drawn through, washing out
many of the erythrocytes. If the washing-out process is watched
under the microscope, the erythrocytes will be seen gliding over or
through the fibrin network, or some of them will be anchored at one
end and if the current is rapid the corpuscles will be pulled out into
pear-shaped forms.

The leucocytes look like big white boulders in the stream, wholly
unmoved by the rushing torrent around them.

TROUBLES IN DARK-FIELD MICROSCOPY

§ 213. If one has available a good light source, a good dark-field
condenser, an immersion objective of suitable aperture for the con-
denser, cover-glasses and glass slips of the proper thickness, and
finally suitable objects for study, one soon learns to get good results;
but with this instrument the technique is far more exacting than
with the bright-field microscope. Troubles which might not be very
noticeable with the bright-field microscope will spoil a dark-field
image. All of the troubles hereafter mentioned have repeatedly
blocked the way of the writer or his pupils, and many others are
likely to occur; but the results when they are good are so satisfac-
tory that no one minds the labor necessary when once a good dark-
field image of a familiar object is seen.

(i) Lack of a dark background. This may be due to either of the
following causes, or possibly a combination of them:

i64

THE DARK-FIELD MICROSCOPE

[Cn. Ill

(a) The aperture of the objective may be too great for the con-
denser so that direct light from the condenser enters the objective.
This is quite likely to happen from forgetting to insert the proper
reducing diaphragm when large apertured immersion objectives are
being used. This is a strong reason for the belief that for dark-
ground work, immersion ob-
jectives of suitable aperture
be furnished by the opticians
(§ 185). If the aperture of
the objective is near the
limit of that of the condenser
there is liable to be a halo on
one side, especially if the
condenser is not perfectly
centered to the objective
(fig. 90). This halo is more
likely to appear with a low
ocular having a large field
than with a high ocular with
a smaller field.

(6) There may be an air
bubble in the immersion liquid

which directs a flood of light into the objective. By moving the prep-
aration this air bubble can be got out of the way, but sometimes it
persists in keeping in the way. In such a case it may be necessary to
focus the objective up and wipe away the immersion liquid and add
a fresh drop. Air bubbles in the immersion liquid between the con-
denser and the glass slip sometimes cause trouble. These can often
be squeezed out by pressing the slip down. In case it does not
remedy the evil, lift up the slip and add new immersion liquid.

(2) Impossibility of getting a good image. This has many causes,
but one is not infrequent. If not enough homogeneous liquid is used
either above or below, when the slip is moved about in searching the
preparation, the immersion liquid gets spread out and there is not
enough to make good contact between the slip and condenser or
between the objective and cover-glass. This condition should be

FIG. 90. FIELD IN A DARK-GROUND
MICROSCOPE WITH A HALO (h) ON THE
RTC;HT SIDE.

CH. Ill] THE DARK-FIELD MICROSCOPE 165

kept in mind and if it seems necessary, add more immersion liquid.
It must be remembered, too, that if the condenser is lowered the
stratum of immersion liquid may be broken and leave the condenser
unimmersed, or if the stratum of liquid is not broken, it is extended,
thus virtually thickening the glass slip. Finally, it sometimes
happens that no immersion liquid is put between the condenser and
the slip. In that case the preparation cannot be lighted.

(3) Violent or moderate motion of the particles of the preparation
when focusing. This is due to the thickness of the preparation. In
focusing for different levels in it the end of the objective comes in
contact with the cover-glass and presses upon it. Furthermore, the
thick preparation lifts the cover up into an unstable position and the
pull of the viscid immersion liquid causes it to rise and fall in focus-
ing even though the objective does not touch the cover-glass. The
remedy is to make the preparations thinner.

(4) Impossible to get the objective in focus. — This may be due
to a cover-glass thicker than the working distance of the objective.
Sometimes owing to the dim light in which one works or to inatten-
tion, the preparation is put on the stage of the microscope with the
cover-glass down next to the condenser instead of up toward the
objective. The glass slip is too thick to focus through when using
a high power.

(5) There may be too small an opening in the stage. In that case
the condenser cannot be raised high enough to touch the glass slip,
and no good image can be obtained even though the space between
them were rilled with the immersion liquid, for that would bring the
focus of the hollow cone of light from the condenser much below the
object. It would be like using far too thick a glass slip.

In other cases the opening in the stage may be just large enough
to receive the condenser top, but leave no room to change its posi-
tion in centering. The only remedy is to use a different condenser
with narrower top or to have the opening enlarged. With some
microscopes there is a removable piece which may be taken out
thus increasing the size of the opening in the stage.

(6) The substage may not be able to rise high enough to bring the
upper end of the condenser at the level of the top of the stage.

1 66 THE DARK-FIELD MICROSCOPE [Cn. Ill

There are two ways to remedy this in some cases. The condenser
may be screwed up in its sleeve, thus bringing it at a higher level
when the substage is run up. The other change is to shorten the
stop for the substage by screwing it downward or by removing it
altogether. Then one has to be careful and not to get the upper end
of the condenser much above the level of the upper face of the stage.
(7) The glass slide may not be in contact with the condenser

(§ 194).

SELECTION OF A DARK-FIELD CONDENSER AND A LAMP

§ 214. Dark-field condensers. — Those now available offer consider-
able choice. In making a selection one should be guided by the work
to be done and by the facilities and space at command. If space is
limited and facilities few, one of the supers tage condensers or one of
the substage condensers with attached light might wisely be chosen
(§ 189, § 200).

In a biological laboratory in which many different persons are to
make use of it on a great diversity of material, one of the parabo-
loid condensers is recommended. This gives fully lighted fields for
objectives of 16 mm. (lox) and higher objectives. It does not re-
quire great skill to get fair results. It gives good results with quite
a wide range of glass slips, and is not so sensitive to exact centering
as the cardioids. In general it will give good results with unskilled
workers, and for those really skilled it will give excellent results for
all powers of the microscope.

If one wishes to get the most perfect results with high powers, then
a condenser of the cardioid form is to be chosen. It gives a very
sharp focus of its hollow cone of light, and serves to light the objects
at its focus in the most perfect manner, and gives a very dark back-
ground. It requires more skill in its use than the paraboloids, but
the results obtained with it are worth the extra trouble.

It is not satisfactory for low powers because of the small spot of
light it gives; but that is unimportant, because for the low powers
the Abbe condenser with a central stop or a dark-field element
(§ 181) answers very well.

CH. Ill] THE DARK-FIELD MICROSCOPE 167

Those made by the American opticians have been found of the
highest excellence, also those made in England and on the Con-
tinent. When one of the cardioids is perfectly centered and lighted,
the clearness of minute details in suitable structures and in minute
living organisms give a certainty to the picture that is unbelievable
to a person who has used only bright-field illumination on them.

Of course, it is desirable to have several: one of the paraboloid,
one of the cardioid, one of the combined, one of the supers tage forms,
and one with the dark-field element (§ 181). This is not commonly
possible except in laboratories.

Whatever form one possesses it is desirable that it be accurately
centered to one microscope, and that that microscope be devoted to
dark-field work. It will then always be ready, and one can work
with it with the same ease and certainty as with a bright-field
microscope.

^ 215. Lamp for dark-field work. — In the selection of a lamp for
dark-field work one must also be guided somewhat by circumstances.
The best lamp that the writer has used, and he has tested them all,
is the 6-volt, io8-watt lamp (figs. 79-82). This gives ample light of
daylight quality. It is no trouble, for when once properly connected
and arranged, it will go on giving its full light as long as the lamp
lasts. It is economical when used with a transformer as it draws
only about one ampere from the no-volt circuit. It is admirably
adapted to photo-micrography and many other purposes where a
brilliant light is needed. It is available also for bright-field work
with the highest powers.

The microscopical training of nearly every worker has been with
bright-field illumination, and consequently the appearances given
with this form of light furnish the standard. Under the dark-field
microscope the same objects have a wholly unfamiliar look, and
indeed, as Edmunds pointed out in 187 7, seem like new things. It
is quite conceivable also that for an investigator who had used the
dark-field microscope only, the bright-field image would be equally
puzzling. In order, then, to interpret these two images of identical
objects, the objects should be studied with both forms of illumi-
nation. The more this is done the less unlike do the objects seem,

i68 THE DARK-FIELD MICROSCOPE [Cn. Ill

until finally one can interpret the appearance whichever form of
illumination is used.

While the combined light- and dark-field condensers (§ 187) are not
perfect for either light- or dark-field work they do serve to show the
identical object first with one and then with the other form of illumina-
tion with t)nly a slight change in the illuminator and no change in the
position of the object. As shown in Chapter XIV, §§ 717, 721, this
result is also easily attainable with the ordinary refracting condensers.

Of all the reasons for non-success with the dark-field microscope,
the most constant one is an inadequate light. Then also the use of
unmodified electric light when it is bright enough to bring out the
minutest details and particles is very hard on the eyes of the observer
unless it is given daylight quality by the use of a daylight glass
filter. From an abundant experience of over 10 years with the dark-
field microscope the writer is positive that the continuous use of
the brilliant, unmodified light would be injurious. Of course, for
a diagnosis requiring a few minutes the unmodified light answers,
but for continuous study for several hours a day the light should be
given daylight quality. This for most observers is not only easier on
the eyes but gives a sharpness of detail that cannot be gained by the
unmodified light.

As stated above, the io8-watt, 6-volt headlight lamp (figs. 79-80)
furnishes abundant light for all uses. The lamp is so constructed
that for photography and projection work the full intensity may be
used by removing the daylight glass filter. Of course, also, any
other filter can be substituted for a special purpose.

COLLATERAL READING

BECK, CONRAD. — The Microscope, 1038, pp. 128-142.
BECK, R. AND J., LT'D. — Dark-ground Illumination (special pamphlet).
CARPENTER, WM. B. — The Microscope and Us Revelations, 1856-1901.
CHAMOT AND MASON. — Handbook of Chemical Microscopy, Vol. T, pp. 84-92.
EDMONDS, J. — On a New Immersion Paraboloid. Mon. Micr. Jour., Vol. 18 (1887),

pp. 78-85.
GAGE, S. H. — Modern Dark-field Microscopy and the History of Its Development.

Trans. Amer. Micr. Soc., Vol. 30 (1920), pp. 95-141. Stain Technology,

Vol. 13 (1938), pp. 25-36.
GORING AND PRITCHARD. — Micrographia. London, 1837.

CH. Ill] THE DARK-FIELD MICROSCOPE 169

HALL, J. C. — Quart. Jour. Micr. Sci., Vol. IV (1856), pp. 205-208. Figures the

"spotted lens."

LISTER, JOSEPH JACKSON. — Trans. Roy. Soc., Vol. 120 (1830), pp. 187-260.
METZNER-ZIMMERMANN. — Wissenschaf tlichen Mikroskopie, 2d ed.
QUEKETT, JOHN. — Practical treatise on the use of the microscope. Editions of

1848, 1852, 1855.

READE, REV. J. B. — See pp. 227-231 of Goring & Pritchard.
SHADBOLDT, G. — Trans. Micr. Soc. London, Vol. Ill (1851), pp. 132, 154.
SII:DENTOPF, H. — Vorgesichte der Spiegelkondensoren, Zeit. wiss. Mikr., Vol.

XXIV (1907), pp". 382-395- ^. o T , __ . TTT

WENHAM, F. H. — Reflecting Condensers, Trans. Micr. Soc. London, Vol. Ill

(1850), pp. 83-90. Quart. Jour. Micr. Sci., Vol. II (1854), pp. 145-158. Trans.

Micr. Soc. London, in Vol. IV (1856), Quart. Jour. Micr. Sci., pp. 55-60.
WRIGHT, A. E. — Principles of Microscopy, Chapter XIV.

CHAPTER IV

THE POLARIZING MICROSCOPE: OPTICS OF THE MICROSCOPE.
§§ 216-272; FIGURES 91-119

A polarizing microscope is one in which a microscope and a polar i-
scope are combined for the purpose of studying microscopic prepara-
tions illuminated by polarized light.

For the biologist the definite information to be gained by using the
polarizing microscope may be stated thus:

(1) Whether the object under the microscope is isotropic, i.e., non-
polarizing, or anisotropic, i.e., polarizing.

(2) Whether the object is uniaxial or biaxial.

(3) Whether it shows interference phenomena, i.e., a black cross
or colors.

(4) Whether the object rotates the plane of polarization.

(5) Whether the object shows pleochroism.

While the full theoretical meaning of all the phenomena may not
be clear, the biologist can be certain tint the different appearances
with p3larizecl light indicate definite physical or chemic.il properties.

§216. Polariscops; polarizer and analyzer. — A polarizer is an
optical device for producing polarized light, and an analyzer is a
device to aid the observer in determining whether the object illumi-
nated with polarized light has any effect upon that light.

The polariscope most frequently used with a microscope consists
of two prisms of Iceland spar (transparent calcite, CaCO.s). The
light traverses them lengthwise. The prisms are cut along a diagonal
and the cut surfaces polished. The two halves of each prism are
then cemented together with Canada balsam or linseed oil.

One prism, the polarizer, is placed between the source of light and
the object, and serves to polarize the light before it reaches the object.

According to the classical theory, ordinary light from the sun or
any other source, is vibrating in all planes transverse to the direc-
tion of propagation of the light. When such light enters the cal-
cite prism polarizer it is divided into two parts. One of these parts,

170

CH. IV]

THE POLARIZING MICROSCOPE

171

called the ordinary ray, is refracted most, and meets the cemented
surface at an angle greater than the critical angle for calcite in con-
tact with the cementing medium. It is, therefore, reflected to the
side of the prism and does not pass on to illuminate the object.

The other ray, called the extraordinary ray, is bent less and can
therefore pass through the cementing medium and the prism to il-
luminate the object. Both rays are polarized, but one is eliminated.
The object is thus illuminated by light vibrating in one definite plane.

It will be noted that only half the light gets through the polarizer to
illuminate the object, hence one must have plenty of light to start with.

Objects illuminated by polarized light look the
same as though they were illuminated by ordinary
light, except those exhibiting pleochroism (§ 233).

The addition of another Nicol prism, the an-
alyzer, so turned that it excludes the light from
the polarizer, serves to show whether the object
under the microscope has produced any change
in the polarized light. This is brought about as
follows: In fig. 91 the diagonals of sections of the
prisms are parallel. In this position the polarized
light passes directly through both prisms. If the
analyzer is rotated 90°, none of the polarized light
transmitted by the first prism can pass through the
second. When so arranged that no light can pass
through both prisms, they are said to be crossed.

FIG. 91. MICRO-POLAR ISCOPE IN POSITION ON THE
MICROSCOPE.

Polarizer The Nicol prism under the stage of the
microscope.

Analyzer The Nicol prism over the ocular.

Stage The stage of the microscope.

Object The object on a slide.

Objective The microscopic objective.

S Set screw for clamping the analyzer to the tube of
the microscope.

Ocular The microscopic ocular in position.

Pointer and Scale The graduated ring and pointer to show the amount of
rotation.

a Handle for raising and lowering the analyzer to arrange it properly with
reference to the eyepoint.

L'E t *,-]

172 THE POLARIZING MICROSCOPE [Cn. IV

§ 217. Polaroid. — Instead of Nicol prisms for the polarizer and
analyzer a substitute has been recently devised. It is called Polaroid,
and is composed of a multitude of fine, elongated crystals imbedded
in a cellulose matrix. This matrix is usually mounted between thin
plates of glass or heavy safety film.

The polaroid can be used with any microscope and shows the
phenomena of polarized light satisfactorily. It is considerably
cheaper than the Nicol prism polariscopes. However, for the most
accurate work with polarized light it is necessary to have an accurately
graduated analyzer, a centering, graduated revolving stage, an ocular
with cross-hairs, means for using the quartz wedge, and a selenite
plate for red of the first order. Such an instrument is shown in
figure 92. (See Martin Grabau, Polarized Light and Its Applica-
tion, 1940. Polaroid Corporation, Cambridge, Mass.) 7

§ 218. Iso tropic and anisotropic objects. — An iso tropic, singly re-
fracting or non-polarizing body is one which does not divide the beam
of light traversing it into two beams vibrating in different planes, but
the entering light vibrates in all planes as when entering. Such an
isotropic substance is glass, and when it is placed between the crossed
polarizer and analyzer the light is not restored. When, however, an
anisotropic, doubly refracting or polarizing object is placed between
the crossed analyzer and polarizer, it divides the light from the
polarizer into two beams and one of them can pass through the
analyzer. The object will then glow with a soft light on a dark
background. Cotton, linen, and other vegetable fibers are aniso-
tropic, and so are many crystals, and animal tissues like muscle and
bone.

TESTING THE POLARIZING MICROSCOPE

Whether the polarizing microscope is simple or elaborate, there
are some definite tests that should be made before accepting the
results obtained by its use.

§ 219. Testing the polarizer and the analyzer. — The polarizer
is usually as perfect as the manufacturers can make it, and so is
the analyzer. It is simple to find out whether the polarizer and
analyzer exclude all light when crossed, and allow it to pass when
in any intermediate position. For this test, and indeed for all the

CH. IV]

THE POLARIZING MICROSCOPE

173

tests, one should work in a dark room, if possible, so that all extra-
neous light is absent.
The condenser is removed, also the objective and the ocular.

FIG. 92. CHEMICAL MICROSCOPE or CHAMOT AND MASON
(Outline Drawing from the Catalogue of the Spencer Lens Co.)

The analyzer.

The set screw to hold the analyzer in place.

Focusing wheel for the coarse adjustment.

Slit in the tube above the objective for the quartz wedge, etc.

Fine adjustment wheel.

i
2
3
4

6 Revolving nose-piece with three objectives in place.

7 Spring clips for fixing the slide in place.

8 Handle of the iris diaphragm of the condenser.
g One of the centering screws.

10 Screw for fixing the stage at any position in its rotation.

11 Fork and contained substage condenser.

12 One of the centering screws for the condenser,
i? The polarizer.

jj Nut for fixing the inclining pillar in place.

75 Wheel for the rack and pinion for raising and lowering the condenser,

16 Mirror.

17 Foot of the microscope.

174 THE POLARIZING MICROSCOPE [Cn. IV

With the polarizer in the zero position and also the analyzer, the
tube of the microscope is lowered as far as possible to bring the
polarizer and analyzer near together. Then light is directed through
the polarizer. If the polariscope is perfect there will be two posi-
tions, o° and 1 80°, when there will be total darkness. In other po-
sitions there will be twilight, and when either element is rotated
45° there will be the brightest light. Apparently it is not possible
to make a polariscope so perfect that there is complete extinction
of light when the nicols are crossed. Some of the light emerging
from the analyzer may not be completely polarized on the one hand;
and on the other, when used with various optical parts, the scattering
and depolarization of a part of the light by the lenses of the
condenser, ocular and objective leave some of the light in a position
enabling it to pass the analyzer.

It is also intelligible that, if the light from the source is excessive,
even more of it will be in condition to pass the crossed nicols. In
any case the field may not be perfectly black when the nicols are
crossed in the most perfect instruments. On the principle of con-
trast, however, the field may appear densely black if there is only a
small amount of polarizing material under the microscope. For
example, cotton fibers polarize so brilliantly that the field about
them appears absolutely black at first. If one moves the slide so
that there are no cotton fibers present it will be seen that the field
is not black but dark gray. (See Beck, part ii, p. 200; Chamot and
Mason, vol. ii, p. 274).

§ 220. Testing the condenser. — After one is convinced that the
polarizer and analyzer are satisfactory, it is well to test the con-
denser. For this the condenser is put in place above the polarizer
and the diaphragm opened wide. The light is then sent up through
the polarizer. No object, objective, or ocular should be in place,
and the analyzer should be at the zero or crossed position. If the
condenser is isotropic, the field will remain just as dark with crossed
nicols as it is without the condenser. Sometimes the condenser
lenses are under strain, then they become polarizing, and the field
cannot be made dark in any position of the polarizer and the ana-
lyzer. Such a condenser, although it may be perfectly good for

CH. IV] THE POLARIZING MICROSCOPE 175

ordinary light observations, should be discarded for polarization ex-
periments, and an iso tropic one used. .

§ 221. Testing the objectives. — Many objectives have one or
more of the lenses under strain, and therefore aniso tropic. With
such objectives the field will not be dark when the nicols are crossed,
and they are not suitable for polariscopic work, although they may be
perfectly good for ordinary microscopic observation. To test an
objective, put it in place, cross the nicols and light the microscope
well. If the objective is isotropic the field will remain dark with
crossed nicols. If the objective is under strain, the field cannot be
made dark. Oculars are not so likely to be under strain, but those
to be used with the polarizing microscope should be tested in the
same way as the objective.

It will be noted in these tests that the position of the analyzer
for the most perfect darkness or extinction is very precise. A
rotation of 5 degrees either way renders the field gray, and it be-
comes lightest at 90°, and again darkest at 180°,

§ 222. Testing the glass slips for the polariscope. — Glass is one
of the isotropic substances, but when under strain, it becomes
anisotropic. The majority of glass slips and corex glass slips tested
have been isotropic, but occasionally one is anisotropic. If the
glass slip on which an object is mounted is polarizing, no exact
estimate of the character of the object being studied can be deter-
mined.

To test the microscopic slips on which objects are to be mounted,
place one on the stage of the polarizing microscope and rotate the
analyzer. If the glass slip is suitable, the appearance will be exactly
as if it were absent, but if no dark field can be obtained when the
polarizer and analyzer are crossed, the slip must be doubly refract-
ing, and should not be used for mounting objects for the polariscope.

Dr. Chamot informed the writer that often the slips used in
chemical microscopy where they had to be heated over the Bunsen
flame, became markedly anisotropic. It is advantageous to have a
glass slip which one knows is polarizing so that the appearance may
be definitely fixed in mind. To prepare such a slip, heat it all over
till it is too hot to hold, then with forceps hold one end in the flame

176 THE POLARIZING MICROSCOPE [Cn. IV

of a Bunsen burner till it is a dull red. Cool slowly by waving in the
air. If it happens to break, try another. When it is cool, put the
end that had been heated under the polarizing microscope, and cross
the polarizer and analyzer. A dark field will not result. Move the
slide till a part that had not been heated very hot is in the field.
Probably a dark field will result when the nicols are crossed. If so,
one can see on the same slip an isotropic and an anisotropic appearance.

§ 223. Centering the revolving stage. — A very practical way to
get it approximately centered is suggested by Dr. Chamot: A disc
of metal with a very small central hole is put into the opening of
the stage of the microscope. One can focus on this, and with the
centering screws put the small hole directly under the crossing point
of the cross hairs. As the centering disc must fit accurately the
opening in the stage, its removal is greatly facilitated by having a
segment removed from the edge; then with the finger nail it can be
easily lifted up.

The other and the most exact method of centering is to use a clear
glass slip, and with a fine pen put a minute dot of ink on the
middle of the slide, or one can, with the aid of a writing diamond,
make a delicate x on the middle of the slide. This is moved about
by the hands until the spot or x is exactly under the crossing point
of the cross hairs of the ocular. The stage is then rotated clear
around and the spot will again be under the crossing point. Now
rotate the stage until the spot or x is farthest from the crossing
point of the cross hairs of the ocular. With the centering screws
move the spot or the x, half the distance toward the crossing point
of the lines in the ocular. Then with the fingers or mechanical stage,
move the slide till the spot or x is directly under the cross hairs.
Revolve the stage. The centering may not be perfect the first trial,
but by continuing one can center the stage so accurately that the
object under examination will remain directly under the cross hairs
of the ocular for that particular objective. If another is turned in
place, the centering may not be quite perfect; but usually it is
approximately so. For perfect centering the stage must be re-
centered for each objective. The rationale of this proceeding is
that the crossing point of the lines in the ocular and the center of

CH. IV] THE POLARIZING MICROSCOPE 177

rotation of the revolving stage must be put on the same axis. If
they are not on the same axis, any spot originally placed directly
under the crossing point in the ocular will describe a circle when the
stage is rotated, and the spot or x will be at the periphery of that
circle. It should be at the center of the circle, that being at the
center of rotation of the stage.

THE PURPOSE OF A POLARIZING MICROSCOPE IN BIOLOGY

§ 224. Physical analysis. — The importance of the physical
analysis of animal and plant structures has long been recognized by
many workers, but this physical analysis has been' carried out only
in a desultory way. Now with thef exact apparatus at a reasonable
cost, and the almost perfect artificial sources of light available, it
seems possible to proceed in a systematic manner to determine just
what and how much the different minute organisms and the tissues
and organs of the higher animals and plants are polarizing.^' There
is certainly a difference in the physical organization of organic as
well as inorganic substances \\hen they behave differently in polar-
ized light, i.e., whether they are isotropic or anisotropic. Every hint
concerning the intimate structure and arrangement of the materials
of biology helps to give a truer insight into their organization. It is
believed, then, that the information gained by the polarizing micro-
scope and the ultra-violet microscope will aid in helping to under-
stand the living world. Fortunately this physical analysis can be
applied to living as well as to dead matter, and it is independent
of the possible changes wrought by the highly artificial staining
processes after treatment with varied chemicals.

It is hoped that in the future the definite determination of the
physical properties of living and fresh animal and plant substances
will be sought for as persistently and faithfully as their staining
reactions.

EXPERIMENTS WITH POLARIZED LIGHT

Make sure that the polarizing microscope, and the glass slips
used for mounting the objects to be studied are suitable for their
purpose (§§ 219-222).

!y8 THE POLARIZING MICROSCOPE [Cn. IV

§ 225. Vegetable material. — - The tissues of plants are as a rule
more strikingly doubly refracting than those of animals, and are
very easy to prepare in the living, fresh and fixed or dried condition.
Potato is one of the best objects to begin with, for both the starch
and the cellulose walls of the vegetable cells are strongly polarizing.
Use a fresh, firm potato. Wash the surface well, then with a sharp
knife or a razor blade make a very thin slice, including some of the
skin. Mount in water or in normal salt solution. Cover and em-
ploy first a 16 mm. (lox) objective, then an 8 mm. (2ox) or a 4 mm.
(4ox) objective. Some of the starch grains are very large, and some
quite small. Some will be free in the mounting liquid and many of
them will be in the cells of the potato. The cellulose cell- walls will
be bright when the nicols are crossed, showing that they are aniso-
tropic. The starch grains, whether large or small, will also be bright
and will show a black cross. This will rotate as the analyzer is
rotated.- This black cross will be met in many cases of doubly
refracting bodies, and is one of the characteristic features of starch.
If one wishes to study dry starch, it is better to mount it i i Canada
balsam. In case one finds it dilicult to get a thin en:u*h section
of the fresh potato, a preparation which will show all the points
may be made by scraping the fresh cut surface, and mounting the
scrapings in water.

§ 226. Cotton and linen fibers. - If cotton or linen fibers are
mounted in water or in balsam, very brilliant polarization is shovvn
with crossed nicols. Plant tissues, whether fresh or .fixed, give good
polarization effects, and show with the greatest clearness the
minutest layers or strands of cellulose, anisotropic crystals, etc. No
one interested in the microscopic structure of plants can afford to
neglect this means of investigation.

§ 227. Mounting specimens for both polarized light and ultra-
violet radiation. — For this, mounting slips which are non-polarizing
on the one hand, and which transmit ultra-violet radiation on the
other, must be used. Such slips are composed of fused quartz or
of corex d. glass (fig. 218). The polished fused quartz slips are very
expensive, costing from $3.50 to $12.00 each. The corex d. polished
slips cost about 75 cts. each. Slips cut from sheets of corex glass

CH. IV] THE POLARIZING MICROSCOPE 179

answer very well even if not polished. Cut from the sheets, the
slips cost about $6.00 per hundred. With ground edges, the cost is
considerably more.

§ 228. Mounting media for polarized light and ultra-violet radia-
tion. — These media, like the mounting slips, must be free from
polarization and entirely transparent to the ultra-violet radiation.
Canada balsam, cedar oil and many of the ordinary media used for
mounting are highly fluorescent in ultra-violet radiation and cannot
be used. Fortunately water and normal salt solution are available
and suited for fresh specimens. For permanent preparations one
may use glycerin. It is slightly fluorescent, but in the thin layers
used answers very well. It also mixes with water, which is a great
advantage for many specimens. For dry or dehydrated objects it
was' found that the mineral oil used in medicine, whether of a
naphtha or of a paraffin base, is transparent to ultra-violet down to
0.3/1. Its refractive index is high (1.48). It is therefore a fairly
good substitute for Canada balsam. It is also good for use in
making the immersion contact with the condenser and slip, and for
the immersion liquid for the oil immersion objectives. (See table,
§ 269.) If used for permanent mounting in place of balsam, the
cover-glass must be sealed as with glycerin mounts (§ 530).

§ 229. Annual tissues and organs; minute animals with polarized
light. — As with plant life, living and minute animals and living and
fresh tissues of animals should be studied with the polarizing microscope
and the appearances later studied with the ultra-violet microscope.

§ 230. For living animals, the water in which they are found in
nature is the best mounting medium. For the higher forms, the
living and fresh tissues should be mounted in some isotonic solution
like normal salt, Ringer's solution, etc. The most perfect isotonic
solution is the body juices in which they are naturally bathed in life.
It is not easy to get these in all cases, hence the use of normal salt,
etc. One must remember also to use quartz or corex slips if the
specimens are to be compared under ultra-violet radiation.

§ 231. Example of isotropic substances. — For an isotropic object
put an ordinary glass slide under the microscope. Cross the nicols.
The field will remain dark.

180 THE POLARIZING MICROSCOPE [Cn. IV

As an example of iso tropic crystals, i.e., those belonging to the
cubic system, make a solution of common salt, sodium chloric! (NaCl).
Place a drop of the salt solution on a slide that has been tested and
found free from strain (§222). As the water evaporates crystals
will be formed. Place the slide under the microscope, shade the
stage well, if not in a darkened room. Cross the nicols. The cubical
crystals of salt will remain dark.

§ 232. Examples of anisotropic substances. — As an example of
uniaxial, anisotropic crystals make a fresh preparation of carbonate
of lime crystals like that described for pedesis (§ 348), or use a prep-
aration in which the crystals have dried to the slide; use a 4 mm.
(4ox) objective, shade the object \\ell, remove the analyzer, and
focus the crystals; then replace the analyzer. Cross the nicols.
In the dark field will be seen multitudes of shining crystals, and if
the preparation is a fresh one in water, part of the smaller crystals
will alternately flash and disappear. By observing carefully, some
of the larger crystals will be found to remain dark with crossed
nicols, others will shine continuously. If the crystals are in such a
position that the light passes through parallel with the optic axis
(§ 232a), the crystals are isotropic like salt crystals and remain
dark. If, hovever, the light traverses them in any other direction,
the ray from the polarizer is divided into two constituents vibrating
in planes at right angles to each other, and one of these will traverse
the analyzer; hence such crystals \ull appear as if self-luminous in a
dark field. The experiment with these crystals from the frog suc-
ceeds well with a 2 mm. homogeneous immersion.

As a further illustration of anisotropic objects, mount some cotton
fibers in balsam (Ch. XI), also some of the lens-paper (§ 54). These
furnish excellent examples of vegetable fibers; striated muscle fibers
are also very well adapted for polarizing objects.

§ 232a. The optic axis of doubly refracting crystals is the axis along which
the crystal is not doubly refracting, but isotropic like glass. When there is but
one such axis, the crystal is said to be uniaxial; if there are two such axes, the
crystal is said to be bi-axial.

The crystals of carbonate of lime from the frog (§ 232) are uniaxial crystals.
Borax crystals are bi-axial.

CH. IV] THE POLARIZING MICROSCOPE 1 8 1

§ 233. Pleochroism, Pleochromatism. — Polarizing or anisotropic
bodies may remove some of the wave lengths of light traversing
them. The wave lengths removed depend upon the plane in which
the light passing through the substance is polarized with respect to
the optic axis of the crystals. For demonstrating the color changes
in pleochroism only one element of the polar iscope is used. As it is
usually easier to remove the analyzer than the polarizer, the polari-
zer is the element left in place.

If the substance under examination is uniaxial, but two colors are
shown, and it is called dichroic.

If the substance is biaxial, then three colors may appear, and the
substance is called trichroic.

An easily prepared dichroic substance is haemin (§ 233a). Find a
large haemin crystal in the preparation and place it at the inter*
section of the cross hairs of the ocular. Note the position of the
stage, then rotate it, and note the changes that take place in a com-
plete revolution, also note the number of decrees of rotation required
to make the changes. With the h'jemin the crystal will be dark twice
in the revolution; and twice it will appear light.

Crystals of acetate of copper show strikingly the dichroic change,
the crystals being a part of the time greenish, and a part of the
time bluish.

A striking and instructive demonstration to show the difference
between pleochromatic and non-pleochromatic objects may be made
by adding part of a cotton fiber to the haemin or the copper when
the specimen is prepared. The cotton will not change in color during
the entire revolution of the stage. If now the analyzer is added the
cotton fiber will be seen to polarize brilliantly.

§ 233a. Haemin, hydrochlorate of haematin (C34 H35 N4 - N4 FeO6 - HC1) is
easily obtained from drie 1 blood. The blood may be fresh or old, it serves, there-
fore to differentiate the red or brown stains of blood from other reddish brown
stains. Hyemin is easily prepared by placing a drop of fresh blood on the middle
of a glass slip and spreading it about in a small area. When dry, a crystal or two
of common salt (sodium chlorid NaCl) is added and a cover-glass put over the
mixture. If old blood is used it should be powdered and several crystals of sail-
added.

With a pipette add enough glacial acetic acid at the edge of the cover-
glass to fill the space under the cover and immerse the blood. Heat in some
way till the acid steams well. It is better not to boil the liquid. Examine under

182 THE POLARIZING MICROSCOPE [Cn. IV

the microscope. If successful the brownish red, rhombic crystals of haemin will be
present in abundance. If not add more acetic acid and heat again. When plenty
of crystals are present, remove the cover-glass and let the preparation dry.
There should be plenty of crystals on the cover-glass as well as on the slide.
When thoroughly dry, add a drop of Canada balsam and mount (§ 534).

Haemin is much used for the detection of blood in medico-legal cases. It can
be obtained from stains no matter how old. Hsemin is dichroic (§ 233) and ani-
sotropic (§ 232), but non-fluorescent.

§ 234. Production of colors. — Many polarizing substances show
the most gorgeous coloration under the polarizing microscope. A
striking example is white human hair. Short pieces mounted in
balsam or other highly refractive medium make a wonderful prepara-
tion. As the pieces are at various angles with the plane of polarization
some are almost sure to glow with great beauty. By rotating the
stage or rotating the specimen by hand different pieces will be in the
right position to give the best effects.

A crystalline substance, sulphonal (CHs^CCSC^Hs^, is excellent
for the dark cross and for the most brilliant coloration. To prepare
a specimen, put a large drop of Canada balsam in the middle of a slip
and warm it till it is quite liquid. Then add about a third as much
dry sulphonal. Heat till the sulphonal melts. Stir with a toothpick
to distribute the crystals evenly, and then warm a cover-glass and
press it down on the melted mixture. Cool the slide by placing it on
a cold body or by putting some alcohol on the back. If the specimen
is successful, and it usually is, it will show a great variety. By rotating
the stage or the analyzer the black crosses will also rotate and the
colors will change to their complements.

COLLATERAL READING FOR POLARIZED LIGHT

BECK, CONRAD. — The Microscope, 1938 ed.

CHAMOT, AND CHAMOT AND MASON. — Chemical Microscopy.

CLARK, C. H. — Practical methods in microscopy (gives sulphonal experiment).

HARDY AND PERRIN. — Principles of Optics.

QUEKETT, JOHN. — Practical treatise on the use of the microscope.

SCHMIDT, W. J. — Die Bausteine des Tierkorpers in polarisiertem Lichte.

SOME OPTICAL PRINCIPLES IN MICROSCOPY

§ 235. Optical facts of prime importance for the microscope. —
In considering the optics of the microscope six fundamental facts

CH. IV]

THE POLARIZING MICROSCOPE

183

concerning light must be kept constantly in mind, for all of them
are involved to a greater or less degree in every microscopic obser-
vation:

(1) Light is composed of radiation which for visual purposes
consists of waves from Ao.4/z to Xo.y/x in length.

(2) Light in a uniform medium extends in straight lines.

X2.,*

Infra-Red

Ultra-Violet

FIG. 93. DIAGRAM OF THE NORMAL SPECTRUM TO SHOW VISIBLE

AND INVISIBLE RADIATION.

The spectrum in this diagram is magnified 50,000 times vertically and hori-
zontally. The visible part of the spectrum extends only from about X.y/x to \.4ju.
D represents the dark sodium lines in the solar spectrum. Incandescent sodium
in a lamp flame shows a bright line at this level.

184 THE POLARIZING MICROSCOPE [Cn. IV

(3) Light may be reflected.

(4) Light is refracted in passing from one medium to another of
different density.

(5) Light may be dispersed or separated into colored bands from
the fact that rays of different wave length are differently bent
(figs. 1 20, 2).

(6) Light may be diffracted, it i.e., bends around small ob-
stacles.

(7) Light may be polarized.

Stated in briefest terms light exhibits the properties of:

(i) Wave motion; (2) Rectilinear propagation; (3) Reflection;
(4) Refraction; (5) Dispersion; (6) Diffraction; (7) Polarization.

§ 236. Wave motion. — From a body like the sun, the electric
arc and other sources of energy, radiation is given off which in most
respects acts as if it consisted of transverse waves, i.e., waves at right
angles to the direction of propagation. The radiation which is
visible forms but a very small segment of the total radiation. In
fig. 93 the visible radiation is shown between wave lengths Xo.4ju and
Xo.7/x, measured in air or in a vacuum. Shorter waves are called
ultra-violet, and longer waves infra-red. The infra-red waves are
shown only up to a length of 2/x, although many of much greater
length exist.

In the ether of space the different visible waves move with equal
velocity, but in the various transparent bodies on the earth, the ve-
locity depends upon the wave length — the shorter the wave, the
slower the motion (§ 245).

§ 237. Light moves in straight lines. — In a uniform medium
light moves in straight lines. Any body in which light can tranverse
freely is said to be transparent. If light meets a body which it
cannot penetrate, it is either reflected (§ 238) or absorbed; if ab-
sorbed, it is changed to some other form of energy, usually heat.

§ 238. Reflection. — If light meets the surface of a body of
different refractive index from the medium which it is already trav-
ersing the light will be changed in its course.

If the surface is smooth and the light is reflected, the incident and
the reflected rays will be in the same plane and will make equal

CH. IV]

THE POLARIZING MICROSCOPE

185

angles on opposite sides of a normal erected at the point of reflection
(fig. 94). The eye can see the light only when in the path of the
lay, or when light is deilected from the ray by dust, etc. (§ 173),

If the surface is irregular,
the reflection will also be
irregular and the light will be
reflected from the point of in-
cidence in the form of a hemi-
sphere (fig. 95), hence light
would reach the eye from any
point in the hemisphere.

§ 239. Refraction. — As or-
dinarily considered, this is the
change in direction which
light undergoes when passing
obliquely from one transparent

FIG. 94. REGULAR OR MIRROR

REFLECTION.

(From Optic Projection).
The angle of incidence i, is equal to the
angle of reflection r; and the incident and
reflected ray are in a plane perpendicular
to the reflecting surface.

niedium into another (figs.

96-98).

A broader statement covering all the phenomena, whether the ray
passes obliquely or normally from one
medium to another, is this: Refraction is
the change in velocity of the waves of
light in passing from one transparent
medium into another.

§ 240. Law of refraction. — The amount
of bending depends upon two factors, — the
relative density of the two media and the ob-
liquity of the incident light. The greater the
obliquity of the incident ray, and the greater
the difference in density, the greater will be
the refraction. The precise law gov-
erning the course and relation of the

ray in the two media is known as the sine law of Snell and Des-

FIG. 95. IRREGULAR OR
DIFFUSE REFLECTION.
(From Optic Projection).
A ray of light meeting a
rough surface, like a piece
of white paper, is scattered
almost equally in all direc-
tions, making a hemi-
sphere of light.

cartes. It is expressed thus:

sin ^

smr

index of refraction, That is, the

sine of the angle of the incident ray with the normal to' the dividing

i86

THE POLARIZING MICROSCOPE

[CH. IV

surface divided by the sine of the angle of the refracted ray with its
normal, gives the relative direction of the ray in the two media, i.e.,

the index of refraction. For
example, in fig. 96, showing the
passage of light to water, the ray
being at 60° with the normal in
air, and 40° 38' in water, the real
relationship in this and in all
other cases is not the relative
size of the two angles, but
the sines of the angles, thus:
sin i or 0.86603

= i-33- That

sin r or 0.65115

is, the sine of the angle in air
is 1.33 times the sine of the
angle in water; and this would
hold true for any other pair of
sines, so that the law is universal

FIG. 96. REFRACTION OF LIGHT IN
PASSING FROM AIR TO WATER.

N Normal at the point of refraction.
sin i In this example sin 60° or 0.86603
sin r In this case sin 40° 38' or 0.65115
- 1.33, average index of refraction for
air and water.

for the wave length of light
giving this index of refraction.

The sine and corresponding
angle are always greater in the
rarer medium and consequently
less in the denser medium. It
follows from this that when the
ray passes from a rarer to a
denser medium and the angle is
made less, the ray must bend
toward the normal. Conversely,
in passing from a denser to a
rarer medium where the angle
is greater, the ray, must bend sinr In this example sin $4° 45' or 0.57000
from the normal. This is a gen- a^lasT*^ ^ °f refraction for air
eral law (see figs. 98, 100-102).

§ 241. Absolute index of refraction. — This is the index of re-
fraction obtained when the incident ray passes from a vacuum into

FIG. 97. REFRACTION OF LIGHT IN

PASSING FROM AIR TO GLASS.
N Normal at the point of refraction.
sin i In this example sin 60° or 0.86603

CH. IV]

THE POLARIZING MICROSCOPE

187

a given medium. As the index of the vacuum is taken as unity, the
absolute index of any substance is always greater than unity. For
many purposes, as for the object of this book, air is treated as if it
were a vacuum, and its index is called unity, but in reality the index
of refraction of air is about 3 ten-thousandths greater than unity.
Whenever the refractive index of a substance is given, the absolute
index is meant unless otherwise stated. For example, when the in-
dex of refraction of water is said to be 1.33, and of crown glass 1.52,
etc., these figures represent the absolute index, and the incident ray
is supposed to be in a vacuum.
§ 242. Relative index of re-
fraction. — This is the index
of refraction between two con-
tiguous media, as, for example,
between glass and diamond,
water and glass, etc. It is ob-
tained by dividing the absolute
index of refraction of the sub-
stance containing the refracted
ray, by the absolute index of
the substance transmitting the
incident ray. For example, the
relative index from water to
glass is 1.52 divided by 1.33.
If the light passed from glass
to water, it would be 1.33

Fir,. g8. REFRACTION OF LIGHT IN
PASSING FROM GLASS TO AIR.
N Normal to the refracting surface.
sin i In this case sin 34° 45' or 0.57000
sin r In this case sin 60° or 0.86603

If figs. 97 and 98 are compared it will
be seen that the ray of light follows
exactly the same path in leaving the
denser medium that it took on entering
it.

divided by 1.52.

By a study of the figures
showing refraction, it will be seen

that the greater th£ refraction the less the angle and consequently the
less the sine of the angle, and as the refraction between two media
is the ratio of the- s\nes of the angles of incidence and refraction

( — — 1, it will be seen that whenever the sine of the angle of refrac-
\sin r/9

tion is increased by being in a less refractive medium, the index of
refraction will show a corresponding decrease and vice versa. That is.

1 88 THE POLARIZING MICROSCOPE [Cn. IV

the ratio of the sines of the angles of incidence and refraction of any
two contiguous substances is inversely as the refractive indices of tlose
substances. The formula is:

(Sine of angle of incident ray\ _ /Index of refraction of refracting mediumX
Sine of angle of refracted ray/ ~ \Index of refraction of incident medium /

Abbreviated f5J5-ij » (j~r™|). By means of this general formula one

can solve any problem in refraction whenever three factors of the
problem are known. The universality of the law may be illustrated
by the following cases:

(A) Light incident in a vacuum or in air, and entering some
denser medium, as water, glass, diamond, etc.

(Sine of angle made by the ray in air \ _ /Index of ref . of denser med.\
Sine of anglelnade by the ray in denser med./ ~ \ Index of ref. of air (i) /

If the dense substance were glass: f^-H = f^-J. If the two media were
water and glass, the incident light being in water the formula would be:

(!EL? ] « ( i^Y If the incident ray were glass and the refracted ray in
sinr/ \i.33/

water: ( ^-H = (•— ^ V And similarity for any two media; and as stated
\smr/ \i.S2/

above if any three of the factors are given the fourth may be readily found.

§ 243. Critical angle and total reflection. — In order to under-
stand the Wollaston camera lucida (fig. 168) and other totally re-
flecting apparatus, it is necessary briefly to consider the critical
angle.

The critical angle is the greatest angle that a ray of light in the
denser of two contiguous media can make with the normal and still
emerge into the less refractive medium. On emerging, it will form
an angle of 90° with the normal, and if the surface is flat the re-
fracted ray will be parallel with the surface separating the two media.

Total Reflection. — In case the incident ray in the denser medium
is at an angle with the normal greater than the critical angle, it will
be totally reflected at the surface of the denser medium, that surface
acting as a perfect mirror. By consulting the figures it will be seen
that there is no such thing as a critical angle and total reflection in
the rarer of two contiguous media.

CH. IV] THE POLARIZING MICROSCOPE 189

To find the critical angle in the denser of two contiguous media: —
Make the angle of refraction (i.e., the angle in the rarer of the two

j- \ o i i Ai_ i . /sin A /index r\

media) 90 and solve the general equation: - — = — ; ; .

y 5 M Vsinr/ \mdex */

(i) Critical angle of water and air: sinr (90°) is i, index of water
1.33, whence

Air

sin i = 0.751 -K This is the sine
of 48° 45', and whenever the ray
in the water is at an angle of
more than 48° 45' it will not
emerge into the air, but be
totally reflected back into the
water.

(2) Critical angle of glass and
air: sin r (90°) is i. Index for

glass is 1.52, whence ^

riG. 99. DISPLACEMENT OF A RAY

sin i\ / I \ . , OF LIGHT IN TRAVERSING AN OBJECT

WITH PLANE FACES.

This figure is to show that while
there is no angular deviation of a ray
of light in traversing a dense medium
with plane faces, there is displacement;
but the emerging ray (r) is parallel
with the entering ray (i).

Air Glass The two media through
which the ray is traveling.

i n Incident ray and normal at the
point of entrance into the glass.

i' Incident ray continued by dotted
lines to show the path which would
have been followed if no glass had in-
tervened.

n'r Normal and refracted ray on em-
ergence from the glass to the air again.

rr Path of the refracted ray traced
backward.

•52"

whence sin i = .875, sine of critical angle in glass covered with water.
The corresponding angle is approximately 61°.
The last shows the advantage of water immersion when a large

= sin o.

which is the sine of 41° -f-. Light
having a greater angle in glass
than 41° is internally reflected as
from a mirror (fig. 94).

(3) Critical angle of glass
covered with water.

/ sin i

\sin r (sin 90° =

/index water (i.33)
\indexglass (1.52)

\ /sin i\
7 °r \ i /

i go

THE POLARIZING MICROSCOPE

[CH. IV

angle of light is desired. With homogeneous immersion there would
be no critical angle for the glass.

§ 243a. Critical angle. — As defined by some physicists the critical angle is
the least angle at which light undergoes total internal reflection at the surface of
the denser medium.

I have followed the rrnre common definition which makes it the greatest angle
at which a ray can emerge into the rarer medium; the emerging angle will then
be 90° and its sine i .000.

§ 244. Table of refractive indices nD. (From Chamot.)
(Temperature 20 to 22 C.)

Index of
Refraction

Name of
Substance

Approximate
Boiling
Point °C.

Approximate
Density

1.32

Methyl alcohol

o 79

1.36

, Ethyl ether

o 71

1-37

Ethyl alcohol

o 79

1.46

Cajeput oil

174

0.92

1.44

Chloroform

i 48

1.47

Glycerine

290

i 61

1.47

Turpentine

i55

o 86

1.48

Castor oil

o 96

1.49

Xylene

136

o 86

1.49

Benzene

o 88

1.50

Clove oil

1.05

1-51

Cedar Wood oil

o 98

i-57

Orthotoluidine

197

I OO

1.625

Carbon bisulphide

I 29

1.52 ±

Canada balsam

1.52-1.59

Glass

1.544-1.553

Quartz

§ 244a. Index of refraction and wave length. — As the shorter waves of the
blue end of the spectrum are more bent than the long waves of the red end in
it indicates that the index of refraction is greater for the blue end than for the
red end. Unless otherwise indicated, the index of refraction (n) is that of the D

line in the spectrum, and if written out entire the index would read —

nor

At the H line it would be expressed thus —

nnr

Specific cases from Watson's Physics:

no for water 1.334 nn for water i .34

no for flint glass 1.584 nH for flint glass 1.614

It is further to be noted that there is not perfect regularity in the increase or
decrease of the index of refraction according to the wave length of the light. The
exact index in each case must be determined experimentally. As will be seen
later, this irregularity makes it possible to construct achromatic instruments

(§ 258).

CH. IV]

THE POLARIZING MICROSCOPE

191

§ 245. The sine law and the velocity of light in different media.

— In the ether of space all wave lengths of light move with equal
velocity, but on the earth the velocity depends on the wave length.
While all wave lengths are retarded by shortening the waves, the
shorter the original wave the greater the retardation. As the re-
fraction of the light is one of the phenomena of this retardation, it

FIG. ioo.

FIG. 101.

FIG. ioo. CRITICAL ANGLE FOR LIGHT PASSING FROM WATER TO

AIR, THE ANGLE IN AIR BRING 90°.
N Normal to the refracting surface.

sin i In this case sin 48° 45' or 0.7519 i , ... , ,

-: — r i • . o - = , in accordance with the general

sin r In this case sin go or i.oooo 1.33

formula :

sin i index r

sin r index i

I Light ray at the critical angle and emerging into the air parallel with the
surface of the water.

d d' Ray of light at an angle greater than the critical one and being internally
reflected back into the water; the angle of incidence and reflection being equal
(fig. 94)-

FIG. 101. CRITICAL ANGLE FOR LIGHT PASSING FROM GLASS TO

AIR, THE ANGLE IN AIR BEING 90°.
N Normal to the refracting surface.

sin i In this case sin 41° + or 0.65780 i , ... A. .

— — T .,. . o — ^~^ > m accordance with the general

stnr In this case sin 90 or i.oooo 1.52

. , sin i index r

formula: -: — = T—J — .

smr index ^

b Light ray at the critical angle and emerging into the air parallel with the
surface of the glass.

d d' Ray of light at an angle greater than the critical angle and being re-
flected back into the glass, the angle of incidence and reflection being equal (Fig.
94).

192 THE POLARIZING MICROSCOPE [Cn. IV

follows that the shorter the wave the greater the bending. This is
shown by the action of the prism (fig. 120, 2), in which the blue is
more deviated than the red.

The retardation of any given wave length (i.e., the relative
shortening of the waves) follows the sine law in passing from one
transparent substance to another. For example, in passing from the
ether to water, the speed in water would be represented by:

or 1.334 for waves at the D Fraunhofer line. (Nichols, South-
sin r

all, Watson.) This means that if the speed in the ether were i,

in water for this wave length the velocity would be . In terms

1-334

of the angle of the light, if the sine of the angle in the ether is i, the

sine of the angle of this wave length in water would be .

1-334

For crown glass the waves opposite the fixed line B, if possessed of

a speed of i in the ether, would have a speed in the glass of .

I-53I

Opposite the H line, with the shorter waves, the speed would be
in crown glass.

That is, then, just as in refraction (§§ 239-240), if the velocity in
one medium and the index of refraction of the two media are known
the velocity in the second medium can be determined; and in
general, knowing any three factors, the fourth can be determined.

While for the discussion of lenses the narrower view of refraction
may suffice, for optical instruments generally it is of fundamental
importance to realize that there is just as much effect on light waves
striking the surface of the refracting body perpendicularly as ob-
liquely. In one case, that of the oblique meeting, the ray is bent
because of the shortening of the waves in passing from a rarer to a
denser medium. If the waves meet the denser substance normally
to its surface, the ray will not be bent, but the shortening of the

CH. IV]

THE POLARIZING MICROSCOPE

193

waves will be the same, leading to an optical shortening of the path
of the ray. This is of prime value when designing optical apparatus
where two optical paths must
be made equal, although the
actual distance in millimeters
may be unequal. The binocular
microscope is a striking example
(figs. 29-31). The shortening
of the path is also very strik-
ingly illustrated by the cover-
glass (figs. 52 B-C, §§ 105-
106).

§ 246. Dispersion by glass,
etc. — This is the separation of
the waves of white light into
groups according to their length;
and these different groups ap-
pear of different colors to the
normal eye. When white light
is dispersed by a glass prism
there results a spectrum or
rainbow with the red at one
extremity and the blue-violet at
the other,
into colors is made possible by

FIG. 102. CRITICAL ANGLE FOR LIGHT
PASSING FROM GLASS TO WATER, THE
ANGLE IN THE WATER BEING 90°.

N Normal to the refracting surface.
sin i In this case sin 61° -f- or 0.8750
sin r In this case sin 90° or i .0000 "*

— — in accordance with the general for-

, sin i index r

mula — — = . — ; .

sin r index i

b Light ray at the critical angle and
emerging into the water at an angle of
90° from the normal.

d d' Ray of light at an angle greater

As this diversion than the critical angle and hence re-
AS mis dispersion . ^ted ba(± ^ ^ glas?> ^ anglg of

incidence and reflection being equal,
the different refrangibility of

the different wave lengths, one would expect that the amount
of bending would be in exact proportion to the wave length.
This is true if one uses a grating and produces a normal spec-
trum (fig. 121). When a prism is employed to produce the dis*
persion, the refraction is not in exact relation to the wave length. In
general, the blue end of the spectrum is expanded arid the red end
contracted. Different kinds of glass and transparent minerals
(quartz, fluorite, etc.) refract differently. This makes achromatism
possible. As pointed out by Newton, if the refraction were in
exact proportion to the wave length, as with gratings, whenever

194

THE POLARIZING MICROSCOPE

[CH. IV

dispersion is overcome, the general refraction would also be over-
come and no achromatic combinations of lenses would be pos-
sible.

§ 247. Diffraction. — This is the bending of light past the edge of
objects. Instead of the light all going in a straight line beyond an
object, especially a narrow strip, some of it extends as if split off
from the main beam at the edge of the obstruction. These dif-
fracted beams may give rise to independent or so-called spurious
images. With low powers the diffracted light does not cause com-
plications, but with high powers the diffraction fringes and diffrac-
tion disc rr.ay produce effects very difficult of interpretation. (See
§ 270 \vhere there is a discussion of the part played by diffracted
light in microscopic images).

LENSES AND IMAGES

§ 248. Lenses. — A lens is a transparent body having one or both
of its opposite sides curved. The curves are most frequently spheri-
cal, and may be either convex
or concave. If both the sur-
faces are curved, the lens may
be considered as composed of
segments of two spheres. These
spheres are of like radius if
the surfaces are similarly
curved, and of unlike radius if
the surfaces are unlike. While a
FIG. 103-104. A CONCAVE LENS SHOW- lens with one plane face may be
ING THE PRINCIPAL, VIRTUAL Focus; AND considered a segment of a single
CONVEX LENS SHOWING THE REAL PRIN- , ..,..,

CIPAL Focus (F F), sphere, optically it is better

to consider two spheres, the

curved surfade from a sphere of finite, and the plane face from a
sphere of infinite radius (fig. 107, 3, 6).

§ 249. Images formed by lenses. — As light entering a dense
transparent body obliquely is bent toward the normal at the point
of entrance, it follows that if the lens has convex faces, the light

CH. IV]

THE POLARIZING MICROSCOPE

195

rays will be made more convergent; if it has concave faces, the
light rays will be rendered more divergent (figs. 103-104). From the
change in the direction of the rays on entering and on leaving a lens,
it is possible to form images of objects
by means of lenses (figs. 105-106).

§ 250. Forms and principal fea-
tures of spherical lenses. — As shown
in fig. 107, lenses may be convex on
both faces, or convex on one face
and plane or concave on the other.
Lenses may also be concave on both
faces or concave on one face and plane
or convex on the other.

If lenses are thick in the middle
and thin on the edge, they make
the rays of light entering them more
convergent. On the other hand,
if they are thin in the middle and
thick on the edge, they make the
light rays entering them more
divergent. In a word, then, thin
edge lenses are called convergent,
and thick edge ones, divergent
lenses. This follows inevitably from
the rule that, on entering a denser
medium, any oblique ray of light
is bent toward the normal, and

on leaving it for a rarer medium, it is bent from the normal
(§ 240).

§ 251. Principal features of spherical lenses. — (i) Principal
axis. This is the straight line passing through the lens and joining
the centers of the two spheres supposedly contributing to the forma-
tion of the lens (fig. 107, 4^ c').

(2) Optic center. This is the point in a lens or near it through
which light rays pass without angular deviation. That is, the ray
passing through the center of the lens continues in a line parallel to

FIG. 105. To SHOW THE
FORMATION OF A REAL AND OF
A VIRTUAL IMAGE BY A CONVEX
LENS. (COMPARE FIG. 11-12).

The size of the image depends
upon its relative distance from
the center of the lens. If it is
farther from the center than the
object, it will be larger than the
object, but if nearer, it will be
smaller (fig. 152).

THE POLARIZING MICROSCOPE

[CH. IV

the original direction as it does in traversing a piece of plane glass
(fig- 99)*

FIG. 106. To SHOW THE FORMATION or A REDUCED VIRTUAL IMAGE BY
A CONCAVE LENS, AND THAT THE IMAGE is LARGER THE NEARER THE OBJECT
Is TO THE PRINCIPAL (VIRTUAL) Focus. (COMPARE FIG. 154-155)-

As shown in the diagrams (fig. 107), the optic center is found by
drawing parallel radii from the two curved surfaces, or from the
curved and plane surface, and joining the ends of the radii. The
center of the lens is at the point where a line connecting the ends of
the radii crosses the principal axis (fig. 107 d.} The reason
light rays traversing the optic center have no angular deviation is
evident, for the radii are perpendicular to the surface of the lens, and
the tangent plane perpendicular to the radius is tangent to the
sphere at the end of the radius. As the tangents of two parallel

CH. IV]

THE POLARIZING MICROSCOPE
1 2

197

FIG. 107.

SPHERICAL LENSES WITH THEIR FORMS AND PRINCIPAL
FEATURES.

(1) Double convex lens showing the two spheres from which it was derived.
c-c' the centers of the two spheres with the principal axis of the lens on the line
joining the centers.

(2) Double concave lens and the two spheres from which it was derived. c-cf
centers of the spheres and axis of the lens.

(3) Plano-convex lens with the sphere from which it was derived. In this case
the axis is on the radius dividing the lens into two equal parts.

(4) Double convex lens showing the two spheres from which it was derived;
rrt parallel radii; tf tangents at the ends of the radii; cc' centers of the two
spheres from which the lens was derived. The line connecting the centers is the
optic axis. The center of the lens (cl) is on this axis.

(5) Double concave lens showing the same features as in (4).

(6) Plano-convex lens showing the same as in (5). In this case the radius of
the curved face is determined as usual, but that of the plane face may be con-
sidered infinity, so that any line perpendicular to the plane face is a part of that
radius. As shown in the figure the center of the lens must be then at the convex
surface of the lens. \

(7) Plano-concave lens the parts are practically like (6).

(8) Thin edge or converging meniscus .lens with the two spheres from which
it was derived. The inner, concave face is from the greater sphere, and the
optic center (cl) is wholly outside the lens.

(9) Thick edge or diverging meniscus lens. In this case the concave face is
from the smaller sphere, and the center of the lens (cl) is on the concave side.

198 THE POLARIZING MICROSCOPE [Cn. IV

radii must themselves be parallel, it follows that a ray of light
passing from one tangential point to the other is traversing a body
with parallel sides at the point of entrance and exit, and hence it
will suffer no angular deviation. The ray may be displaced as in
traversing any thick transparent body (fig, 99). With meniscus
lenses the optic center (fig. 107, 8, 9) is on an extension of the line
joining the centers of curvature, and wholly outside the lens.

(3) Secondary axis. This is any line which passes through the
optic center of the lens and is oblique to the principal axis.

(4) Principal focal point. The principal focal point or focus of
a lens or of a lens system like an objective, a simple microscope, etc.,
is the point on the principal axis vshere rays of light parallel to the
principal axis before entering the lens or lens system, cross the prin-
cipal axis after leaving the lens or objective (figs. 103-104). The
focus is also called the burning point. With a concave mirror it is
the point on the principal axis where rays parallel with the principal
axis before meeting the mirror, cross the principal axis after reflec-
tion from the concave surface. This point is situated half-way be-
tween the face of the mirror and the center of curvature.

ABERRATION OF LENSES

§ 252. Spherical aberration. — This is a defect of spherical lenses
shown in fig. 108. That is, the parallel ray at the edge crosses the
principal axis or comes to a focus nearer the center of the lens than a
ray near the axis. If, then, the full aperture is filled, as shown in the
figure, with rays parallel with the axis, there will be a series of foci,
those of the border rays being nearer the lens than those near the
middle of the lens (fig. 108, fi, f2, f3).

§ 253. Correction of spherical aberration. — It is possible by
selecting convex and concave lenses of different material and hence
of different refractive power, to overcome the spherical aberration
of the convex lens with an equal and opposite aberration in a con-
cave lens without overcoming the converging action of the convex
lens. Consequently rays will all come to one focus. Such a lens
combination is said to be aplanatic or spherically corrected,

CH. IV]

THE POLARIZING MICROSCOPE

199

FIG. 108. SPHERICAL ABEK-
RATION IN LENSES.

Axis The principal optic
axis.

123 Ray i at the edge
comes to a focus at / i; ray
2 at/2, and ray 3 at/3, that
is, the nearer the optic axis,
the longer the focus; and the
nearer the edge of the lens,
the shorter the focus.

If the correction were not quite sufficient so that the border rayc
still came to a focus slightly nearer the lens than the middle rays,
the combination would be under-corrected.
If the concave lens were too strong, the
border rays of the convex lens would
come to a focus farther from the lens
than the middle rays, and the com-
bination would be said to be over-
corrected. Sometimes under-correction
or over-correction is designed to com-
pensate for parts of the optical appara-
tus which the rays will meet later, or for
aberrations produced before the light
reaches the objective. The common
ahd almost universal example is the
spherical aberration introduced by the
cover-glass over the specimen (fig. 109).

§ 254. Cover-glass correction. — By referring to fig. 109 it will
be seen that the effect of the cover-glass is precisely like the spherical
aberration due to the unequal refraction of the different zones of a
convex lens; that is, the border rays are more bent than those nearer
the axis, as the obliquity of the rays is greater (§ 240).

Now to overcome this there must be introduced into the objective
an under-correction just sufficient to balance the effect of the cover-
glass. If the lenses are fixed in position in the objective it will
be evident that one njust select a cover-glass which is of the exact
thickness to satisfy the correction of the objective. The makers of
objectives are now very precise in stating exactly how thick the
covers should be for their objectives, and it is the part of wisdom
to pay heed to their statements if one hopes to get the best
results.

If one's objectives are adjustable (§§ 149-150), it is possible to
arrange the combinations so that quite a range of cover-glass thick-
ness or mounting medium thickness can be used and still get the best
optical effect by balancing the aberrations (§ 256).

206

THE "POLARIZING MICROSCOPE

CCH. iv

- Slide

FIG. 109. SPHERICAL ABERRATION INTRODUCED BY THE COVER-GLASS.

Axis The principal optic axis extending through the condenser and up through
the object and microscope.

Slide The glass slide on which the object is mounted.

Object The object to be studied; it is mounted on the slide.

Balsam The medium in which the object is mounted. It has practically the
same refractive index as the cover.

Cover-glass The thin glass plate over the object.

I 2 3 The light rays extending obliquely upward from the object.

3 2 i Light rays traced backward to their apparent origin, the most oblique
ray (j) being most bent, thus rendering its origin apparently highest.

rrr Points of refraction of the three oblique rays.

§ 255. Tube-length. — The length of the tube on the micro-
scope must be made of the standard for which the objective used
was corrected or aberrations will appear.

If the tube is shorter than the objective was corrected for, the
effect is the same as thinning the cover-glass. That is, it introduces
under-correction. This makes it possible to compensate for too
thick a cover by shortening the tube (§§ 150, 256).

When homogeneous immersion liquid is used one does not have to
trouble about the exact thickness, but care must be taken not to
use so thick a cover that the free working distance will be too short

(§ 101).

By consulting the catalogues of microscope manufacturers one can
find for what tube-length and thickness of cover-glass their unad-
justable objectives are corrected. For example, in the last editions
of the catalogues of the Bausch & Lomb Optical Company of
Rochester, and of the Spencer Lens Company of Buffalo, it is stated

CH. IV]

THE POLARIZING MICROSCOPE

OCULAR

201

that the tube-length is 160 millimeters and, as shown in the accom-
panying figure (fig. no), it includes the parts from the upper end of
the draw-tube to the nut into which
the objective is screwed.

The cover-glass thickness is given
as o.i 8 millimeter, and the user is
warned that for the higher powers
a variation in thickness from this
standard of 0.03 or 0.04 mm.
would deteriorate markedly the per-
fection of the image. The state-
ment is furthermore made that
with the homogeneous immersions
no harm would result from varying
thickness of cover-glass, but on the
other hand great care must be
exercised there to use the correct
tube-length or aberrations will be
introduced.

§ 266. Table showing cause of spherical aberration in the microscope
and means of correction. —

OBJCCTIVK

•DENSER

FIG. 1 10. THE MICROSCOPE
SHOWING TUBE-LENGTH

Under-correction produced by:

1. Too weak a concave element in the

objective.

2. Too close an approximation of the

lenses of the objective.

3. Too short a tube, that is, the ocular

and objective are too close to-
gether.

4. Use of too thin a cover-glass.

Over-correction produced by:
i a. Too strong a concave element in

the objective.
2a. Too great a separation of the

lenses of the objective.
3a. Too long a tube, that is, the ocular

and objective are too far apart.

4a. Use of too thick a cover-glass.

Any defect can be neutralized by applying the right amount of
what would produce the opposite condition. For example, the over-
correction produced by too thick a cover-glass can be corrected by:
(4) using a thinner cover-glass; (3) shortening the tube; (2) putting
the lenses of the objective closer together; (i) using a weaker con-
cave element in the objective.

202 THE POLARIZING MICROSCOPE [Cn. IV

If there is under-correction from too short a tube it can be neutral-
ized by: (3a) lengthening the tube; Ua) using a thicker cover-glass;
(aa) separation of the lenses of. the objective; (la) using a stronger
concave element in the objective. And similarly with under-
correction or over-correction from any cause; opposites neu-
tralize.

§ 257. Chromatic aberration. — Spherical aberration which has
just been discussed is present in lenses even when the light is of one
wave length; chromatic aberration, on the other hand, appears in
addition when composite light traverses a lens. That is, every wave
length of necessity is differently refracted; the shortest waves most,
the longest waves least. If then a single beam of white light trav-
erses a lens, the different wave lengths will be refracted differ-
ently and the blue-violet waves made to cross the axis first, the red
waves last. There will be then a series of colored foci extending
along the axis, as shown in fig. in. Every simple lens, then, whose
aperture is filled with composite light, will show both spherical and
chromatic aberration, and the greater the aperture and the shorter
the focus the more pronounced will be both forms of aberration. In
order that perfect images may be produced, both aberrations must
be eliminated.

Fortunately the visible spectrum does not include a greater range
of wave lengths (fig. 93), and if it were markedly less, the optician
would find his task greatly lightened. As shown in fig. 210, the
brightest region of the spectrum to the eye is really limited, and the
old opticians made good instruments for visual purposes by over-
coming the aberrations in large part in this very limited region; but
with the requirements of photography and for the most complete
visual study of the phenomena and objects of nature by means of
optical instruments, greater and still greater demands were made for
optical instruments including at least the whole visible spectrum,
and for some purposes extending into the infra-red and the ultra-
violet.

§ 258. Correction of the aberrations of lenses. — From the very
law of refraction bound up with the different wave lengths of visible
light it would seem impossible to obtain the refraction necessary to

CH. IV]

THE POLARIZING MICROSCOPE

203

produce images (figs. 105-111) without at the same time dividing the
light up into its colors. If the refraction of each wave length were
in exact proportion to its length,
as with a diffraction grating, it
would be impossible to pro-
duce achromatic images. Newton
thought the refraction was always
as with a grating, and he ex-
plained the satisfactory images
produced by lenses on the ground
that the narrow part of the
spectrum most brilliant to the eye
overwhelmed the dimmer parts
so that the colored images on
both sides of the visual image were
ignored.

If one compares, however, the
spectrum produced by the diffrac-
tion grating (fig. 121) with that
produced by a glass prism (fig.
122), it will be seen that the
refraction of the different wave
lengths (dispersion) differs very
markedly in the two cases, al-
though the total length of the spectrum is the same in both.

The red is much contracted and the blue expanded with the glass
prism. One can then have what might be called a mean refraction
with the glass prism, the refraction of the individual groups of wave
lengths not being in proportion to the lengths. Now it is from this
irregularity of the refraction in different parts of the spectrum, and
because the irregularity differs with different transparent substances,
that it is possible to have the refraction necessary to produce images
without having the light dispersed into colors at the same time.
This is shown in fig. 112, 2, where a smaller prism of flint glass pro-
duces the same amount of dispersion as a larger prism of crown glass.
If these prisms are with their edges opposite, the spectrum produced

FIG. in. CHROMATIC ABERRATION
WITH COMPOSITE LIGHT.

White light A beam of white light
composed of all the colors meeting a
lens and the different wave lengths
being differently refracted breaks
the composite light up into its con-
stituent colors.

Red Blue The long waved red
light is less refracted than the
shorter waved blue light. After
crossing at the foci the blue light is
on the outside of the diverging
cone.

fb,fr The focus of the blue light

b) is nearer the lens than the focus
of the red light (fr).

Axis The optic axis of the lens.

The dispersion or separation into
colors differs with different trans-
parent substances, and is not in
proportion to the mean refraction.

2O4

THE POLARIZING MICROSCOPE

[CH. IV

by the flint glass will be brought together by the crown glass and
white light will result; but as the mean refraction of the larger

FIG. IT 2. ACHROMATISM BY COMBINING DIFFERENT KINDS OF GLASS.

(1) White light (W) traversing two equal crown glass (CC) prisrns with their
bases opposite. The dispersion into a spectrum by the first prism is overcome by
the second prism and the light is recombined into a white beam (W1), which is
displaced as if it had traversed a piece of plane glass.

Red Blue The red and the blue edges of the spectrum. The blue is more
refracted than the red.

(2) White light (W} traversing a flint glass prism (F) and being dispersed into
the spectral colors. The spectrum formed by the flint prism is recombined by the
crown glass prism (C), but the emerging ray of white light (W2) is refracted mark-
edly toward the base of the crown glass prism, showing the possibility of an
achromatic image. The arrows show the direction in which the light is extending.

crown glass prism is greater than that of the flint glass prism , the
ray of white light will not extend parallel with the original direction,
but be bent toward the base of the crown glass prism. As a lens
may be considered an infinite number of prisms combined, it be-
comes intelligible from this how it is possible to produce colorless
images by combining flint-glass concave and crown glass convex
lenses; or other pairs of lenses where the dispersion and refraction
give comparable results.

In making the color corrections for the lenses, the spherical cor-
rections were also made; the extent of both corrections attained up
to the present is discussed below.

§ 259. Corrections in Achromatic and Apochromatic objectives. —
(i) Spherical aberration. In achromatic objectives the spherical
aberration is corrected for one color only, in apochromatic objectives
for two colors. (2) Chromatic aberration. In achromatic objectives
correction is made for two colors; in apochromats for three colors.

Cn. IV] THE POLARIZING MICROSCOPE 205

In the apochromats it was found impossible to make the high
corrections necessary even with all the new glasses made available

FIG. 113. ACHROMATIC COMBINATIONS OF CROWN AND FLINT GLASS LENSES.
(From Lewis Wright's Optical Projection).

CCCCCC Thin edge or converging crown glass lenses.

F F F F F F Thick edge or diverging flint glass lenses. The flint glass over-
comes the dispersion without overcoming the mean refraction, hence all these
combinations are converging.

by the Jena glass works; but with the new forms of glass and a
natural mineral, fluorspar, fluorite, calcium fluoride, with its very
low index of refraction and small dispersion, it was found possible to
make the fundamental advance in microscope objectives represented
by the apochromatic objectives.

The possibility of bringing three colors to one focus makes the
apochromatic objectives especially valuable for photography. The
visual and actinic foci are coincident, and if the apparatus is well
constructed, there is never any difficulty in getting sharp pictures,
for the photographic image is sharpest when it appears sharpest to
the normal eye.

§ 260. Compensation oculars. — As the front lens of objectives of
high power (figs. 20, 21) is not a combination but a single lens,
aberrations are inevitably introduced which must be eliminated by a
subsequent part of the optical train. The most striking and trouble-
some defect is the so-called difference of chromatic magnification,
that is, the differently colored constituent images forming the final
image are of different magnitudes, the blue one being larger than the

2O6

THE POLARIZING MICROSCOPE

[CH. IV

red one. This defect is more easily corrected in the ocular than in
the subsequent combinations of the objective. The ocular is then
constructed to give a red image sufficiently large to bring its mag-
nification up to that of the blue image, and hence the final image as
seen by the eye is correct.. The low power apochromats could be
corrected for this, but for the sake of using the same oculars on all

FIG. 114. POSITIVE COMPENSATION OCULAR.
(From Spitta, p. no).

C F C The field lens is composed of two double convex crown lenses and one
double concave, flint glass lens.

C The eyelens is of crown glass, and is separated from the field combination
the right distance to give the necessary excess magnification of the red image to
make it balance the blue image which was over magnified by the objective.

Red Blue The red and blue rays limiting the image. It is seen here that the
rays are not parallel but divergent, as they extend^ above the ocular. When pro-
jected by the eye to the virtual image, the rays cross, throwing the red one to the
outside, thus giving a larger image than is given by the blue ray, and the orange
haze at the margin of the field when looking through the ocular toward the win-
dow or the sky.

§ 259a. It is interesting to note that the wonderful optical qualities of fluor-
spar were known to Sir David Brewster, and recommended by him for aid in
achromatization (Brewster's work on the microscope, 1837, P- m); and before
1860 our own Charles A. Spencer used fluorspar in one of the combinations of his
objectives (Proc. Acad. Nat. Sci., Phila., Vol. LVI (1904), p. 475J Trans. Amer.
MJcr, Soc.; 1901, p. 23).

CH. IV]

THE POLARIZING MICROSCOPE

207

powers the defect is left or purposely introduced into all the apochro-
mats. It will be seen from the above statement that for projection
or for photography the apochromats cannot be used satisfactorily
without the ocular to complete the corrections. (See figs. 114-115.)
The over-correction of the ocular necessary to give the greater
magnification to the red constituent of the image leads to the posi-
tion of the red on the outside of the projected (virtual) beam; hence

FIG. 115. HUYGENIAN OCULAR SHOWING THE ORDINARY AND THE COMPENSATING

ACTION.

(From Spitta, p. 106).

Ordinary action. (H).

If the rays are traced on the left, it will be seen that the field lens (C) brings
the rays to a focus at the diaphragm (D), and that they cross and pass on to the
eyelens slightly divergent; but in passing through the eyelens (C), the red and
blue constituents are made parallel to each other, and are projected into the field
of vision in close parallel (virtual) bundles and hence appear achromatic.

Compensating action (C).

For this the field lens is of flint glass (F), and the eyelens of crown glass (C).
Or the eyelens may be an over-corrected combination. The result is the same,
viz., the red image is magnified more than the blue image by the ocular, and this
balances the excess magnification of the blue image by the objective, and in the
projected (virtual) image the red is on the outside, producing the orange haze
at the margin of the field when looking through the ocular, toward a window, or
the sky.

208 THE POLARIZING MICROSCOPE [Cn. IV

in looking through a compensation ocular toward the window or the
sky, an orange haze appears around the margin. As the ordinary
Huygenian ocular has an under-corrected eyelens, the blue constit-
uent will be on the outside of the projected (virtual) image and
there appears a blue haze around the edge of the field (Spitta, pp.
112-113).

ANGULAR AND NUMERICAL APERTURE

§ 261. Angular aperture. — By this is meant the angle of light
which passes from the object to the objective and becomes effective
in producing the microscopic image (fig. 116). It has been known for
a very long time that the clearness of the image, other things being
equal, depends upon the width of the angle of light coming from the
object; and that the resolution of details depends very largely upon
the angular aperture of the objective. The difficulty of overcoming
the aberrations also becomes greater as the angle is increased; and
it was the triumph of the early American opticians, Spencer and
Tolles, that they were able to make the corrections for high powers
with very large angular aperture.

§ 262. Numerical aperture. — With the introduction of immersion
systems into modern microscopy, it was seen and pointed out with
great distinctness by Spencer and Tolles that the aperture of such
immersion objectives might exceed 180° of light in air. For the
average microscopist, however, this seemed an impossibility. By
referring to figs. 100-102 the matter becomes very easily intelligible,
for it is seen that light in water in passing into air spreads out so that
an angle in water of 48° 45' on each side of the normal (97° 30')
spreads out into an angle of 180° in air; therefore light at an angle
of 97° 30' in water is equal to 180° in air, and if the water immersion
objective receives and transmits for the formation of the image an
angle of light in the water greater than 97° 30', its angle is greater
than an air angle of 180°. The critical angle for glass to air is 41° on
each side of the normal, and a total "angle of 82° in the glass would
spread out to form the whole 180° in the air. Therefore, if with
homogeneous immersion objectives an angle above 82° is transmitted

CH. IV]

THE POLARIZING MICROSCOPE

209

by the objective for the formation of the image, the angle is so much
greater than 180° in air.

The confusion was reduced to order by Abbe, to whom makers
and users of optical instruments owe so many
debts. He applied the simple laws of trigonom-
etry, using the sine function of the angle,
and taking into consideration the medium of
the lowest refractive index between the object
and the objective. If it were air, unity was
taken; if water, the index of water — 1.33; if
glass, 1.52; and if any other immersion fluid,
the refractive index of that fluid. By thus
considering the index of refraction of the
medium immediately in front of the objective,
it becomes possible to make comparisons
which are rigidly exact, and express in terms
which do not seem to be impossibilities, like an
angle in excess of 180° entering a flat surface.

The nomenclature introduced by him and
now universally employed is Numerical Aper-
ture, and includes in its significance both
the angle of the light and the index of refrac-
tion of the medium from which the light passes
into the objective. The formula is N.A. =
n sin ut in which n is the index of refraction
of the air for dry, the water for water immersion and the cedar oil
for homogeneous immersion; and u, is the sine of half the angle of
the light entering the microscope objective, no matter what medium
is between the object and objective.

As there are three factors in this formula, if one knows any two of
them the third is readily found.

§ 264. Significance of numerical aperture. — It is now universally
agreed that, the corrections in chromatic and spherical aberration
being the same, the power to define minute details depends directly
on the numerical aperture; the greater the numerical aperture, the
greater is the resolution (see also §§ 271-272).

FIG. 1 1 6. ANGULAR
APERTURE OF AN OB-
JECTIVE.

Axis, The princi-
pal optic axis of the
objective.

B The object just
outside the principal
focus.

ADC Diameter of
the front of the objec-
tive and base of the
angle of aperture.

A B D Half the
angle of aperture (u)',
AD representing the
sine of u (see § 262).

2IO

THE POLARIZING MICROSCOPE

[CH. IV

§ 264a. Table of the usual group of American objectives with their numerical

aperture (N.A .) and the method of obtaining it. Compiled

from the manufacturers1 catalogues.

Achromatic
objectives with
initial magnifi-
cation at 1 60 mm.

Angular
aperture

(3»)

Natural --ine
of half the
angular aper-
ture (u)

I if lex of
refraction
of the me-
dium in
front of the
objective

Numerical

(N.A. =

aperture
n sin «)

32 mm. (x4)

n°3o'

sin 5°45'
= 0.10019 sin u

;i = i. oo

N.A. = n sin

u — o.io -\-

16 mm. (xio)

29°

sin i4°3o'
= 0.25038 sin u

n = i.oo

N.A. = n sin

u = o 25 -f

8 mm.

(X2I Or X20)

60°

sin 30°
= 0.5000 sin u

n = i.oo

N.A. = n sin

u = o 50

4 mm.

(X43 or 44)

83°

sin 4i°3o'
=* 0.66262 sin u

n - i.oo

N.A. = n sin u = 0.66 -j-

3 mm.
(x6o)

ii6°3o'

sin 5 8° 1 5'
- 0.85035 sin 11

n =1.00

N.A. = n sin

u = 0.85 +

i.o mm. 1.8 mm.
oil immersion
(xpy or X95)

iio°3o'

sin 55°i5'
— 0.82165 sin u

n - 1.52

N.A. = n sin

U =s I 25 —

Table of a dry, a water immersion and a homogeneous immersion objective

to give a comparison of the angular aperture required

in each to give a uniform N.A. of 0.50.

Dry Objective

60°

sin 30°
= 0.5000 sin u

n = i.oo

N.A.

= n sin u — 0.50

Water immer.
Objective

44° 20'

sin 22°io'
= 0.37594 sin u

n = T.33

N.A.

= n si.i u — o 50

Homo, immer.
Objective

38°28'

bin 1 9° 1 4'
— 0.328947 sin u

n - 1.52

N.A

= n sin u — 0.50

§ 264b. The values for the index of refraction: n, i.oo for air; n, 1.33 for water;
and n, 1.52 for homogeneous immersion liquid used in determining numerical
aperture, are not strictly accurate nor are the sines and numerical apertures; they are
approximate round numbers. It will be seen also that in each case the sine of half
the angle of aperture may be found by dividing the N.A. by the index of refraction
(n) of the medium in front of the objective, for air by i.oo, water by 1.33, and
homogeneous liquid, 1.52, It follows also that with dry objectives the N.A. will
always be the sine of half the angle of aperture.

§ 265. Why a homogeneous immersion condenser is required. — If the

definition of minute details requires adequate numerical aperture, it is evident
that it is of fundamental importance that the substage condenser be able to
supply the light at the adequate aperture.

CH. IV] THE POLARIZING MICRSOCOPK 211

Assuming that the substage condenser is properly constructed, the
question is, can it illuminate the object with the proper numerical
aperture? ^

By referring to § 262, and to figures 100-102, it is evident that an
object mounted on a glass slide and separated from the condenser by
a stratum of air can get light from the condenser only up to the
critical angle, that is 41°, on each side of the normal, or a total of 82°,
corresponding to a numerical aperture of i. The objective maybe capa-
ble, however, of receiving and utilizing a numerical aperture of 1.40.

If now the condenser also has a numerical aperture of 1.40 and it
is connected to the slide by means of homogeneous immersion liquid,
the entire aperture will illuminate the object and can enter the
homogeneous immersion objective.

If the substage condenser is in immersion contact with the glass
slip by means of water, then, as shown in figs 100-102, 73, the object
can be illuminated with a light cone of 122°, that is, an aperture of
n sin «°, in this case 1.52X0.875 = 1.33 N.A. If the greatest
possible aperture is required, as in dark-field illumination (§ 190)
and for some of the most exacting work with the bright-field micro-
scope, then the condenser must be in homogeneous immersion con-
tact with the glass slip (figs. 73, 84).

§ 266. Determination of the aperture of objectives with an aper-
tometer. — Excellent directions for using the Abbe Apertometer may
be found in the Jour. Roy. Micr. Soc., 1878, p. 19, and 1880, p.
20; in Dippel, Czapski and Spitta, Chapter XIV. The following
directions are but slightly modified from Carpenter-Dallinger, pp.
394-396. The Abbe apertometer involves the same principle as that
of Tolles, but it is carried out in a simpler manner; it is shown in
fig. 117. As seen by this figure it consists of a semicircular plate of
glass. Along the straight edge or chord the glass is beveled at 45°,
and near this straight edge is a small, perforated circle, the perfora-
tion being in the center of the circle. To use the apertometer the
microscope is placed in a vertical position, and the perforated circle
is put under the microscope and accurately focused. The circular
cd^e of the apertometer is turned toward a window or plenty of
artificial light so that the whole edge is lighted. When the objective

212 THE POLARIZING MICROSCOPE [Cn. IV

is focused on the perforated circle, the draw-tube is removed and
in its lower end is inserted the special objective which accompanies

FIG. 117. ABBE APEKTOMETER.

As shown in the figure the face bears two series of figures. Those at the top
give the numerical aperture, and the lower ones give the angular aperture. It
will be noted that there is no angular aperture greater than that represented
by a numerical aperture of i , the sine of 90°.

the apertometer. This objective and the ocular form a low power
compound microscope, and with it the back lens of the objective,
whose aperture is to be measured, is observed. The draw-tube is
inserted and lowered until the back lens of the objective is in focus, —
" In the image of the back lens will be seen stretched across, as it
were, the image of the circular part of the apertometer. It will
appear as a bright band, because the light which enters normally at
the surface is reflected by the bevel part of the chord in a vertical
direction so that in reality a fan of 180° in air is formed. There are
two sliding screens seen on either side of the apertometer; they
slide on the vertical circular portion of the instrument. The images
of these screens can be seen in the image of the bright band. These
screens should now be moved so that their edges just touch the periphery
of the back lens. They act, as it were, as a diaphragm to cut the fan
and reduce it, so that its angle just equals the aperture of the objec-
tive and no more."

Determination of numerical aperture (N.A.} by means
of a thick plate glass. (H. P. Gage.')

For this the apparatus needed is: (i) A microscope with the ob-
jectives and the condenser to be tested.

CH. IV] THE POLARIZING MICROSCOPE 213

(2) A strong illuminating device like one of the dark-field lamps
(figs. 79-82, 46), or direct sunlight may be used if it is available.

(3) A slip of plate glass 5 to 10 mm. thick and face of 37 X 75
mm. One face of the glass should be given a matt surface with the
finest carborundum or emery (§ 95a). The other face should be left
smooth.

(4) A pinhole, opaque disc about 10 mm. in diameter should be
cemented to the middle of the smooth surface with Canada balsam.
This then should be covered, and in balsam, something like a tissue
section (§ 533). Tin foil or dense black paper may be used for the
opaque disc, and a sewing needle or small pin can be used to make
the central opening.

(5) Fine dividers and a scale such as is used in determining the
magnification of the microscope (§ 364) for measuring the diameter
of the light cone.

The thickness of the glass slip should be known. It is most
easily and accurately determiried by one of the micrometer calipers
(figs. 219-220). The refractive index of the glass slip must also
be known. If a refractometer is at hand, it takes but a few minutes
to find out the refractive index. If a refractometer is not available
an index of 1.515 maybe assumed as a sufficiently close approximation.

§ 267. Obtaining the data for determining the N.A. of objectives.
— Place the slip of plate glass on the stage of the microscope with
the pinhole disc up. Focus the pinhole with the objective to be
tested, then clamp the slip so that it will not move. Make the
microscope horizontal. Remove the ocular and point the tube di-
rectly toward the lamp. Remove the condenser and the mirror.
Mutually arrange the lamp and the microscope till there is seen a
circle of light on the ground surface of the slip. With the fine ad-
justment, focus sharply the pinhole. One can tell when the sharpest
focus is gained by the diameter of the circle of light for then it will
be greatest. Measure the diameter with the dividers. If one works
at night or in a darkened room greater exactness will be possible.

If a homogeneous immersion objective is to be tested, homo-
geneous liquid must be used to make immersion contact with the
cover-glass.

214 THE POLARIZING MICROSCOPE [Cn. IV

The data thus obtained give all that is needed for finding the
numerical aperture, n sin «=N.A., for the diameter of the light cone
gives the diameter, A-C., of the cone and hence the base of the
angle (fig. 116). The thickness of the plate glass gives the height
(fig. 116, A-C, B-D}. The refractive index by observation is 1.515.

§ 268. Aperture of a condenser. — For this the plate glass slip is
turned over bringing the pinhole down. It is put in immersion
contact with the top of the condenser. The microscope is made
vertical, and with the plane mirror a strong light is reflected to the
condenser. The pinhole should be at the focus of the condenser.
To do this it may be necessary in order to get sufficient distance
between the top of the condenser and the pinhole, to place a glass
slip under the plate glass. The slip must be in immersion contact
both with the plate glass and with the condenser. It is well to try
first a slide not over i mm. thick. In order to make sure that the
pinhole is at the focus of the condenser, the body tube of the
microscope is removed and light is thrown straight down through
the plate glass and pinhole to the condenser. By turning the plane
mirror at the proper angle the image of the pinhole will be seen and
one can tell whether or not the pinhole is in focus by the sharpness
of the edges. If it is not in focus because too low, then a glass slip
must be added. If one has already added a glass slip the pinhole
may be too high.

Measuring the diameter of the light cone. It is not easy to see
the light cone by looking directly down for the light in line of the
pinhole is so brilliant. By looking obliquely the border of the cone
can be seen.

§ 268a. Determination of the N.A. after the above data have been
secured. — Referring to fig. 116, let BA and BC be the limiting rays
of the light cone from the objective or condenser after passing the
pinhole. The thickness of the plate glass, BD, and the diameter of
the bright disc A.C. have been measured.

The angular aperture of the objective or condenser in glass is

ABC, and the half angle is ABD. — is the tangent of this half

CH. IV] THE POLARIZING MICROSCOPE 215

angle, i.e., — , or if the thickness of the glass is t and the diameter of
2

a' d

the light spot (AC) d, then tangent - equals — • From trigonometric

a' a'

tables the value of sin — is found corresponding to tangent — . This
2 2

is multiplied by the index of refraction, and the result will be the
numerical aperture.
Examples. Suppose the thickness of the plate glass slip is 10 mm.;

a 5
the diameter of the light disc 5 mm., then tan - is or

2 IO X 2

.25. The angle whose tangent is .25 is i4°2/ and the sine of this
angle is .2425. If this is multiplied by the refractive index of the
plate glass: .2425 X 1.515, equals .3673, the numerical aperture of the
dry objective in glass. To find the angular aperture in air, find
the angle corresponding to the sine .3673. It is the sine of 21° 33',
that is, half the air angle. If this is multiplied by 2 there will
result the total air angle (21° 33' X 2 = 43° 6')-

In an actual experiment it was found that the thickness of the
plate glass was 6.7 mm.; its refractive index 1.5135. The diameter
of the disc of light obtained from a condenser in immersion contact

22.4
with the plate glass, is 22.4 mm. Then as above: — equals

2 X 0.7

1.672, which is the tangent of 59° 7'. The sine of 59° 7' - .8582.
This multiplied by the refractive index of the plate glass gives:
.8582 x 1.5135 equals 1.30, the N.A. of the condenser. It will be
noted that the ray proceeding at an angle of 59° 7' from the axis in
glass would be totally reflected at the glass surface and would never
have got into the plate glass in the first place if it had not been in
immersion contact with the condenser. Also the sine of 59° 7', .8582
multiplied by the refractive index of the glass gives a number, 1.30,
that is, greater than unity, greater than is possible for any angle,
and no ray corresponding to this can exist in air.

§ 269. Refractometer tests upon various liquids. — In order to
investigate adequately the optical properties of the liquids used for

2l6 THE POLARIZING MICROSCOPE [Cn. IV

homogeneous immersion and other purposes in microscopy, it is
necessary to employ a refractometer, and to test all at the same
temperature. The one used by the writer for the data given in the
following table was loaned to him by Dr. Chamot of the chemical
department.

The refractive indices are all at 20° centigrade and for the D or
sodium line of the solar spectrum (fig. 123); and the average separa-
tion into colors between the lines F in the green-blue, and C in the
red of the solar spectrum.

For homogeneous immersion liquid nothing has been found up to
the present as satisfactory as thickened cedar-wood oil from Junipe-
rus mrginiana. Practically all modern homogeneous immersion objec-
tives are designed for use with this immersion liquid, which has an
average refractive index at the D line of: nD = 1.51565. The av-
erage dispersion of these 8 samples is: vF — vC ~ 0.01080.

The first substance purposely employed for homogeneous immer-
sion with objectives by Tolles was Canada balsam from the balsam-
fir (Abies balsamea). Its index of refraction is somewhat greater and
its dispersion somewhat smaller than that of thickened cedar-wood
oil, but in case cedar oil is not available it might still be used with
successful results.

It will be seen by consulting the table that there is no single
liquid which can take the place of cedar-wood oil for immersion
purposes. Different workers have found the viscidity of the cedar
oil a disadvantage for, in examining preparations in thin liquids,
the cover-glass is likely to be pulled about by the adhesion of the'
cedar oil to the objective. In looking for a substitute in which the
viscosity would be less, the heavy mineral oils of the paraffin and
naphthalene series have come into use (§ 309). Their refractive in-
dex and dispersion are somewhat different from cedar oil so that they
do not make a perfect substitute alone, but mixed with alpha-bromo-
naphthalene they can be brought to the same refractive index, but
not exactly to the same dispersion. Such a mixture answers for most
purposes and they have the advantage of not being volatile and of
having little viscidity. Their odor, however, is not so pleasant as
cedar oil. In passing, it might be said that the homogeneous im-

CH. IV]

THE POLARIZING MICROSCOPE

3X7

mersion objectives may be used without any immersion liquid, or
with water, with castor oil, glycerin, etc. It should never be for-
gotten, however, that for the best effects one must employ an im-
mersion liquid for which the optician corrected the objective.

Table Showing the Index of Refraction at the D line ("D} and the Mean Dis-

n—

persion (nF-nC) between the Fixed Lines F and C, and the v-valite

With Various Homogeneous Liquids and Other Substances.

Name of Substance

Index of
Refraction

Mean Dis-
persion

v- Value of
Dispersion
»D-i

(»/))

(nF-nC)

»F-nC

(A) Homogeneous Cedar Oil

I ^IQO

o 01125

46.13

(B) " " "

5l6l

o 01072

48. 14

.<a66

o 01065

48 50

(D) " " "

.5130

o 01089

47. 10

(E) " " "

.5132

o 01066

48.13

(F) " " "

. Ci2Q

o 01082

4.7 37 -4-

(G) " " "

.5145

o 01085

4.7 .4.2 —

(Ga) " " " . ..

( . *i78cr.

{0.01070

/4.8 40

Averages for Cedar Oil

\ 5i78cl.
.51565

o 01066
0.01080

148 52
47-74

(H) BrN 18 % in mineral oil, Naph. .
(J) BrN 17.25% in mineral oil, P..
Aqua distillata (II >O)

-5152
•5i5i
.33338

0.01082
o 01270
0.00582

47.60
40-55
57-44

(Tap water) (H2O)

.33365

o 00626

53.30

Alpha-Bromo-Naphthalene (d0H7Br.)
Canada balsam, Pennock's paper-
filtered

•5586

. t>2O2

o 013485
o 00958

41.40
54 30

Canada balsam thinned with xylene
Carbon tetrachlorid (CC14)

.51578
.4614

0.00928
0.00983

55 58
46 89

Castor oil (Oleum ricini) , . .

4.7QC

o 00892

C2 7Q

Cedarwood oil (Florida extra)

• S°35

0.01064

47 32

Cedarwood oil very thick ....

. "s2O^

o 01072

48.53

Chloroform (CHC13)

,4462

0.00892

50.02

Clove oil (Oleum caryophylli) old . .
Glycerin (C3H5(OH)3)

•5399
.4720

0.01725

0.00816

3I-30
57-73

Nujol (mineral oil)

.4789

o 00885

54.11

Petrolatum liquidum, Parf

.48525

o 00875

55-47

Petrolatum liquidum, Naph

.4840

o 00898

53.89

Sandal wood oil

.52

O.OII

Turpentine (commercial)

•4749

0.01071

44 3°

Xylene (CgHio) pure

i . 4965

0.01532

32.41

Xylene (commercial)

I . 4934

0.01533

32.12

If the immersion cedar oil gets too thick, the best substance for
thinning it is the thin cedar-wood oil (Florida extra).

2i8 THE POLARIZING MICROSCOPE [Cn. IV

§ 270. Diffracted light in microscopy. — As most microscopic ob-
servation depends upon directed light from some source like the sun
or a lamp sent to and through the object by a mirror only or by the
aid of a condenser or a mirror and condenser, the phenomena of
diffraction are present. It is evident that if the objects observed
were self-luminous the conditions would be different from those
existing when the object must be viewed with direct light from some
outside source.

In traversing small orifices or slits and objects with minute details
the spreading out of diffracted light is a necessary accompaniment.
The diffracted rays are shown by broken lines in the accompanying
figures from Wright (fig. 118). As seen from these, there may be
two systems of diffracted rays, one from the object and another
from the border of the objective, and these two systems of diffracted
rays act differently.

The r61e played by the diffracted light has been variously inter-
preted by opticians. By Abbe and his adherents diffracted light is of
supreme importance, and microscopic vision is a thing by itself (sui
generis) and not to be interpreted by ordinary geometric optics.
Certain very striking experiments have been devised to show the
accuracy of this hypothesis, but, as pointed out by many, the or-
dinary use of the microscope never involves the conditions realized
in those experiments.

While the supreme importance ascribed by some to the diffracted
light may not be accepted, no one will deny the presence of diffrac-
tion phenomena in microscopic vision. If, furthermore, the dif-
fracted rays are brought by the microscope to the final focus with
the undiffracted light passing from the object through the micro-
scope, the image will be conceivably more perfect than as if the
diffracted rays produce secondary images, or mere blur.

§ 271. Depths of focus and aperture. — It is known to all workers
with the microscope that with objectives of low aperture it is pos-
sible to change the focus rather markedly up or down without
seeming to lose in sharpness, while with objectives of great aperture
a sharp focus is almost immediately lost in focusing up or down
beyond a point. The reason for this is made strikingly evident by

CH. IV]

THE POLARIZING MICROSCOPE

219

FIG. n 8. DIFFRACTED LIGHT IN MICROSCOPY.
(From Wright's Principles).

Object (grating) lighted with a narrow beam (/) from the condenser and giving
off diffracted rays which are brought to a focus with the dioptric beam (I) above
the objective in part (full lines); and in part forming diffracted beams on each
side above the objective (broken lines). These diffracted beams not brought to
the same focus as the dioptric beam cause imperfections or confusion in the image.

Small diaphragm (C D) below the condenser focused on the grating, A B, and
from this point the dioptric beam (solid white) and diffracted light (broken lines)
extend through the objective and finally focus at B' A'. By looking at the eye-
point with a magnifier the image of the back lens shows not only the diaphragm
image (Dr C'), but secondary images of the same (Df C* and D" C). See small
figure in the middle also.

22O

THE POLARIZING MICROSCOPE

[Cn. IV

FIG. 119. EFFECT OF APERTURE ON DEFINITE Focus AND ON OVERCOMING

OPACITIES.

(From Wright's Principles of Microscopy, p. 77).

(1) To show the definiteness of the focus (/) with a large aperture. Either
above or below this is a large diffusion circle (a b) due to the size of the section of
the aperture.

(2) Indefiniteness of the focus due to the fact that a cross section of the aper-
ture considerably above or below the true focus (/), gives so small a diffusion
circle (a or 6), that it can hardly be distinguished from the true focus.

(3) Low aperture and an opacity in the path of the light. Tt is so large rela-
tively here that a clear image would be impossible.

(4) The same opacity in a larger aperture.

(5) The same opacity in a still larger aperture. There is now enough of the
beam outside the opacity to make the object visible.

CH. IV] THE POLARIZING MICROSCOPE 221

fig. 119 i, 2. Let /be the most perfect focus; if one turns to a or
b the appearance is almost unchanged in the low apertured objective
(2), but the diffusion circle is very marked in the high apertured
objective (i). Furthermore, the brilliancy of the image must be
markedly greater with the larger aperture (Wright, p. 77).

§ 272. Aperture and the effect of opacities. — Between the retina
and the object there are many possibilities of opacities in the image-
producing beam of light — for example, the eye lashes, particles of
dirt in the tears over the cornea, besides particles on the glass sur-
faces. Figure 119 3, 4, 5 show graphically the relative obscuration
which must result with the same opacity in beams of different aper-
ture. In (3) the shadow is so great that almost the entire aperture is
obscured, and vision made difficult or impossible. In (4) with a
larger aperture the shadow is not so overwhelming, and in (5) with
the large aperture there is still possibility of fairly good vision in
spite of the shadow.

It is believed that the inevitable narrowing of the beam in high
power magnification and the presence of opacities in the eye form
the bar to resolution, and that if the apparatus and the eye could, on
the one hand, be free from opacities to throw shadows and thus
obscure the image, or on the other hand the terminal beam could be
opened up to make the aperture greater, the eye could discriminate
beyond the limits heretofore ascribed to it (Wright, Ch. XVI).

As the higher the power of the ocular the smaller is the eyepoint
(figs. 24-25), it is evident that any obscurities have a greater effect
with the high ocular. The rule to use as low an ocular as possible
is a good one to follow with bright field illumination (Wright, p.
227).

For dark-field illumination, the high power oculars are mostly
better (§ 181).

Consult Carpenter-Dallinger and Beck, Part II, Chamot and Spitta,
and Sir. A. E. Wright for further information.

CHAPTER V

MICRO-SPECTROSCOPE; POCKET SPECTROSCOPE
§§ 273-302; FIGURES 120-124

RADIATION FROM THE SUN AND OTHER SOURCES

§ 273. Visible and invisible radiation. — From any primary
source of light-energy like the sun, the electric arc, etc., not only is
given off the energy which to the eye is appreciated as light, but
wave lengths of energy both longer and shorter than those affecting
the eye. As shown in fig. 93, the segment of the energy spectrum
which is visible to the eye is exceedingly limited, being included
between about Xo.4/x and \O.^/JL. Under special illumination, waves
shorter than Ao.4/4 and longer than Xo.7/z can be seen, but the
extension into the infra-red or the ultra-violet is slight, and is not
used for ordinary visual purposes.

It is fortunate for optical instruments that the visible spectrum is
so limited. Indeed, if the visible spectrum were even more limited,
as shown by the use of monochrorratic light, it would be easier to
obtain perfect images, for the aberrations arising from the different
wave lengths would be avoided.

The spectroscope has for its object the giving of information con-
cerning the visible spectrum, and it has proved of great help indeed.
It should not be forgotten, however, that the color effects produced
by the spectroscope are not the only ones and in some ways not the
most important. What it really does is to divide the wave lengths
into groups, and in absorption phenomena the important thing
is that some wave lengths are not present or are cut out by the
absorbing medium and hence there are present dark bands in the
spectrum (absorption bands). These absorption bands could be seen
and their significance appreciated by a person wholly color blind
— and there is occasionally such a person.

§ 274. A micro-spectroscope, spectroscopic or spectral ocular, is
a direct-vision spectroscope combined with a microscope ocular of

CH. V] MICRO-SPECTROSCOPES 223

the Huygenian form. At the usual position of the ocular diaphragm
is substituted a special slit mechanism. The spectroscope part of
the combination consists of an Amici prism of considerable dis-
persion placed in a tube hinged to the top of the ocular and fastened
by a spring. This makes it possible to swing the spectroscope aside
and look into the ocular in the usual way. In making spectroscopic
observations, the spectroscope is brought over the ocular in the line
of sight.

The spectroscope is made complete by the eyelens of the ocular
and the slit mechanism in place of the ocular diaphragm. This slit
should be parallel to the apices of the prisms which are located at
the focal point of the eyelens. Light traversing the slit is rendered
approximately parallel by the eyelens of the ocular.

At the diaphragm level is a prism for reflecting horizontal rays
vertically. This device is for a comparison spectrum side by side
with the spectrum of the object on the stage of the microscope.

Finally, near the top is a lateral tube with mirror for the purpose
of projecting a scale of wave lengths upon the spectrum under
observation.

In this Amici prism the excess dispersion is given by the flint
glass prism or prisms, and the parallelization by the crown glass
prisms; and following the rule that the shortest waves are bent
most, the colors have the position indicated in figure 124. But if
one looks into the direct vision spectroscope or holds the eye close
to the single prism (fig. 120), the colors will appear reversed as if the
red were more bent. The explanation of this is shown in fig. 120, 2,
where it can be readily seen that if the eye is placed at £, close to
the prism, the different colored rays appear in the direction from
which they reach the eye and consequently are crossed in being
projected into the field of vision and the real position is inverted.
The same is true in looking into the micro-spectroscope. The actual
position of the different colors may be determined by placing some
ground-glass or some of the lens-paper near the prism and observing
with the eye at the distance of distinct vision.

§ 274a. The author wishes to acknowledge the aid rendered by Professor
JS. L. Nichols in giving the explanation offered in § 274.

224

MICRO-SPECTROSCOPES

.1 *

FIG. 120. DIAGRAM OF A DIRECT-VISION MICRO-SPECTROSCOPE.

1 The spectroscope is shown in position on the microscope, the tube of the
microscope being much shortened to save space.

Stage, the stage of the microscope on which is a watch glass with sloping sides.

Objective The objective of the microscope.

S S' S" Screws for clamping the apparatus and for changing the position
of parts.

Slit The slit of the spectroscope between the ocular lenses in the position of
the ocular diaphragm, i.e. where the real image of the object to be examined is
formed.

Hinge The hinge on which the prism can be turned off the ocular.

Amid prism The direct- vision prism composed of a middle flint and two
crown-glass prisms.

Red Yellow Blue Arrangement of the colors as they emerge from the prism.

Scale tube and Mirror The mirror to throw light into the scale tube and
project an image of the Angstrom scale into the field,

2 Prism showing that with the eye close to the prism the colors seem re-
versed'from the position actually occupied.

3 Comp. prism The prism introduced under the slit and serving to reflect
up into the microscope a spectrum for comparison with that extending along
the axis of the microscope from below. C L Liquid in the tube whose spec-
trum is to be compared with that of the liquid in the watch glass on the stage
of the microscope.

4 The slit mechanism and comparison prism (/>).

5 S Set screws for changing the width and length of the slit.

CH. V]

MICRO-SPECTROSCOPES

225

VARIOUS KINDS OF SPECTRA

By a spectrum is meant the colored bands appearing when the
light traverses a dispersing prism or conies from a diffraction grating,
or is affected in any way to separate the different wave lengths of
light into groups. When daylight or some good artificial light is thus
dispersed one gets the appearance so familiar in the rainbow.

§ 275. Continuous spectrum. — In case a good artificial light,
as the electric light, is used, the various rainbow or spectral colors
merge gradually into one another in passing from end to end of the
spectrum. There are no breaks or gaps.

§ 276. Line spectrum. — If a gas is made incandescent, the spec-
trum it produces consists, not of the various rainbow colors, but of
sharp, narrow, bright lines, the color depending on the substance.

All the rest of the spectrum is dark. These line spectra are very

V B G Y O R

-^-T— * — r

FIG.

G F E *D C B

121. NORMAL SPECTRUM OF DAYLIGHT SHOWING THE SEGMENTS OF COLOR,
V B G Y 0 R, AND THE DARK LINES, HGFEDCB A.

In the normal spectrum produced by a grating the refraction is directly pro-
portional to the wave length of the light; here the red is a broad band and the
violet-blue narrow. (Compare the prismatic spectrum where the red is narrow
and the blue broad.)

Xo.4ju Xo.yju, the wave lengths between which the radiation is visible (see fig.
144).

V B G Y <

G F E D C B

FIG. 122. PRISMATIC SPECTRUM OF DAYLIGHT.

As glass does not refract the different wave lengths in direct proportion to their
frequency, the width of the bands of color are strikingly unlike those of a normal
spectrum. That is, in a glass spectrum the blue- violet forms a relatively broad
band and the red a narrow one.

226 MICRO-SPECTROSCOPES [CH. V

strikingly shown by metallic vapors heated to incandescence, e.g.
sodium. These spectra are usually obtained by heating some salt
of the substance (see § 287).

§ 277. Absorption spectrum. — By this is meant a spectrum in
which there are dark lines or bands. The most striking and interest-
ing of the absorption spectra is the Solar Spectrum, or spectrum of
sunlight. If this is examined by a good spectroscope it will be found
to be crossed by dark lines, the appearance being as if one were to
draw pen marks across a continuous spectrum at various levels,
sometimes apparently between the colors and sometimes in the midst
of a color. These are the so-called Fraunhofer lines. Some of the
principal ones have been lettered with Roman capitals, A, B, C, D,
E, F, G, H, commencing at the red end. The meaning of these lines
was for a long time unknown, but it is now known that they corre-
spond with the bright lines of a line spectrum. For example, if
sodium is put in the flame of a spirit or Bunsen lamp it will vapor-
ize and become luminous. If this light is examined there will be
seen one or two bright yellow bands corresponding in position with
D of the solar spectrum (figs. 121, 123). If now the spirit-lamp
flame, colored by the incandescent sodium, is placed in the path of
the electric light, and it is examined as before, there will be a con-
tinuous spectrum, except for dark lines in place of the bright sodium
lines. That is, the comparatively cool yellow light of the spirit-
lamp cuts off or absorbs the intensely hot yellow light of the electric
light; and although the spirit flame sends a yellow light to the spec-
troscope, it is so faint in comparison with the electric light that the
sodium lines appear dark. It is believed that in the sun's atmos-
phere there are incandescent metal vapors (sodium, iron, etc.), but
that they absorb the light from the sun which corresponds with
their own wave lengths, and hence the dark lines. If the incan-
descent vapors could be seen by themselves without the intense
light behind them, they would give bright lines as shown by the
bright sodium lines seen in the alcohol or Bunsen flame.

§ 278. Absorption spectra from colored substances. — While
the solar spectrum is an absorption spectrum, the term is more com-
monly applied to the spectra obtained with light which has passed

CH. V]

MICRO-SPECTROSCOPES

227

through or has been reflected from colored objects which are not
self-luminous.

It is the special purpose of the micro-spectroscope to investigate
the spectra of colored objects which are not self-luminous, i.e., blood
and other liquids, various minerals, as monazite, etc. The spectra
obtained by examining the light reflected from these colored bodies
or transmitted through them possess, like the solar spectrum, dark
lines or bands, but the bands are usually much wider and less

A 3 C

) ]

Mill 1

: F

•T

tilt

(

; *, . I I..

lift

0
r-*
C»
C»

fi*

-t

AUUe-

FIG. 123. SPECTRA TO SHOW DIFFERENT KINDS OF ABSORPTION BANDS.

Solar Spectrum The spectrum of daylight showing the dark, fixed lines
(Fraunhofer lines) A B C D E F G, and the wave lengths in microns, .70, .60
.50, .40.

Sodium The spectrum of incandescent sodium. With this spectroscope it is
a single bright yellow band (D) at about Xo.5Q;u, all the rest of the spectrum
being dark.

Perman. potash The spectrum of a solution of permanganate of potash. It
has five absorption bands, two being especially dark and sharply outlined.

Methaetnoglobin The spectrum of methaemoglobin with several absorption
bands, the two in the yellow-green being darkest. The blue end of the spec-
trum is also greatly shortened.

These spectra have the blue end at the right instead of at the left (compare
figs. 1 21-122.

sharply defined. Their number and position depend on the sub-
stance or its constitution (fig. 123), and their width, in part, upon
the thickness of the body. With some colored bodies, no definite
bands are present. The spectrum is simply restricted at one or

228 MICRO-SPECTROSCOPES [Cn. V

both ends and various of the other colors are considerably lessened
in intensity. This is true of many colored fruits.

§ 279. Angstrfan and Stoke's law of absorption spectra. — The
waves of light absorbed by a body when light is transmitted through
some of its substance are precisely the waves radiated from it when
it becomes self-luminous. For example, a piece of glass that is
yellow when cool gives out blue light when it is hot enough to be
self-luminous. Sodium vapor absorbs two bands of yellow light (D
lines) ; but when light is not sent through it, but the vapor itself is
luminous and is examined as a source of light, its spectrum gives
bright sodium lines, all the rest of the spectrum being dark (fig. 123).

§ 280. Law of color. — The light reaching the eye from a colored
solid, liquid, or gaseous body lighted with white light will be that
due to white light less the light waves that have been absorbed by
the colored body. For example, a thin layer of blood under the
microscope will appear yellowish green, but a thick layer will appear
pure red. If now these two layers are examined with a micro-
spectroscope, the thin layer will show all colors, but the red end
will be slightly, and the blue end considerably, restricted, and some
of the colors will appear considerably lessened in intensity. Finally,
there may appear two shadow-like bands, or, if the layer is thick
enough, two well-defined dark bands in the green (§ 295).

If the thick layer is examined in the same way, the spectrum will
show only red with a little orange light, all the rest being absorbed.
Thus the spectroscope shows which colors remain, in part or wholly,
and it is the mixture of this remaining or unabsorbed light that gives
color to the object.

§ 281. Complementary spectra. — While it is believed that
Angstrom's law (§ 280) is correct, there are many bodies on which it
cannot be tested, as they change in chemical or molecular constitu-
tion before reaching a sufficiently high temperature to become lumi-
nous. There are compounds, however, like those of didymium,
erbium, and terbium, which do not change with the heat necessary
to render them luminous, and with them the incandescent and ab-
sorption spectra are mutually complementary, the one presenting
bright lines where the other presents dark ones (Daniell).

CH. V] MICRO-SPECTROSCOPES 229

ADJUSTING THE MICRO-SPECTROSCOPE

§ 282. The micro-spectroscope, or spectroscopic ocular, is put in
the place of the ordinary ocular in the microscope, and clamped to
the top of the tube by means of a side screw for the purpose.

§ 283. Adjustment of the slit. — In place of the ordinary dia-
phragm with circular opening, the spectral ocular has a diaphragm
composed of two movable knife edges by which a slit-like opening of
greater or less width and length may be obtained at will by the use
of screws for the purpose. To adjust the slit, depress the lever
holding the prism-tube in position over the ocular, and swing the
prism aside. One can then look directly into the ocular. The lateral
screw should be used, and the knife edges approached till they ap-
pear about half a millimeter apart. If now. the Amici prism is put
back in place and the microscope well lighted, one will see a spectrum
by looking into the upper end of the spectroscope. If the slit is too
wide, the colors will overlap in the middle of the spectrum and be
pure only at the red and blue ends; and the Fraunhofer or other
bands in the spectrum will be faint or invisible. Dust on the edges of
the slit gives the appearance of longitudinal streaks on the spectrum.

§ 284. Mutual arrangement of slit and prism. — In order that the
spectrum may appear as if made up of colored bands going directly
across the long axis of the spectrum, the slit must be parallel with
the refracting edge of the prism. If the slit and prism are not thus
mutually arranged, the colored bands will appear oblique, and the
whole spectrum may be greatly narrowed. If the colored bands are
oblique, grasp the prism tube and slowly rotate it to the right or to
the left until the various colored bands extend directly across the
spectrum.

§ 285. Focusing the slit. — In order that the lines or bands in
the spectrum shall be sharply defined, the eyelens of the ocular
should be accurately focused on the slit, The eyelens is movable,
and when the prism is swung aside it is easy to focus the slit as
one focused for the ocular micrometer (§ 375). If one now uses
daylight there will be seen in the spectrum the dark Fraunhofer
lines (figs. 121, 123).

230 MICRO-SPECTROSCOPES [Cn. V

To show the necessity of focusing the slit, move the eyelens down
or up as far as possible, and the Fraunhofer lines cannot be seen.
While looking into the spectroscope move the ocular lens up or down,
and when it is focused, the Fraunhofer lines will reappear. As the
different colors of the spectrum have different wave lengths, it is
necessary to focus the slit for each color if the sharpest possible
pictures are desired.

It will be found that the eyelens of the ocular must be farther from
the slit for the sharpest focus of the lines at the red end, than for
the sharpest focus of those at the blue end. This is because the
wave length of the red is markedly greater than for blue light
(figs. 93, 124).

Longitudinal dark lines on the spectrum may be due to irregular-
ity of the slit or to the presence of dust. They are most trouble-
some with a very narrow slit.

§ 286. Comparison or double spectrum. — In order to compare
the spectra of two different substances one must examine their
spectra side by side. This is provided for in the better forms of
micro-spectroscopes by a prism just below the slit, so placed that the
light entering it from the mirror at the side of the drum shall be
totally reflected in a vertical direction, and thus parallel with the
rays from the microscope. The two spectra will be side by side, with
a narrow dark line separating them. If now the slit is well focused
and daylight is sent through the microscope and into the side to the
reflecting or comparison prism, the colored bands and the Fraun-
hofer dark lines will appear directly continuous across the two
spectra. The prism for the comparison spectrum is movable and
may be thrown entirely out of the field if desired. When it is to
be used, it is moved about halfway across the field so that the two
spectra shall have about the same width.

§ 287. Scale of wave lengths. — In the Abbe micro-spectroscope
the scale is in a separate tube near the top of the prism and at right
angles to the prism-tube. A special mirror serves to light the scale,
which is projected upon the spectrum by a lens in the scale-tube.
By means of this scale, the wave lengths of any part of the spectrum
may be read off directly, after the scale is once set in the proper

CH. V] MICRO-SPECTROSCOPES 231

position, that is, when it is set so that any given wave length on the
scale is opposite the part of the spectrum known by previous
investigation to have that particular wave length. The point most
often selected for setting the scale is opposite the sodium line, where
the wave length is, according to Angstrom, 0.5892/4. In adjusting
the scale, one may focus very sharply the dark sodium line of the
solar spectrum and set the scale so that the number 0.589 is opposite
the sodium or D line ; or a method that is frequently used and serves
to illustrate §§ 276-277, is to saturate some asbestos cloth in a
strong solution of common salt (sodium chlorid, NaCl) or bicar-
bonate of soda (NaHCO3). Heat in a Bunsen or alcohol flame and
the incandescent sodium will give the bright D lines.

If now ordinary daylight is sent through the comparison prism, the
bright lines of the sodium will be seen to be directly continuous
with the dark lines at D in the solar spectrum (fig. 123). By re-
flecting light into the scale-tube the image of the scale will appear
on the spectrum, and by a screw just under the scale-tube, but
within the prism-tube, the proper point on the scale (0.589^) can be
brought opposite the sodium band. All the scale will then give the
wave lengths directly. Sometimes the scale is oblique to the spec-
trum. This may be remedied by turning the prism-tube slightly one
way or the other. It may be due to the wrong position of the scale
itself. If so, grasp the milled ring at the distal end of the scale- tube
and, while looking into the spectroscope, rotate the tube until the
lines of the scale are parallel with the Fraunhofer lines. It is
necessary in adjusting the scale to be sure that the larger number,
0.70, is at the red end of the spectrum.

The numbers on the scale should be clearly denned. If they do
not so appear, the scale-tube must be focused by grasping the outer
tube of the scale-tube and moving it toward or from the prism-tube
until the scale is distinct. In focusing the scale, grasp the outer
scale-tube with one hand and the prism-tube with the other, and
push or pull in opposite directions. In this way one will be less
likely to injure the spectroscope.

§ 288. Designation of wave length. — Wave lengths of light are
designated by the Greek letter X followed by the number indicating

232 MICRO-SPECTROSCOPES [Cn. V

the length in some fraction of a meter. See fig. 93 where the visible
spectrum is indicated as lying between wave lengths Xo.y/z and
0.4/4. In this book the micron (JJL) is taken as the unit as with other
minute measurements. Other units are also employed, especially
smaller ones so that the wave lengths will appear as whole numbers
instead of decimal fractions. (See §§ 380-382).

§ 289. Lighting for the micro-spectroscope. — Opaque objects are
illuminated by placing the microscope in a strong light, or by re-
flecting light upon them by a mirror, or by the use of a bull's-eye or
other condenser. The light from one of the dark-field lamps is
excellent. For transparent objects, the amount of the substance and
the depth of the color must be considered. As a general rule, it is
well to use plenty of light, as that from a substage condenser with a
large opening in the diaphragm or with the diaphragm entirely open.
For very small objects and thin layers of liquids, it may be better
to use less light. One must try both methods in a given case, and
learn by experience.

The direct and the comparison spectra should be about equally
illuminated. One can manage this by putting the object requiring
the greater amount of illumination on the stage of the microscope.

Furthermore, one should be on his guard against confusing the
ordinary absorption bands with the Fraunhofer lines when daylight
is used. With lamplight the Fraunhofer lines are absent.

§ 290. Objective to use with the micro-spectroscope. — If the
material is of considerable bulk, a low objective (16 mm. lox) is to be
preferred. This depends on the nature of the object under exami-
nation, however. In case of individual crystals one should use suffi-
cient magnification to make the real image of the crystal entirely fill
the width of the slit. The length of the slit may then be regulated
by the screw on the side of the drum, and also by the comparison
prism. If the object does not fill the whole slit, the white light
entering the spectroscope with the light from the object might ob-
scure the absorption bands.

In using high objectives with the micro-spectroscope one must
very carefully regulate the light and sometimes shade the object.

§291. Focusing the objective. — For focusing the objective the

CH. V] MICRO-SPECTROSCOPES 233

prism-tube is swung aside, and then the slit made wide by turning
the adjustable screw at the side. If the slit is open one can see
objects when the microscope is focused as with an ordinary ocular.
After an object is focused, it may be put exactly in position to fill
the slit of the spectroscope, then the knife edges are brought together
till the slit is of the right width; if the slit is then too long it may
be shortened by using one of the mechanism screws on the side, or
if that is not sufficient, by bringing the comparison prism farther
over the field. If one now replaces the Amici prism and looks into
the microscope, the spectrum is likely to have longitudinal shimmer-
ing lines. To get rid of these, focus up or down a little so that the
microscope will be slightly out of focus.

§ 292. Amount of material necessary for absorption spectra and
its proper manipulation. — The amount of material necessary to
give an absorption spectrum varies greatly with different substances,
and can be determined only by trial. If a transparent solid is under
investigation, it is well to have it in the form of a wedge, then suc-
cessive thicknesses can be brought under the microscope. If a liquid
substance is being examined a watch glass with sloping sides forms
an excellent vessel to contain it, then successive thicknesses of the
liquid can be brought into the field, as with the wedge-shaped solid.
Frequently only a very weak solution is obtainable; in this case it
can be placed in a homeopathic vial, or in some glass tubing sealed
at the end, then one can look lengthwise through the liquid and get
the effect of a more concentrated solution. For minute bodies like
crystals or blood corpuscles, one may proceed as described in the
previous section. (See also § 302.)

MICRO-SPECTROSCOPE EXPERIMENTS

§ 293. Put the micro-spectroscope in position, arrange the slit
and the Amici prism so that the spectrum will show the various
spectral colors going directly across it (§ 284), and focus the slit.
This may be done either by swinging the prism-tube aside and pro-
ceeding as for the ocular micrometer (§375), or by moving the
eyelens of the ocular up and down while looking into the micro-spe^

234 MICRO-SPECTROSCOPES [Cn. V

troscope until the dark lines of the solar spectrum are distinct. If
they cannot be made distinct by focusing the slit, then the light is
too feeble or the slit is too wide. With the lever move the com-
parison prism across half the field so that the two spectra shall be of
equal width. For lighting, see § 289.

§ 293a. Pocket spectroscope. — Many of the purposes for which a micro-spec-
troscope was specially designed, can almost as well be accomplished by a much
cheaper, pocket, direct-vision spectroscope (Bleile, Trans. Amcr. Micr. Soc. 1900,
p. 8). To use this with a microscope, it is clamped in some kind of an adjustable
holder like the lens holder, (figs. 17, 127), the ocular of the microscope is removed,
and the pocket spectroscope is put over the top of the tube and in line with the
optic axis of the microscope. As the slit mechanism and the parallelizing lens
form a part of the spectroscope, one has simply to open the slit the right amount
and focus it by pulling out the tube of the spectroscope. All the other opera-
tions are the same as for the larger micro-spectroscope. The object to be ex-
amined is put in the center of the field of the microscope. For this, of course the
ocular should be in place. After the ocular is removed, one can adjust the pocket
spectroscope so that the object sends the light transmitted through it to the spec-
troscope. If one has a research lamp like figure 80, the iris of the lamp can be
made smaller and larger by closing or opening it. Also the image of this opening
focused on the object by the condenser can be made larger or smaller by chang-
ing the distance between the lamp and the microscope.

The real image of the object should be at the level of the slit of the spectro-
scope. This is easily determined by using a piece of ground glass over the upper
end of the tube, and focusing until the real image is sharp on the ground glass.

This opening or closing of the iris of the lamp and varying the distance between
the lamp and the microscope condenser enables one to enclose the object and
exclude outside objects almost as effectively as the arrangements in the micro-
spectroscope for shortening and narrowing the slit (§ 291.)

§ 294. Absorption spectrum of permanganate of potash. — Make
a solution of permanganate of potash by putting a few crystals in a
watch glass of water. The solution should be of such strength that
a stratum of 3 to 4 mm. thickness will be transparent. Place the
watch glass under the microscope. Use a 16 mm. (lox) or lower
objective and open widely the condenser diaphragm; light strongly.
Look into the spectroscope and slowly move the watch glass into the
field. Note carefully the appearance with the thin stratum of liquid
at the edge and then as it gradually thickens on moving the watch
glass still farther along. Count the absorption bands and note
particularly the red and blue ends. Compare with the comparison
spectrum (fig. 123). For strength of solution see § 292.

§ 295. Absorption spectrum of blood. — Obtain blood from a
recently killed animal, or flame a needle, and after it is cool, prick

in. V] MICRO-SPECTROSCOPES 235

the finger two or three times in a small area; then wind a handker-
chief or a rubber tube around the base of the finger and squeeze the
finger with the other hand. Some blood will ooze out of the pricks.
Rinse this off into a watch glass partly filled with water. Continue
to add the blood until the water is quite red. Place the watch glass
of diluted blood under the microscope in place of the permanganate,
using the same objective, etc. Note carefully the spectrum. It
would be advantageous to determine the wave length opposite the
center of the dark bands. This may easily be done by setting the
scale properly, as described in § 287. Make another preparation,
but use a homeopathic vial instead of a watch glass. Cork the vial
and lay it down upon the stage of the microscope. Observe the
spectrum. It will be like that in the watch glass. Remove the cork
and look through the whole length of the vial. The bands will be
much darker, and if the solution is thick enough, only red and a
little orange will appear. Reinsert the cork and incline the vial so
that the light traverses a very thin layer, then gradually elevate the
vial and the effect of a thicker and thicker layer may be seen. Note
especially that the two characteristic bands unite and form one wide
band as the stratum of liquid thickens. Compare with the follow-
ing.

Add to the vial of diluted blood a drop or two of ammonium sul-
phide, such as is used for a reducing agent in chemical laboratories.
Shake the bottle gently and then allow it to stand for ten or fifteen
minutes. Examine it and the two bands will have been replaced by
a single, less clearly defined band in about the same position. The
blood will also appear somewhat purple. Remove the cork to
admit fresh air, then shake the vial vigorously, and the color will
change to the bright red of fresh blood. Examine it again with
the spectroscope and the two bands will be visible. After five or
ten minutes another examination will show but a single band. In-
cline the bottle so that a thin stratum may be examined. Note that
the stratum of liquid must be considerably thicker to show the single
absorption band than was necessary to show the two bands in the
first experiment. Furthermore, while the single band may be made
quite black on thickening the stratum, it will not separate into two

MICRO-SPECTROSCOPES

[CH. V

bands with a thinner stratum. In this experiment it is very instruc-
tive to have the watch glass of arterial blood under the microscope
and the vial of blood to which has been added the ammonium sul-
phide in position for a comparison spectrum.

The two-banded spectrum is that of oxy-hemoglobin, or arterial
blood; the single-banded spectrum of hemoglobin (sometimes called
reduced hemoglobin) or venous blood. The respiratory oxygen
is present, in the two-banded spectrum but absent from the single-
banded spectrum. When the bottle was shaken the hemoglobin
took up oxygen from the air and became oxy-hemoglobin, as occurs
in the lungs, but soon the ammonium sulphide took away the respir-
atory oxygen, thus reducing the oxy-hemoglobin to hemoglobin.
This may be repeated many times (fig. 124).

FIG. 124. ABSORPTION SPECTRUM OF ARTERIAL AND OF VENOUS BLOOD.
(From Gamgee and McMunn).

1 Absorption of arterial blood, oxy-hemoglobin. There are two definite
bands between wave lengths 0.60^ and 0.50;*, that is, in the yellow-green, and
the blue end of the spectrum is cut down markedly.

2 Single dark band of the venous blood, hemoglobin, in the yellow-green. The
blue end of the spectrum is less cut off than with arterial blood.

A B C D E F G 11 Fixed lines of the solar spectrum .90, .80, .70, .60, .50, .40;
wave lengths in microns in the different regions. These spectra have the red
end at the left instead of to the right, as is now more usual (figs. 120-122).

§ 296. Met-hemoglobin. — The absorption spectrum of met-
hemoglobin is characterized by a considerable darkening of the blue
end of the spectrum and of four absorption bands, one in the red
near the line C and two between D and E, nearly in the place of the
two bands of oxy-hemoglobin; finally there is a somewhat faint,
wide band near F. Such a met-hemoglobin spectrum is best ob-
tained by making the solution of blood in water of such a concentra-
tion that the two oxy-hemoglobin bands run together, and then

CH. V] MICRO-SPECTROSCOPES 237

adding three or four drops of a o.i % aqueous solution of perman-
ganate of potash. Soon the bright red will change to a brownish
color, when it may be examined (fig. 123). Instead of the perman-
ganate one may use hydrogen dioxide (H^C^).

§ 297. Carbon monoxide hemoglobin (CO-hemoglobin). — To
obtain this, kill an animal in illuminating gas, or one may allow
illuminating gas to bubble through some blood already taken from
the body. The gas should bubble through a minute or two. The
oxygen will be displaced by carbon monoxide. This forms quite a
stable compound with hemoglobin, and is of a bright cherry-red
color. Its spectrum is nearly like that of oxy-hemoglobin, but the
bands are farther toward the blue. Add several drops of ammonium
sulphide and allow the blood to stand some time. No reduction will
take place, thus forming a marked contrast to solutions of oxy-hemo-
globin. By the addition of a few drops of glacial acetic acid a dark
brownish-red color is produced.

§ 298. Carmine solution. — Make a solution of carmine by put-
ting o.i gram of carmine in 100 cc. of water and adding 10 drops of
strong ammonia. Put some of this in a watch glass or in a small
vial and compare the spectrum with that of oxy-hemoglobin or
carbon-monoxide hemoglobin. It has two bands in nearly the same
position, thus giving the spectrum a striking similarity to blood.
If now several drops, 15 or 20, of glacial acetic acid are added to the
carmine, the bands remain and the color is not markedly changed,
while with either oxy-hemoglobin or CO-hemoglobin the color is
decidedly changed from the bright red to a dull reddish-brown, and
the spectrum, if any can be seen, is markedly different. Carmine
and 0-hemoglobin can be distinguished by the use of ammonium
sulphide, the carmine remaining practically unchanged while the
blood shows the single band of hemoglobin (§ 295). The acetic acid
serves to differentiate the CO-hemoglobin as well as the O-hemo-
globin.

§ 299. Colored bodies not giving banded spectra. — Some quite
brilliantly colored objects, like the skin of a red apple, do not give a
banded spectrum. Take the skin of a red apple, mount it on a slide,
put on a cover-glass, and add a drop of water at the edge of the

23<S MICRO-SPECTROSCOPES [Cn. V

cover. Put the preparation under the microscope and observe the
spectrum. Although no bands will appear, in some cases at least,
yet the ends of the spectrum will be restricted and various regions
of the spectrum will not be so bright as the comparison spectrum.
Here the red color arises from the mixture of the unabsorbed waves,
as occurs with other colored objects. In this case, however, not all
the light of a given wave length is absorbed; consequently there are
no clearly denned dark bands; the light is simply less brilliant in
certain regions and the red rays so predominate that they give the
prevailing color.

§ 300. Nearly colorless bodies with clearly marked absorption
spectra. — In contradistinction to the brightly colored objects with
no distinct absorption bands are those nearly colorless bodies and
solutions which give as sharply denned absorption bands as could be
desired. The best examples of this are afforded by solutions of the
rare earths, didymium, etc. These in solutions that give hardly a
trace of color to the eye give absorption bands that almost rival the
Fraunhofer lines in sharpness.

§ 301. Absorption spectra of minerals. — As example, take some
monazite sand on a slide and either mount it in balsam, or cover and
add a drop of water. The examination may be made also with the
dry sand, but it is less satisfactory. Light well with transmitted
light and move the preparation slowly about. Absorption bands will
appear occasionally. Swing the prism tube off the ocular, open the
slit, and focus the sand. Get the image of one or more grains di-
rectly in the slit, then narrow and shorten the slit so that no light
can reach the spectroscope that has not traversed the grain of sand.
The spectrum will be satisfactory under such conditions. It is fre-
quently of great service in determining the character of unknown
mineral sands to compare the spectra with known minerals. If the
absorption bands are identical, it is strong evidence in favor of the
identity of the minerals.

§ 302. While the study of absorption spectra gives one a great
deal of accurate information, great caution must be exercised in
drawing conclusions as to the identity or even the close relationship
of bodies giving approximately the same absorption spectra. The

CH. V] MICRO-SPECTROSCOPES 239

rule followed by the best workers is to have a known body as control
and to treat the unknown body and known body with the same
reagents, and to dissolve them in the same medium. If all the
reactions are identical, then the presumption^ is strong that the
bodies are identical or very closely related. For example, while one
might be in doubt between a solution of oxy- or CO-hemoglobin and
carmine, the addition of ammonium sulphide serves to change the
double to a single band in the 0-hemoglobin, and glacial acetic
acid enables one to distinguish between the CO-blood and the car-
mine, although the dmmonium sulphide would not enable one to
make the distinction. Furthermore, it is unsafe to compare objects
dissolved in different media. Different objects as " cyanine and
aniline blue dissolved in alcohol give a very similar spectrum, but in
water a totally different one." " Totally different bodies show ab-
sorption bands in exactly the same position (solid nitrate of uranium
and permanganate of potash in the blue) " (MacMunn). The rule
given by MacMunn is a good one: " The recognition of a body
becomes more certain if its spectrum consists of several absorption
bands, but even the coincidence of these bands with those of another
body is not sufficient to enable us to infer chemical identity, what
enables us to do so with certainty is the fact, that the two solutions
give bands of equal intensities in the same parts of the spectrum
which undergo analogous changes on the addition of the same re-
agent. It should be borne in mind that the position of a band may
be changed greatly through increased or diminished dissociation,
and that the absorption bands given by a crystal may be quite
different from those given by the same material in solution and
furthermore that the absorption spectra are sometimes different in
different directions through the crystal " (Chamot, p. 112). This
is easily demonstrated if one has a centering, revolving stage on the
microscope (fig. 92).

For the use of the spectroscope with the ultra-violet microscope for
determining the character of fluorescent light, see § 314.

CHAPTER VI

THE ULTRA-VIOLET MICROSCOPE AND PHYSICAL ANALYSIS.
§§303-324; FIGURES 125-130

THE ULTRA-VIOLET MICROSCOPE

The necessity of sunlight for green plants and its beneficial effect
upon animal life have been the common knowledge of mankind for
unnumbered generations.

The knowledge is relatively recent, however, that the radiation
from the sun and from artificial sources is composed of waves of
varying length, and that only a narrow band of that radiation is
visible (fig. 93). The knowledge is still more recent that the short
waves beyond the visible spectrum (ultra-violet, x-rays, etc.), have a
profound effect upon living things, and that the appearances given
to various structures by these invisible waves promise to give much
definite information concerning their physical make-up.

So difficult is the complete understanding of the function and
structure of organic nature that it seems worth while to make use of
every means available to aid in making the structure and the
function more completely understood. From what is already known,
it is certain that the ultra-violet radiation has not only a profound
physiological effect upon animals and plants, but also gives definite
appearances to various structures and thus throws light upon their
chemical and physical constitution.

The effect that ultra-violet radiation has upon living things, is
primarily upon the individual structural units or cells of which they
are composed. If then the structural units can be studied before,
during and after the ultra-violet treatment by means of a suitable
microscope (an ultra-violet microscope) in which all powers can 'be
used, it is hoped that enough of the changes can be discovered to
enable the biologist to gain some understanding of the changes that
occur in the animal or plant as a whole.

240

CH. VI] THE ULTRA-VIOLET MICROSCOPE 241

It is already certain knowledge that the ultra-violet microscope is
of use in what may be called the physical analysis of living things,
and of their individual tissues and organs, for, some of them at
least, when excited by the short, invisible, ultra-violet radiation,
emit visible radiation. In that case the tissues or organs glow with a
soft radiance of their own, and become visible by their own lumi-
nescence. When they do thus become luminous, they are said to
fluoresce.

Some structures do not fluoresce when radiated by ultra-violet.
Such structures, then, remain dark in ultra-violet. (See §§ 317-318.)
Although a structure or organism does not fluoresce, it may be
profoundly affected by the ultra-violet radiation. For example, it
has been known for decades that bacteria and other microscopic
forms are killed by ultra-violet in excess. Physicians also have
learned that in applying ultra-violet to animals and to men, the
amount must be limited or serious results occur. Every one knows
about sun-burn, and how painful it may be. Mountain climbers
found long ago that sun-burn was almost sure to occur on the snow-
and ice-covered peaks if one did not take the precaution to smear
the face with some substance opaque to ultra-violet and to protect
the eyes by colored glasses. We think now that it is not so much the
visible light that does the damage, but the invisible, ultra-violet.

§ 303. Ultra-violet or fluorescence microscope. — This is a mi-
croscope under which small objects can be submitted to ultra-violet
radiation and the results observed.

For such observations there must be, first, some source in which
the ultra-violet is abundant. Best for the microscope is the lately
perfected high-pressure capillary mercury arc (loo-watt H4 lamp)
of the General Electric Vapor Lamp Co. (fig. 129 A).

The carbon arc using specially filled cored carbons is also good.

These sources also produce a great amount of visible radiation,
which must be excluded by the use of suitable screens so that only
the ultra-violet radiation is present in the radiation utilized. It is
also highly important to know just what wave lengths pass through
the screen so that any observed effect on the object can be ascribed
to the wave length, or wave lengths producing the effects. For

242

THE ULTRA-VIOLET MICROSCOPE

[CH. VI

example, if the object is fluorescent, it is desirable to know what
wave-lengths excite the fluorescence.

FIG. 125. ULTRA-VIOLET MICROSCOPE.

1 Incline ocular. The usual straight ocular is satisfactory.

2 Housing of the prism to give the inclination.

3 Wheel of the coarse adjustment.

4 Wheel of the fine adjustment.

5 Nosepiece with three objectives.

6 Spring clips for holding the specimen in place.
7-7 Mechanical stage movement screw heads.

8 Joint in the pillar for inclination of the microscope.

9 Jointed pillar.

10 Centering screw head for the condenser.

1 1 Aluminum vapor mirror for reflecting ultra-violet radiation.

1 2 Tightening screw head for the mirror mounting.

§ 30i. Aluminum vapor mirror. — Some kind of a reflector is necessary to
change the direction of the illuminating beam in microscopic work. The

CH. VI]

THE ULTRA-VIOLET MICROSCOPE

243

ordinary mirror with silvered back does not answer for ultra-violet reflection
for so great a number of the short ultra-violet wave lengths are absorbed by
the silver and the glass. Formerly a quartz prism was used as reflector, This
is heavy, expensive and rather difficult to manipulate. Since the publication
of the 1 5th edition there has been developed a practical means of making
vapor deposits of metals. A first surface mirror on ordinary glass of aluminum
has given the author as good results as ever could be obtained by the quartz

FIG. 126. ALUMINUM VAPOR MIRROK; DARK-FIELD SUBSTAGE CONDENSES, AND
FRONT LENS OF THE MICROSCOPE OBJECTIVE.

1 Beam of fluorescent light entering the front lens of the microscope objective.

2 Front lens of the objective.

3 Beam of ultra-violet radiation at so great an angle that it cannot enter the
objective.

4 Corex glass slip in immersion contact with the top of the dark-field con-
denser.

5 The hollow cone of ultra-violet radiation. The central part of the cone is
stopped by the opaque diaphragm at the base or lower end of the quartz dark-
field element of the condenser.

6 Lower quartz element of the dark-field condenser.

7 The aluminum-vapor mirror on the front of glass or the back of quartz.

8 Beam of ultra-violet radiation.

244

THE ULTRA-VIOLET MICROSCOPE

[CH. VI

prism reflector, and is easier to manipulate. To toughen the soft aluminum
film there is first deposited a chromium film and on this the aluminum. In
spite of the toughening by the chromium, the surface becomes more or less
scratched and dimmed by long usage and much cleaning. To have the ad-
vantages of a back or second surface mirror on a hard transparent substance, by
the efficient aid of Dr. H. P. Gage of the Corning Glass Works and the "The
Evaporated Films Company of Ithaca, N. Y." the author has secured alumi-
num vapor reflectors with the aluminum film on the back of polished quartz
discs. These are especially brilliant reflectors of the ultra-violet and the
protection of the aluminum film by the quartz covering will, it is believed,
preserve the aluminum reflecting surface with its original brilliance indefi-
nitely. From determinations by George B. Sabine of the physics department of
Cornell University, the percentage of reflection averaged for the second-surface,
quartz mirror, waves 238 to 377 m/j, 89%. Such mirrors are mounted in the us-
ual way for the fork of the mirror holder, or the unmounted mirror can be
stuck to the glass mirror with beeswax.

It may be stated in passing, that such aluminum mirrors are excellent for
all microscopic work including photography with the microscope.

§ 305. As the ultra-violet is invisible, it is necessary to use some
means for knowing when the radiation is directed from the source to
the reflector, whence by proper manipulation of the reflector it is sent
up through the condenser to the object on the stage of the microscope.
A card smeared with a strong emulsion of anthracene in cane sugar
and dried, makes a good detector of ultra-violet from its strong
fluorescence. See also § 315 to make sure the object is well illumi-
nated.

FIG. 127.

BULL'S-EYE, QUARTZ CONDENSER (Q) FOR USE WITH THE
ULTRA-VIOLET MICROSCOPE.

§ 306. Ultra-Violet condenser. — The substage condenser and the
bull's-eye condenser (fig. 128) must be made of quartz to insure the
transmission of the ultra-violet radiation.

CH. VI]

THE ULTRA-VIOLET MICROSCOPE

245

§ 307. Quartz dark-field element. — The upper element of the
condenser should be for dark-field (§ 181) illumination. The object
will then be radiated with the ultra-violet at so great an angle that
none of it will get directly into the microscope objective (§§ 171, 178).
If, however, the object is fluorescent, the luminous object will send
visible light to the objective and thence on to the eye of the ob-
server. The relatively small amount of ultra-violet which is de-
flected by the object into the objective will not cause sufficient

FIG. 128. ARRANGEMENT OF PARTS TOR THE ULTRA-VIOLET MICROSCOPE.

1 Outline of the lamp-house

2 The quartz condenser on adjustable stand for directing the ultra-violet
radiation from the mercury lamp to the quartz prism of the ultra-violet micro-
scope.

3 Ultra- Violet microscope. Here it is shown with the usual vertical tube
for the ocular.

4 Sliding screen carrier. The dark part is the red-purple screen, and
the white part the corex, ground glass to transmit the full mercury radiation.
It is also shown over the window in the lamp-house.

5 Card with a coating of anthracene to aid in directing the ultra-violet
upon the quartz prism.

6 The mercury lamp head opposite the quartz tube of the lamp.

7 Piece of uranium glass on the table. It is one of the most perfect detec-
tors of ultra-violet radiation.

8 Metal shelf for holding the lamp-house.

9 Table for the microscope, etc.

10 Regulating mechanism for the current with the mercury arc. The feet of
the lamp and of the table have pads of thick felt under them to diminish vibra-
tion.

246 THE ULTRA-VIOLET MICROSCOPE [Cn. VI

fluorescence of the glass or of the Canada balsam used in sealing the
lenses to interfere. If the regular top element of the condenser is
left in place, the amount of ultra-violet getting past the object and
into the objective is likely to cause so much fluorescence in the
objective that the appearance is like looking into a bright fog.

§ 308. Ultra- Violet transmitting slips. — For mounting slips one
must use a non-fluorescing substance like quartz or corex D glass
of the Corning Glass Works (fig. 218). The corex slips, if cut from
sheets, cost about $6.00 per hundred while the quartz slips cost from
$3.50 to $10.00 each. As the quartz and the corex appear so much
like glass, the author has had them made in a special size, 65 X 25
mm. (fig. 218). He found it wise to mark them with a writing
diamond also.

For the cover-glasses, those ordinarily used answer well. They
transmit the fluorescent light from the object, and help to eliminate
any of the ultra-violet which is deflected toward the objective.

§ 309. Immersion media. — Non-fluorescing liquids must be used
or the objects will appear in a bright fog. Fortunately for an im-
mersion liquid there is available the medicinal mineral oil (petrola-
tum, nujol, etc.). Both those with a paraffin and those with a
naphthalene base are wholly non-fluorescing for the ultra-violet most
useful for microscopy (fig. 129), that is, the ultra-violet from the
mercury or carbon arc which is transmitted through the screens
used to eliminate the visible light.

Fortunately also, the water in which the small organisms live is
non-fluorescing, and the isotonic solutions used in physiological
experiments are likewise non-fluorescing, so that all desired physio-
logical tests can be made with living things and living tissues.

§ 310. Permanent mounting media. — Canada balsam is fluoresc-
ing and therefore not available. The medicinal mineral oil answers
very well, but the cover-glass must be sealed with shellac or other
sealing cement. The steps for permanent preparations - are exactly
as for balsam mounting (§§ 534-535) except that the mineral oil is
used instead of the balsam. Its index of refraction (nD 1.48) is
almost as great as Canada balsam and cedar oil, so that it can be
substituted with little loss in optical results.

CH. VI]

THE ULTRA-VIOLET MICROSCOPE

247

§ 311. Ultra-violet lamp. — The recently developed loo-watt
H4 capillary arc (fig. 129 A) is the most satisfactory source of ultra-
violet for the microscope and for photomicrography (see § 470).
It is also good for naked-eye fluorescence phenomena shown by chloro-
phyll solutions, colored cloth and masses of any substance (see p. 257).

FIG. 129 A. CAPILLARY MERCURY ARC LAMP FOR FLUORESCENCE AND
FOR PHOTOMICROGRAPHY.

(/) Lamp House The metal container of the capillary mercury arc bulb (j).

SC Screw for holding the tube containing the electric cable to the socket.
It serves to regulate the height of the mercury arc. This is in a tube fastened to
a metal plate that can be moved toward or from the front of the lamp house,
to change the position of the mercury arc.

Lamp This word is placed just above the polarized socket of the cable going to
the lamp socket.

S This is placed under the middle of the sloping screen to shut off the radiation
which would extend upwards to the eyes of the observer.

C Free opening of the lamp house when both visible and invisible radiation
is desired.

RP CA Red-Purple Corex A filter to transmit radiations between 250 m/x

248

THE ULTRA-VIOLET MICROSCOPE

[Cn. VI

and 410 m/£. The clear opening and the ultra-violet filter are in a sliding carrier
so that either may be used at will. The actual size is indicated by the lo-cm. scale
at the bottom of the lamp house.

(2) Alternating current regulator for the capillary arc.

Ln Plug for connecting with the line.

Lamp Polarized plug for connecting with the cable to the lamp.

R Part of the regulator.

The oval openings on the two sides of the electric cables near the top are for
screws to hold the regulator in place. For the inexperienced, it would be wise to
have the manufacturers add the cables with plug and socket to the regulator.

(j) Transparent view of the capillary mercury arc bulb with its glass case.

CA Capillary arc within its glass sheath. (Two-thirds natural size.)

(4) Block for holding the filter in photomicrography.

S Screen or filter 5 to 7.5 cm. square.

n-n Two of the four headless nails to hold the screen upright.

W Wooden block and (L) lead on the bottom.

250

280

310

330

370

410

Mi Mi-Microns

FIG. 129 B. SIMPLE CURVE TO SHOW THE TRANSMISSION OF ULTRA-VIOLET AND
VISIBLE RADIATION OF THE CORNING CLEAR COREX D GLASS AND OF THE RED-
PURPLE CoRiix A GLASS.

In the curves the base-line or abscissa shows the wave length in millimicrons,
and the vertical line the transmission percentage for the different wave lengths
of the radiation.

RP CA. Red-Purple Corex A. It shows transmission between 250 m/i and
410 mjLt, the maximum being between 280 mju and 370 m/z. As there is some
transmission from 390-410 mju, there is a limited amount of visible radiation in
this filter.

CC D. Clear Corex D. This curve also shows transmission commencing at
250 m/x with the maximum ultra-violet at 360 m/x. From that point onward there
is' between 90 and 100% transmission.

CH. VI] THE ULTRA-VIOLET MICROSCOPE 249

It takes about five minutes for the lamp to emit the full amount
of ultra-violet. In working with the high pressure mercury arc one
should wear goggles with glass opaque to ultra-violet, or should be
careful not to look at the full light. The eyes might be injured.
The crystalline lens of the eye is highly fluorescent. If much ultra-
violet got into the eyes everything would appear as in a bright
fog.

§ 314. Spectroscope used with the ultra-violet microscope. —
This enables one to determine the wave lengths of visible light
present in the fluorescent radiation from the object. Sometimes
all the colors of the rainbow are represented. As the fluorescent
light is rarely pure white, some of the colors will be more intense
than others. Sometimes only a part of the colors are represented,
and in some cases there will be absorption bands. (See Fluorescence
of the Uranyl Salts by Nichols, pp. 120-121.) In case one does
not possess a micro-spectroscope, a pocket spectroscope (§ 2Q3a)
will answer fairly well.

To make sure that all the light passing through the spectroscope
comes from a definitely fluorescing object, one must be careful to
have the object limited by the slit, or be the only object lighted
(§§ 291, 293a).

EXAMPLES OF THE USE OF THE ULTRA-VIOLET MICROSCOPE

§ 315. Anthracene crystals. — This is an example of mixed
crystals, both of which are highly fluorescent. It illustrates the
manner of arranging the microscope on the one hand, and of detect-
ing mixtures on the other.

The room is darkened, or one works at night. The mercury arc
lamp is lighted (§ 313), and the microscope put opposite the lamp-
house window at about 22 cm. distance. The quartz bulPs-eye
condenser is placed at about 8 to 10 cm. from the lamp window. To
determine quickly and certainly when the microscope, the bull's-
eye condenser and the radiation from the lamp are in line and prop-
erly arranged, one can use a card smeared with anthracene in a
strong solution of cane sugar. This fluoresces so brilliantly that

250 THE ULTRA-VIOLET MICROSCOPE [CH. VI

one can adjust the different elements accurately. The ultra-violet
beam should be focused on the specimen. At first one can focus
the light on the reflector. Then to get the beam up through the
condenser to the specimen, one can use a corex slip mount of the
anthracene in sugar. When the radiation reaches it, it will fluoresce.
Then by looking through the microscope with a 16 mm. (lox) ob-
jective and a sx or other low ocular in place, focus the crystals,
and by adjusting the prism or the bull's-eye quartz condenser or
both, get the brightest light possible. This is the procedure up
to the present for all specimens, the anthracene slide acting as a
guide or indicator. For the actual study of any specimen, the under
side of the corex slip should be in immersion contact with the top of
the condenser, using the non-fluorescing immersion petrolatum, (§ 309).

If one studies the anthracene preparation, a part of the crystals
will fluoresce blue, and a part yellow-green. The yellow-green
crystals are in excess of the blue ones. The blue crystals represent
the pure anthracene, and the yellow-green ones contain chrysogen
(Nichols and Howe, Fluorescence of the Uranyl Salts, p. 12). By
moving the screen carrier, one can see the appearance in the full
mercury arc radiation.

§ 316. Elastic tissue, physical analysis. — This tissue is abun-
dantly present in all adult vertebrates with the possible exception of
three of the lowest forms: Amphioxus, myxine and the lamprey.
It is present in increasing quantity as the animal series advance in
the zoological scale until finally in adult man almost all of the con-
nective substance in the body contains a greater or less amount of
elastic tissue.

It is relatively late in appearance in the embryo, and steadily
increases in amount with the added years. Like all the other tissues
of the body, it is surrounded by and in a kind of matrix of collage-
nous or white fibrous connective tissue. It is easily obtained in a
nearly pure form in the elastic ligament of the neck (ligamentum
nuchae) in grazing animals. The only drawback of this tissue for
illustrating the advantages of physical analysis is that there is no
striking difference in appearance to the naked eye, nor in its reac-
tions when alive and when dead.

CH. VI]

THE ULTRA-VIOLET MICROSCOPE

251

§ 317. Fresh material. — A piece of the ligamentum nuchae of a
recently killed beef animal is secured and its satiny appearance
noted by the naked eye.

With a sharp razor or safety razor blade wet with normal salt
solution, make as thin a section as possible free-hand across the end
of the ligament or a part of it. Place on a corex slip and add a drop
of normal salt solution. Cover with an ordinary cover-glass. Make
a similar section lengthwise of the ligament, and mount on a corex
slip as before.

FIG. 130 A, B. SKETCHES TO SHOW THE APPEARANCE OF A CROSS SECTION OF
ELASTIC TISSUE: A. UNDER THE ULTRA-VIOLET MICROSCOPE. B. UNDER THE
DARK-FIELD MICROSCOPE.

They look like positive and negative images of the same thing. Compare with
C, D.

If the mercury arc has been lighted for five minutes or more so
that the full amount of ultra-violet is being given off, hold the piece
of ligamentum nuchae in the path of the ultra-violet. It will
fluoresce with a white light slightly tinged with blue. The appear-
ance is striking and, when once seen, will not be forgotten. The
sections will also fluoresce so that they stand out on the corex slips.

§ 318. Microscopic examination of the fresh sections. — Making
sure that the ultra-violet is passing up through the quartz con-
denser by the use of an anthracene specimen (§ 315), put the cross
section of the elastic tissue in place and in immersion contact with
the top of the condenser. Use first a 16 mm. (lox) objective, and

252 THE ULTRA-VIOLET MICROSCOPE [Cn. VI

later an 8 mm. (2ox) objective or a higher one. When the micro-
scope is in focus, it will be seen that the cut ends of the elastic
fibers glow with a soft bluish-white radiance. Between the cut
ends the specimen will appear dark. Note carefully the location
of one of the light areas, or set the pointer of the ocular (fig. 40)
upon it. Then move the screen carrier along until the visible light
from the mercury lamp passes to the object. One will get a dark-
field image, and what was dark with the ultra-violet radiation will
appear brilliantly light in the dark-field picture. The elastic fibers
by contrast will appear dark. That is, the two appearances are the
positive and the negative images of each other (fig. 130 A, B).

Remove the cross section, and put under the microscope the
longitudinal section of the elastic tissue. Use the ultra-violet and
the visible light of the mercury arc by moving the ultra-violet filter
in place and then the corex glass. Here the elastic fibers will be
seen in their length, and will fluoresce just as did the cross sections.
The ordinary connective tissue will also behave as in the cross
section.

§ 319. For the spectral colors. — Make sure that the fluorescent
light is as brilliant as possible with the ultra-violet filter in place.
Remove the ocular and put in its stead the spectroscope. This will
show the colors making up the fluorescent light. The spectrum will
show all the colors, but the brightest part will be in the blue-green.

§ 320. Elastic tissue with the polarizing microscope. — Use the
same preparations as for the ultra-violet experiment. Put them
under the polarizing microscope, and cross the nicols. The elastic
tissue does not polarize under ordinary conditions, therefore it will
remain dark with crossed nicols. The white fibrous tissue does
polarize, that is, is anisotropic when the fibers are at right angles to
the axis of the microscope, but not when seen in cross section or in
oblique positions. Then the fibers will remain dark with crossed
nicols.

Comparing the polariscopic picture with the dark-field appearance,
it will be seen that the dark areas are relatively very large and the
bright part very small. This is due to the fact that most of the
fibers of the ordinary connective tissue are not at right angles to

CH. VI]

THE ULTRA-VIOLET MICROSCOPE

the microscope axis, and hence join the elastic tissue in producing
dark areas (fig. 130, B, C).

C D

FIG. 130. C D SKETCHRS TO SHOW THE APPEARANCE OF A CKOSS SECTION
OF ELASTIC TISSUE: C UNDKR THE POLARIZING MICROSCOPE, D. STAINED AND
UNDER THE BRIGHT-FIELD MICROSCOPE.

In C the connective tissue with fibers at right angles to the axis of the mi-
croscope polari/e light while the elastic tissue and the connective tissue not at
right angles to the microscope axis remain dark. Compare with the true amount
of ordinary connective tissue shown light in B and dark in D.

In D. the ordinary connective tissue was stained blue and the elastic tissue
pink with Mallory's connective tissue stain.

§ 321. Fixed Elastic Tissue with the ultra-violet microscope. —

It was found that elastic tissue fixed two or three days in a mixture
of Mueller's fluid and formalin (Mueller's 90 cc., strong formalin
10 cc.), washed in water for half a day and then imbedded in
paraffin in the usual manner, and sectioned with a micro-
tome, gave all the reactions shown by the fresh material. The
sections should be thin, 5/1 to ni. No albumen fixative should be
put on the slide for it fluoresces. The slips for mounting should be
of corex or quartz. After the sections are dry on the slip, the
paraffin is removed by xylene and the sections covered with an
ordinary cover-glass on which *is a large* drop of the petrolatum.
Seal the cover with shellac. Examine exactly as for the fresh ma-
terial, both with the ultra-violet and the polarized light. The
appearances are practically the same as for the fresh material.

THE tfLTRA-VIOLET MICROSCOPE

[CH. VI

PHYSICAL ANALYSIS OF STRUCTURE
IN ULTRA-VIOLET AND IN VISIBLE RADIATION

COMPARISON WITH STAINING
DATE AND NAMK

Living

NAKKD-EYK
Appearance

Fresh

Fixed

ULTRA-VIOLFT

Living

Microscope with Corex
Glass Filter and

Fresh

Dark-Field

Fixed

Living

MICRO-SPECTROSCOPE

For Colors in

Fresh

Fluorescence

Fixed

Living

POLARIZING
Microscope

Fresh

Fixed

MICRO-INCINERATION
FOR MINERAL CON-
TENTS

Fixed

VITAL AND OTHER STATNING
In Comparison with
Physical analysis

§ 322. Comparison by different methods. — It is of great interest
to compare physical appearances under the polarizing and ultra-violet
microscope with neighboring sections of the same tissue stained in
various ways; also of incinerated specimens. Such a comparison

CH. VI]. THE ULTRA-VIOLET MICROSCOPE 255

gives one a conception of how complex are the structures of the body,
and how limited is the information gained by any single method of
treatment (see fig. 130 AB, CL).

Fcr the staining see under elastic stains in § 582.

Fcr the orcein stain, ccunterstain with methylene blue to bring
out the nuclei, and note that these are confined to the ordinary
connective tissue, none being found in the clastic tissue.

ULTRA-VIOLET WITH MICROSCOPIC ANIMALS. CILIATED
EPITHELIUM, ETC.

§ 323. Minute animals and ultra-violet. — To test the effect of
ultra-violet on minute animal life, make a preparation of the living
forms found in an infusion (§§ 210, 543). Use corex for a slip, and
the water in which the animals naturally live for a normal mounting
medium. Make immersion contact with the condenser and allow
the ultra-violet passing through the red-purple corex (fig. 129) to act
on the organisms for, say five minutes, then examine them with the
visible light of the daylight lamp, (fig. 79, or 80). Have a similar
preparation under the usual dark-field microscope as control. Com-
pare the appearance of the two preparations. Continue the short
exposures to ultra-violet and find out how long the animals live, and
what changes take place. To prevent the drying out of the mount-
ing liquid, seal the cover with oil as for a fresh blood preparation
(§ 21 1). Do the same for the control.

For a preparation of ciliated cells, scrape the roof of the throat of
a live frog. Mount on a corex slip using aqueous humor, or the
blood of the frog for a mounting medium. SealAh& cover as for
blood. Prepare a control in the same way. ExpSe to ultra-violet
as directed for the minute aninials, ajjd note any effect.

GREEN PLANT TISSUE UNDER 4$£ U£*RA- VIOLET MICROSCOPE

§ 324. Fluorescence of plant structures. — The fact that the
green substance of plants fluoresces red [was discovered by Sir
David Brewster in 1833. (See Stokes, Philis. Trans., Vol. 142, pp.
463-464.) Brewster used a solution of chforophyll in alcohol, and

256 THE ULTRA-VIOLET MICROSCOPE [Cn. VI

that is still the ordinary method of showing the amazing change in
color when chlorophyll is submitted to ultra-violet radiation,

It is not necessary to put the chlorophyll in solution to get the
red fluorescence. This was strikingly shown by the chloroplastids
obtained from the leaves of the snapdragon (antirrhinum) which
had become macerated in a flower vase. Following this hint, sec-
tions were made free-hand of the green leaves of many different
plants, mounted on corex slips in water, and examined under the
ultra-violet microscope. All of them showed the red chlorophyll
bodies. It was found later that the easiest and most effective way
to get sections of the most favorable thickness, and to secure isolated
chloroplastids, was to put the blade of grass or the other chlorophyll-
bearing structure on a corex slip in some water and scrape it with a
moderately sharp scalpel or other knife. The fragments thus ob-
tained show everything. One of the most strikingly beautiful and
instructive preparations was made in this way by placing a blade
of grass on a corex slip and scraping it. Some of the fragments
showed the individual chloroplastids in their cells. Other chloro-
plastids were free. All fluoresced a beautiful red. The cellulose
veins extend lengthwise, and fluoresce a brilliant bluish white.
The appearance was then like a ribbon with brilliant, narrow, white
stripes, and broad red ones.

Under the polarizing microscope with crossed nicols the cellulose
glowed with a brilliant white, but the chloroplastids did not polar-
ize. However, the light given off by the cellulose veins and the cell
walls of the plastids and the parenchyma of the tissue is enough
to bring out the green color of the chlorophyll. Occasionally in
these scraped preparations some of the chloroplastids are iso-
lated, and if one is studied with a high power, it will be seen that
the wall polarizes, and that the chlorophyll is green even with
crossed nicols.

It is believed that the physical analysis by means of the ultra-
violet microscope, the polarizing and the dark-field microscope will
prove of as much help to the botanist as to the animal histologist.

CH. VI] THE ULTRA-VIOLET MICROSCOPE 257

ULTRA-VIOLET FOR NAKED EYE DEMONSTRATIONS

Unless one has paid attention to such matters, it is unbelievable
that the same object under different kinds of light or radiation
should appear so strikingly different. For a good example, take a
cheap, red bandanna handkerchief. Look at it by daylight, by
kerosene light, and by the ordinary mazda light. Then hold it in
the beam of ultra-violet, next in the visible mercury light. Chloro-
phyll has already been cited. Quinine in water does not show at all
in daylight, but in the ultra-violet it glows with a wonderful blue-
white radiance.

Figured dress goods, cheap, brightly colored handkerchiefs, neck-
ties, etc., give a change in appearance which is truly marvelous.

These naked-eye appearances with different radiation make the
thoughtful person appreciate how many facts must be taken into
consideration in order to gain a true conception of the appearance
of what seem the simplest things in nature and art. After such an
exhibition one feels deeply the need of caution in one's statements,
and the danger of being dogmatic about anything, unless all the
conditions and the circumstances are thoroughly understood.

COLLATERAL READING

In the first place should be mentioned the fundamental contribution of
George Gabriel Stokes. Philos, Trans, of the Royal Society, Vol. 142, p. 555 et.
sq. Change in the Refrangibility of Light. On p. 470 in a note at the bot-
tom he says: "I do not like this term (David Brewster's Internal Dispersion)
and am almost inclined to coin a word and call the appearance Fluorescence, from
fluor-spar as the analagous opalescence is derived from the name of a mineral. "
This term has received universal approval.

On p. 503 in describing fluorescence he says: "We may express the result
extremely well by saying that the fluid or solid medium (which fluoresces) is self-
luminous so long as it is under the influence of the active light."
NICHOLS, EDWARD L. AND HOWES, HORACE L. in collaboration with MERRITT,
ERNEST, WTLBER, D. T. AND WICK, FRANCES G. — Fluorescence of the
Uranyl Salts. The Carnegie Institution of Washington, 1919. A funda-
mental monograph. The historical summary is very helpful.
NICHOLS, EDWARD L., HOWES, H. L. AND WILDER, D. T. — Cathode Lumines-
cence of Incandescent Solids. Carnegie Institution of Washington, 1928.
The definitions in the field of fluorescence are of great help. The main part
of the monograph deals with what may be called the fluorescence of hot
solids.

258 THE ULTRA-VIOLET MICROSCOPE [Cn. VI

HERSCHEL, WILLIAM. — Annalen der Physik Bd. 7, 1801. pp. 137-157. Dis-
covery of infra-red.

RITTER, JOHANN WiLHELM. — Annalen der Physik Bd. 12, 1803, pp. 409-415.
Says he found the ultraviolet blackened chlorid of silver Feb. 22, 1801.

POLICARD. — Built, d'histologie, 1925, Ultraviolet in histology.

GATES, FREDERICK L. — Study of bacteriacidal action of ultraviolet. Studies
from the Rockefeller Institute, Vol. 73, pp. 9-26.

RUSSELL & RUSSELL. — Ultra- Violet radiation and actinotherapy. 3d, edition,
1928.

KING, MCKENZIE. — Practical Ultra- Violet Light Therapy, 1926.

LUCKIESH, M. — Artificial Sunlight, combined with radiation for health and
for vision. 1900.

DANCKWORTT, P.W. — Lumineszenz- Analyse im filtrierten Ultravioletten Licht.
Zweite, erwerterte Auflage. 1929. American Journal of Physical Therapy,
1924-1931. British Journal of Actinotherapy, now British Journal of Physical
Medicine. Strahlentherapie Bd. 1-41. Quarterly Cumulative Index Medicus,
1916-3 r . For current work on all medical topics including ultra-violet. Journal
of the Optical Society of America.

GAGE, H. P. - Hygienic Effects of Ultraviolet Radiation. Trans. Illuminating
Engineering Soc. Vol. XXV, 1930.

MAUGHAM, GEORGE H. AND DYE, J. A. — Biological measurements of ultra-
violet sources. American Journal of Physical Therapy (1924-1931). De-
cember, 1930, January, 1931.

CALKINS, GARY N.— Effect of ultraviolet rays. Biol. Built. LVII, pp. 59-68.
For fluorescing substances see all of the above and especially, Stokes and
Danckwortt.

LUCAS, FRANCIS F. — The architecture of living cells — Recent advances in methods
of biological research — Optical sectioning with the ultra violet microscope.
Technical publications of the Bell Telephone System, Monograph B 514, Oct.
1930,

LUCAS, F. F. AND STARK, MARY B. — A study of livirg germ cells ... by means of
the ultra violet microscope. Jour. Morphology. Vol. 52, (1931) pp. 91, 115.
Many photo-micrographs.

MORGAN, DR. ANNA H. — Field Book of Ponds and Streams. N. Y. 1930.

KIDLKY, GRANT AND TRTPP. — Fluorescence in Ultra-violet Light, being Vol. 7 of
a series of monographs on applied chemistry. Second edition, 1935.

CHAPTER VH

INTERPRETATION OF APPEARANCES
§§325-358; FIGURES 131-142

§ 325. Appearances which seem perfectly unmistakable with a
low power may be found erroneous or very inadequate with high
powers; for details of structure which cannot be seen with a low
power may become perfectly evident with a higher power or a more
perfect objective. On the other hand, the problems of microscopic
structure become more and more complex with increased precision
of* investigation and more perfect optical appliances, for structures
that appeared intelligible with a less perfect microscope may show
complexities in their details of structure with the more perfect
microscope which open up an entirely new field for interpretation.
Further, if the specimen is viewed with the dark-field microscope,
the polarizing and the ultra-violet microscope, wholly new appear-
ances are almost sure to arise (§ 357, Ch. Ill, IV, V, VI).

One must always be on the lookout for errors in judgment induced
by color effects due to purely optical means and to color in the speci-
men, and also to avoid confusing refraction, reflection, and diffrac-
tion effects with pigments, or actual structures of any kind. It is
not infrequent in searching for malarial pigment in the red blood
corpuscles to mistake the dark-looking crenations on the corpuscles
for the pigment sought (§ 326).

The need of the most careful observation and constant watchful-
ness lest the appearances may be deceptive is thus admirably stated
by Dallinger. (See Carpenter-Dallinger, p. 427): " The correctness of
the conclusions which the microscopist will draw regarding the nature
of any object from the visual appearances which it presents to him
when examined in the various modes now specified will necessarily
depend in a great degree upon his previous experience in microscopic
observation and upon his knowledge of the class of bodies to which
the particular specimen may belong. Not only are observations of

259

26o INTERPRETATION OF APPEARANCES [Cn. VII

any kind liable to certain fallacies arising out of the previous notions
which the observer may entertain in regard to the constitution of
the objects or the nature of the actions to which his attention is di-
rected, but even the most practised observer is apt to take no note
of such phenomena as his mind is not prepared to appreciate. Errors
and imperfections of this kind can only be corrected, it is obvious, by
general advance in scientific knowledge; but the history of them
affords a useful warning against hasty conclusions drawn from a too
cursory examination. If the history of almost any scientific investi-
gation were fully made known, it would generally appear that the
stability and completeness of the conclusions finally arrived at had
been only attained after many modifications, or even entire altera-
tions of doctrine. And it is therefore of such great importance as
to be almost essential to the correctness of our conclusions that they
should not be finally formed and announced until they have been
tested in every conceivable mode. It is due to science that it should
be burdened with as few false facts [artifacts] and false doctrines
as possible. It is due to other truth-seekers that they should not
be misled, to the great waste of their time and pains, by our errors.
And it is due to ourselves that we should not commit our reputation
to the chance of impairment by the premature formation and publi-
cation of conclusions which may be at once reversed by other ob-
servers better informed than ourselves, or may be proved fallacious
at some future time, perhaps even by our own more extended and
careful researches. The suspension of the judgment whenever there
seems room for doubt is a lesson inculcated by all those philosophers
who have gained the highest repute for practical wisdom; and it is
one which the microscopist cannot too soon learn or too constantly
practise."

The general law for the whole matter is to study the object in
every way possible (§ 358).

For the experiments, §§ 327-340, no condenser is to be usedy except
in a part of § 340.

§ 326. "The distinction between a dark element which is referable to pigment
and a dark element which is referable to the deflection of light can generally be
made by watching the effect produced by the alteration of the focus. Where the

CH. VII]

INTERPRETATION OF APPEARANCES

261

dark element corresponds to a point from which light is deflected a change of the
focus will be associated with a change from dark to bright. Where pigment is in
question a change of focus will substitute only a more diffuse for a less diffuse
dark element." (Wright, p. 44.)

§ 327. Dust or Cloudiness on the Ocular. — Employ the 16 mm.
IQX objective, 4x or 5x ocular, and fly's wing as object.

Unscrew the field lens and put some particles of lint from dark
cloth on its upper surface. Replace the field lens and put the ocular
in position (§ 85). Light the field well and focus sharply. The
image will be clear, but part of the field will be obscured by the
irregular outline of the particles of lint. Move the object to make
sure this appearance is not due to it.

Grasp the ocular by the milled ring just above the tube of the
microscope and rotate it. The irregular objects will rotate with the
ocular. Cloudiness or particles of dust on any part of the ocular
may be detected in this way.

Unscrew the field lens and remove the lint before proceeding.

§328. A small bright field. — With low objectives (25-50 mm.
[5x~3.2x]) if too small a diaphragm is used and put close to the
object, only the central part of the field will be illuminated, and
around the small light circle will be seen a dark ring (fig. 132). If

FIGS. 131, 132. THE MICROSCOPIC FIELD COMPLETELY AND ONLY PARTLY

ILLUMINATED.

A The field completely illuminated; a net micrometer is used as object.
B The field is only partly illuminated; the same net micrometer is used as
object, but not all of it appears in the partially lighted field.

262 INTERPRETATION OF APPEARANCES [Cn. VII

the diaphragm is lowered or a sufficiently large one employed, the
entire field will be lighted (fig. 131). (See also § 131 for diaphragms
with the condenser).

§ 329. Relative position of objects or parts of the same object. —
The general rule is that objects highest up come into focus last in
focusing up, first in focusing down.

§ 330. Objects having plane or irregular outlines. — As object
use three printed letters in stairs mounted in Canada balsam (fig.
133). The first letter is placed directly upon the slide, and covered
with a small piece of glass about as thick as a slide. The second
letter is placed upon this and covered in like manner. The third

FIG. 133. LETTERS IN STAIRS TO DETERMINE RELATIVE POSITION BY
FOCUSING UP AND DOWN.

letter is placed upon the second thick cover and covered with an
ordinary cover-glass. The letters should be as near together as
possible, but not overlapping. Employ the same ocular and ob-
jective as above (§327).

Lower the tube till the objective almost touches the top letter;
then look into the microscope and slowly' focus up. The lowest letter
will first appear and then, as it disappears, the middle one will ap-
pear and so on. Focus down, and the top letter will first appear,
then the middle one, etc. The relative position of objects is de-
termined exactly in this way in practical work.

For example, if one has a micrometer ruled on a cover-glass 0.15-
0.25 mm. thick, it is not easy to determine with the naked eye which
is the ruled surface. But if one puts the micrometer under a micro-
scope and uses a 4 mm. (4ox) objective, it is easily determined. The
cover should be laid on a slide and focused till the lines are sharp.
Now, without changing the focus in the least, turn the cover over.
If it is necessary to focus up to get the lines of the micrometer

CH. VII] INTERPRETATION OF APPEARANCES 263

sharp, the lines are on the upper side. If one must focus down, the
lines are on the under surface. With a thin cover and delicate lines
this method of determining the position of the rulings is of consider-
able practical importance.

§ 331. Determination of the form of objects. — The procedure is
exactly as for the determination of the form of large objects. That
is, one must examine the various aspects. For example, if one were
placed in front of a wall of some kind, one« could not tell whether it
was a simple wall or whether it was one side of a building unless in
some way one could see more than the face of the wall. In other
words, in order to get a correct notion of any body, one must ex-
amine more than one dimension, — two for plane surfaces, three for
solids. So for microscopic objects, one must in some way examine
more than one face. To do this with small bodies in a liquid the
bodies may be made to roll over by pressing on one edge of the
cover-glass. And in rolling over the various aspects are presented
to the observer. With solid bodies, like the various organs, correct
notions of the form of the elements can be determined by studying
sections cut at right angles to each other. The methods of getting
the elements to roll over, and of sectioning in different planes, are in
constant use in histology, and the microscopist who neglects to see
all sides of the tissue elements has a very inadequate and often a
very erroneous conception of their true form.

§ 332. Transparent objects having curved outlines. — The success
of these experiments will depend entirely upon the care and skill
used in preparing the objects in lighting and in focusing.

Employ a 4 mm. (4ox) or higher objective and a lox ocular for
all the experiments. It may be necessary to shade the object (§ 155)
to get satisfactory results. When a diaphragm is used, the opening
should be small and it should be close to the object.

§ 333. Air bubbles. — Prepare these by placing a drop of thin
gum arabic mucilage on the center of a slide and beating it with a
scalpel blade until the mucilage looks milky from the inclusion of air
bubbles. Put on a cover-glass but do not press it down.

§ 334. Air bubbles with central illumination. — Shade the object,
and with the plane mirror light the field with central light (fig. 20).

264

INTERPRETATION OF APPEARANCES

CH. VII]

Search the preparation until an air bubble is found appearing
about i mm. in diameter, get it into the center of the field, and if
the light is central the air bubble will appear with a wide, dark,

circular margin and a
small, bright center. If
the bright spot is not
in the center, adjust
2 +^f> the mirror until it is.

This is the simplest
and surest method of
telling when the light
is central or axial when
no condenser is used
(§ no).

Focus both up and
down, noting that, in
focusing up, the central
spot becomes very clear
and the black ring very
sharp. On elevating
the tube of the micro-
scope still more, the
center becomes dim,
and the whole bubble
loses its sharpness of
outline.

§ 335. Air bubbles with oblique illumination. — Remove the
substage of the microscope and all the diaphragms. Swing the
mirror so that the rays may be sent very obliquely upon the object
(fig. 134). The bright spot will appear no longer in the center, but
on the side away from the mirror (fig. 136 A).

§ 336. Oil globules. — - Prepare these by beating a small drop of
clove or other oil with gum arabic mucilage on a slide and covering
as directed for air bubbles (§ 333), or use a drop of milk in a drop
of water.

§ 337. Oil globules with central illumination. — Use the same

FIGS. 134-135. OBLIQUE ILLUMINATION WITH A
MIRROR AND WITH A CONDENSER.

j The light is shown to be oblique with ray c\
rays A B are central. The arrows indicate the
path of the rays. (For the objective see expla-
nation of figure 44.)

2 Abbe condenser with an eccentric dia-
phragm (j) admitting light only on one side.

Axis The principal optic axis. Ob Objective.

S A xis Secondary axis.

CH. VII]

INTERPRETATION OF APPEARANCES

265

diaphragm and light as above (§ 334). Find an oil globule appearing
about i mm. in diameter. If the light is central, a bright spot will
appear in the center. Focus up and down and note that the dark
ring is narrower than with air and that the bright center of the oil
globule is clearest last in focusing up.

§ 338. Oil globules with oblique illumination.
— Remove the substage, etc., as above, swing the
mirror to one side and light with oblique light.
The bright spot will be eccentric, and will ap-
pear to be on the same side as the mirror (fig.

FIG. 136. SMALL
AIR BUBBLE (A)
AND OIL GLOBULE
(O) WITH OBLIQUE
LIGHT.

The arrow indi-
cates the direction
of the light.

§ 339. Oil and air together. — Make a prepara-
tion exactly as described for air bubbles (§ 333),
and add at one edge a little of the mixture of oil
and mucilage (§ 336); cover and examine.

The substage need not be used in this ex-
periment. Search the preparation until an air
bubble and an oil globule, each appearing about
i mm. in diameter, are found in the same
field of view. Light first with central light, and
note that, in focusing up, the air bubble comes
into focus first and that the central spot is smaller than that
of the oil globule. Then, of course, the black ring will be
wider in the air bubble than in the oil globule. Make the light
oblique. The bright spot in the air bubble will move away from the
mirror, while that in the oil globule will move toward it (fig. 136).

As the air bubble is of less refractive index than the mucilage, it
will act like a concave lens (fig. 137), while the oil globule, having a
greater refractive index than the mucilage, will act as a convex lens
(fig. 137, § 33Qa).

It is possible to distinguish oil and air optically, as described
above, only when quite high powers are used and very small bubbles
are selected for observation. If a 16 mm. (lox) objective is used
instead of a 4 mrn. (4ox), the appearances will vary considerably from
that given above for the higher power. It is well to use a low as
well as a high power. Marked differences will also be seen in the

266

INTERPRETATION OF APPEARANCES

[CH. VII

appearances with objectives of small and of large aperture, as the
larger aperture takes in more oblique rays and hence the black

margin is narrowed (§ 341).

FIG.

137. AIR BUBBLES AND
GLOBULE IN WATER.

Axis The principal optic axis.

F, F The principal foci of the air
and oil. As the air is less refractive
than water its focus is virtual. The
focus of the oil globule is real, as its
refraction is greater than water.

It should be remembered
that the image in the compound micro-
scope is inverted (fig. 18); hence the
bright spot really moves toward the
mirror for air, and away from it for
oil.

£ 340. Air and oil by reflected
light. — Use the same prepara-
tion as in § 339. Cover the dia-
OIL phragm or mirror so that no
transmitted light can reach the
preparation. The oil and air
will appear like globules of silver
on a dark ground. The part
that was darkest in each with
transmitted light will be lighted,

and the bright central spot will be somewhat dark. Use also the
condenser and dark-ground illumination.

Experiments in which the substage condenser is used (§§ 341-
348).

§ 341. Distinctness of outline. — In refraction images this de-
pends on the difference between the refractive power of a body and
that of the medium which surrounds it. The oil and air were very
distinct in outline, as both differ greatly in refractive power from the
medium which surrounds them, the oil being more refractive than
the mucilage and the air less (fig. 137).

Place a fragment of a cover-glass on a clean slide, and cover it
(fig. 138). Use it as object and employ the 16 mm. (lox) objective
and 8x or lox ocular. The fragment will be outlined by a dark
band. Put a drop of water at the edge of the cover-glass. It will
run in and immerse the fragment. The outline will remain distinct,
but the dark band will be somewhat narrower. Remove the cover-
glass, wipe it dry, and wipe the fragment and slide dry also. Put a
drop of 50% glycerin on the middle of the slide and mount the

CH. Vll]

INTERPRETATION OF APPEARANCES

267

FIG. 138. FINE
FORCEPS FOR PLAC-
ING COVER-GLASSES
ON SPECIMENS.

fragment of cover-glass in that. The dark contour will be much
narrower than before.

Draw a solid glass rod out to a fine thread. Mount one piece in
air, and the other in 50% glycerin. Put a
cover-glass on each. Employ the same optical
arrangement as before. Examine the one in air
first. There will be seen a narrow, bright band,
vuth a wide, dark band on each side (fig. i3Qa).
The one in glycerin will show a much wider
bright central band, \\ith the dark borders cor-
respondingly narrow (fig. i3Qb). The dark
contour depends also on the numerical aperture
of the objective — being \\ider with low aper-
tures. This can be readily understood when it
is remembered that the greater the aperture the
IT. ore oblique the rays of light that can be received,
and that the dark band simply represents an
area in which the rays are so greatly bent or
refracted (fig. 137) that they cannot enter the objective and con-
tribute to the formation of the
image; the edges are dark
simply because no light from
them reaches the observer.

If the glass rod or any other
object were mounted in a
medium of the same color and
refractive power, it could not
be distinguished from the
medium.

The effect of the immersing liquid on the contour bands around
any transparent object is made of practical use in the determination
of the refractive index of crystals and other bodies. When the
crystal and liquid are of the same index there will be no band, and
the more they differ, the wider will be the band. As shown in
§§ 333~~34°> lighting with oblique light, also focusing up and down,
will indicate whether the crystal is of greater or less index than the

FIG. 139.

GLASS RODS IN AIR AND
IN GLYCERIN.

a Glass rod in air and viewed by
central transmitted light.

b Glass rod mounted in 50% gly-
cerin; the dark border is narrower than
when mounted in air.

268 INTERPRETATION OF APPEARANCES [Cn. VII

liquid. For this method a series of liquids of known index of re-
fraction must be at hand. For a complete discussion, see Chamot,
p, 185, Chamot and Mason, vol. I, p. 366.

A very striking and satisfactory demonstration may be made by
painting a zone or band of eosin or other transparent color on a
solid glass rod, and immersing the rod in a test tube or vial of cedar
oil, clove oil, or turpentine. Above the liquid the glass rod is very
evident, but under the liquid it can hardly be seen except where
the red band is painted on it. This is a good example of a color
image and of a refraction image to the naked eye (§ 152).

§ 341a. Some of the rods have air bubbles in them, and then there results
a capillary tube when they are drawn out. It is well to draw out a glass tube
into a fine thread and examine it as described. The central cavity makes the
experiment much more complex.

§ 342, Highly refractive. — This expression is often used in de-
scribing microscopic objects (medulla ted nerve fibers, for example),
and means that the object will appear to be bordered by a wide,
dark margin when it is viewed by transmitted light. And from the
above (§ 341), it would be known that the refractive power of the
object and the medium in which it was mounted must differ con-
siderably.

§ 343. Doubly contoured. —
This means that the object is
bounded by two usually parallel,
dark lines with a lighter band
between them. In other words,
FIG. 140. SOLID GLASS ROD COATED t^e Object ;s bordered by (i) a
WITH COLLODION TO SHOW DOUBLE , , ,. / s r u <. r i j
CONTOUR. dark hne> (2) a "§"* ban(1, and

(3) a second dark line.

This may be demonstrated by coating a fine glass rod (§ 341) with
one or more coats of collodion or celloidin and allowing it to dry,
and then mounting in 50% glycerin as above (§ 341). Employ a
4 mm. (4ox) or higher objective, light with transmitted light, and it
will be seen that where the glycerin touches the collodion coating there
is a dark line, next this is a light band, and finally there is a second
dark line where the collodion is in contact with the glass rod (fig. 140).

CH. VII] INTERPRETATION OF APPEARANCES 269

§ 343a. The collodion used is a 6 % solution of soluble cotton (parlodion, collo-
dion, pyroxylin) in equal parts of sulphuric ether and 95%, or, preferably, ab-
solute alcohol. It is well to dip the rod two or three times in the collodion and
to hold it vertically while drying. The collodion will gather in drops, and one
will see the difference between a thick and a thin membranous covering (fig. 140).

§ 344. Optical section. — This is the appearance obtained in ex-
amining transparent or nearly transparent objects with a microscope
when some plane below the upper surface of the object is in focus.
The upper part of the object, which is out of focus, obscures the
image but slightly. By changing the position of the objective or
object, a different plane will be in focus and a different optical sec-
tion obtained. The most satisfactory optical sections are obtained
with high objectives having large aperture. : ,

Nearly all the transparent objects studied may be viewed in
optical section. A striking example will be found in studying mam-
malian red blood corpuscles on edge. The experiments with the
solid glass rods (fig. 139) furnish excellent and striking examples of
optical sections.

§ 345. Currents in liquids. —Employ a 16 mm. (lox) objective,
and as object put a few particles of carmine, starch, or chalk dust on
the middle of a slide and add a drop of water. Grind the carmine or
other substance well with a scalpel blade; leave the preparation
uncovered. If the microscope is inclined, a current will be pro-
duced in the water, and the particles will be carried along by it.
Note that the particles seem to flow up instead of down; why is
this? How would it appear to flow with an erecting microscope?

§ 346. Velocity under the microscope. — In studying currents or
the movement of living things under the microscope, one should not
forget that the apparent velocity is as unlike the real velocity as the
apparent size is unlike the real size. If one consults (fig. 51, it will
be seen that the actual size of the field of the microscope with the
different objectives and oculars is inversely as the magnification.
That is, with great magnification only a small area can be seen.
The field appears to be large, however, and if any object moves
across the field, it may appear to move with great rapidity, whereas
if one measures the actual distance passed and notes the time, it will
be seen that the actual motion is quite slow. One should keep this

270 INTERPRETATION OF APPEARANCES [Cn. VII

in mind in studying the circulation of the blood. The truth of
what has just been said can be easily demonstrated in studying
the circulation in the gills of necturus, or in the frog's foot, by
using first a low power in which the field is actually of considerable
diameter (fig. 51; Table, §94) and then using a high power.
With the high power the apparent motion will seem much more
rapid. For spiral, serpentine, and other forms of motion, see Car-
pen ter-Dallinger, p. 433.

§ 347. Pedesis or Brownian movement. — Employ the same
object as above, but a 4 mm. (4ox) or higher objective in place of
the 1 6 mm. (lox). Make the body of the microscope vertical so
that there may be no currents produced. Use a small diaphragm
and light the field well. Focus and there will be seen in the field
large motionless masses, and between them small masses in constant
motion. This is an indefinite, dancing, or oscillating motion.

This indefinite but continuous motion of small particles in a
liquid is called Brownian movement or pedesis; also, but improperly,
molecular nlovement, from the smallness of the particles.

The motion is increased by adding a little gum arabic solution or a
slight amount of silicate of soda or soap; sulphuric acid and various
saline compounds retard or check the motion. One of the best ob-
jects is lamp-black ground up in water with a little gum arabic.
Carmine prepared in the same way, or simply in water, is excellent;
and very finely powdered pumice-stone in water has for many years
been a favorite object. Pedesis is exhibited by all solid matter if it
is finely enough divided. For high powers, and with the dark-field
microscope a very dilute mixture of carbon ink in water is excellent.
For the dark-field microscope the chylomicrons of the blood show
the Brownian movement admirably (§ 212).

Compare the pedetic motion with that of a current by slightly
inclining the tube of the microscope. The small particles will con-
tinue their independent leaping movements while they are carried
along by the current. The pedetic motion makes it difficult to ob-
tain good photographs of milk globules and other small particles.
The difficulty may be overcome by mixing the milk with a very
weak solution of gelatin and allowing it to cool (10 % gelatin is good).

CH. VII] INTERPRETATION OF APPEARANCES 271

Until recently no adequate explanation of this movement had
been offered. At the present time it is believed to be due to the
kinetic activity of matter, and in itself to be one of the best proofs
of that activity. This is what is said by Rutherford: "The
character of the Brownian movement irresistibly impresses the
observer with the idea that the particles are hurled hither and
thither by the action of forces resident in the solution, and that
these can only arise from the continuous and ceaseless movement of
the invisible molecules of which the fluid is composed." " What-
ever may be the exact explanation of this phenomenon, there can
be but little doubt that it results from the movements of the mole-
cules of the solution, and is thus a striking, if somewhat indirect,
proof of the general correctness of the kinetic theory of matter."
Nature, Vol. 81, 1909, pp. 257-263; Science, N. S., Vol. 30, 1909,
pp. 289-303.

By the aid of the ultra-microscope it has been shown that the
particles in smoke, etc., exhibit the pedetic movement even more
strikingly than do those in liquids.

§ 348. Demonstration of pedesis with the polarizing microscope.
— The following demonstration shows conclusively that the pedetic
motion is real and not illusory (Ranvier, p. 173).

Open the abdomen of a dead frog (an alcoholic or formalin speci-
men is satisfactory). Turn the viscera to one side and observe the
small whitish masses at the emergence of the spinal nerves. With
fine forceps remove one of these and place it on the middle of a clean
slide. Add a drop of water, or of water containing a little gum
arabic. Rub the white mass around in the drop of liquid and soon
the liquid will have a milky appearance. Remove the white mass,
place a cover-glass on the milky liquid, and seal the cover by paint-
ing a ring of castor oil all around it, the ring being half on the slide and
half on the cover-glass. This is to avoid the production of currents
by evaporation.

Put the preparation under the microscope and examine with first
a low power, then a high power (4 mm. 4ox). In the field will be
seen multitudes of crystals of carbonate of lime; the larger crystals
are motionless, but the smallest ones exhibit marked pedetic movement.

272 INTERPRETATION OF APPEARANCES [Cn. VII

Use the micro-polariscope, light with great care, and exclude all
adventitious light from the microscope by shading the object (§ 155)
and also by shading the eye. Focus sharply and observe the pedetic
motion of the small particles, then cross the polarizer and analyzer,
that is, turn one or the other till the field is dark. Part of the large
motionless crystals will shine continuously and a part will remain
dark, but small crystals between the large ones will shine for an
instant, then disappear, only to appear again the next instant. This
demonstration is believed to furnish absolute proof that the pedetic
movement is real and not illusory.

For the help given by the micro-spectroscope see Ch. V.

§ 349. Use of dark-ground illumination for interpreting appear-
ances. — Dark-ground illumination is almost invaluable for bringing
out details of structure and for showing movement in living things.
The granules and different parts in living cells and minute organisms
are of so nearly the same refractive index that it is exceedingly diffi-
cult to differentiate them with the ordinary methods of illumination.
On the other hand, with dark-ground illumination the different
structures stand out with the greatest clearness.

§ 350. Specimens to use for dark-ground illumination. — (i) Or-
ganisms from hay infusion. Use for the infusion a small fruit jar or
other glass dish. Go to a stream or pond and from a shallow, stag-
nant pool along the edge take some of the surface of the mud and
put it into the jar with some of the water. Add some of the dead
grass found along the edge of the pond; cut up into short pieces.
Set in a warm, dimly lighted or dark place for a day or longer. This
should soon be alive with all sorts of minute living things.

If it is not easy to get the water, mud and dead grass, fairly
good results are obtained by putting some ordinary hay in water of
any kind.

With fine forceps take a leaf or piece of stem of the dead grass and
put it on a slide. Move it around and press it down so that a good
drop of liquid and debris will be on the slide. Remove the grass and
cover the liquid with a 0.15 mm. cover-glass. This should be
studied fresh with a 4 mm. (4ox) objective, lox ocular, and trans-
mitted light. Then put in place the dark-ground illuminator, center

CH. VII] INTERPRETATION OF APPEARANCES 273

it and add some homogeneous liquid to the top of the condenser and
run it up till the liquid is in contact with the under side of the slide.
Put a drop of homogeneous liquid on the cover-glass and use a
homogeneous immersion objective in which the aperture has been
cut down to 0.85 N.A. or less.

(2) Saliva. Put a drop of saliva on a slide and cover it with a
0.15 mm. cover-glass. Examine as in (i).

Note the pedetic or Brownian movement of the granules in the
rounded salivary corpuscles, the minute granules in the broad oval
epithelium, etc.

(3) Fresh blood. — For preparing and studying this, follow the
directions given in § 211.

§ 351. Difference of appearance due to difference of focus. —
If one takes a geometrical pattern like that shown in fig. 141 and
looks at it in the ordinary way, the appearance is that of white spots
on a dark field. If now the head is held closer and closer to the
picture, an inversion \\ill take place and the appearance is of dark
spots in a ^hite field. This illustrates how difficult it is to deter-
mine the real appearance under the microscope of objects having
geometrical patterns, especially if there are
several of them superimposed, as with the
wire gauze experiment (§ 355). The image
is often just as satisfactory in one focus
as in another, although the appearance
changes very markedly in the two posi-
tions.

§ 352. Comparing two microscopic fields
side by side. — It is so difficult to carry
in the mind the exact appearance of any PA^RNI4ITO GSH°O™£
structure or complex pattern, that many FERENCE OF APPEARANCE
efforts have been made to have the micro- D*PENDING ON THE
scopic images side by side so that they can (From
be looked at at the same time. This has
been accomplished by using two microscopes and projecting two
fields side by side, as can be done by having two microscopes like the
one shown in fig. 182.

274

INTERPRETATION OF APPEARANCES

[CH. VII

Another method is by means of a comparison ocular (fig. 142).
Then two objects under two microscopes have the images side by
side in the ocular, half the field being taken up by one object and

FIG. 142. COMPARISON OCULAR FOR PLACING HALF THE FIELDS OF

Two MICROSCOPES SIDE BY SIDE. (Rl R*).
(Bausch & Lomb Optical Co., from Chamot).

T1 To fit into the tube of the left microscope.

T2 To fit into the tube of the right microscope.

P Prisrn to reflect the beam from the right microscope to the prism 722,
whence it is reflected up through the ocular (O) into the right half of the field
shown above in the face view.

Pl Rl The prism and left half of the field shown in face view in the diagram
at the top.

half by the other; then the eye can compare two structures side by
side.

§ 353. Muscae volitantes. — These specks or filaments in the
eyes due to minute shreds or opacities of the vitreous humor, some-

CH. VII] INTERPRETATION OF APPEARANCES 275

times appear as part of the object as they are projected into the
field of vision. They may be seen by looking into the well-lighted
microscope. They may also be seen by looking at brightly illumi-
nated snow or other white surface. By studying them carefully it will
be seen that they are somewhat movable and float across the field
of vision, and thus do not remain in one position as do the objects
under observation. Furthermore, one may, by taking a little pains,
familiarize himself with the special forms in his own eyes so that the
more conspicuous at least may be instantly recognized.

§ 354. Miscellaneous observations. — In addition to the above
experiments it is very strongly recommended that the student follow
the advice of Beale, p. 248, and examine first with a low power then
with a higher power; mounted dry, then in water; lighted with re-
flected light, then with transmitted light, the following: potato,
wheat, rice, and corn starch (easily obtained by scraping the potato
and the grains mentioned); bread crumbs; portions of feather
(portions of feather accidentally present in histological preparations
have been mistaken for lymphatic vessels — Beale, 288) ; fibers of
cotton, linen, and silk (textile fibers accidentally present have been
considered nerve fibers, etc.); the scales of butterflies and moths,
especially the common clothes moths; the dust swept from carpeted
and wood floors; tea leaves and coffee grounds; dust found in living
rooms and in places not frequently dusted (in the last will be found
a regular museum of objects).

§ 355. Wire gauze experiment. — For a very striking illustration
of the need of care in interpretation with naked eye observation,
take two pieces of wire gauze such as is used for milk strainers, or
some slightly coarser. Place these over each other and look through
them toward the light. Where there is but a single layer the weave
is evident, but where the two pieces overlap the appearance is very
puzzling, and changes constantly as one piece is rotated, bringing the
threads and meshes at an angle. One could hardly believe that the
structure is so simple when looking through two layers of the gauze.

If it is necessary then to see all sides of an ordinary gross object,
to observe it in various positions and with varying illumination and
under various conditions of temperature, moisture, and in single as

276 INTERPRETATION OF APPEARANCES [Cn. VII

well as multiple layers to obtain a fairly accurate and satisfactory
knowledge of it, so much the more is it necessary to be satisfied
with the interpretation of appearances under the microscope only
after applying every means of investigation at command. Even
then only such details of the image will be noted and understood as
the brain behind the eye has been trained to appreciate.

§ 365a. Experiment with wire gauze. — F6r this very striking, naked-eye
experiment with the wire gauze the author is indebted to a suggestion from
Dr. Chamot.

§ 356. Inversion of the microscopic image. — As all the images
produced by the modern compound microscope are inverted unless
they are erected by a special arrangement of prisms, one must learn
to interpret the appearances in an inverted image with the same
certainty as in erect images seen by the naked eye or through the
simple microscope. It may be remarked in passing that with the
compound microscope the image is actually erect on the retina of
the eye (figs. 2, 18).

With the compound microscope it soon becomes as easy to move
the slide in the right direction to see a desired part as it is to make
the proper motions when examining an object with the naked eye,
although the motions are directly opposite in the two cases. Indeed,
so natural does it become for the worker with the compound micro-
scope to make the proper motions for the object giving the inverted
image, that if he uses a compound microscope with an erecting prism
he almost invariably moves the preparation in the wrong direction.
With the simple microscope, however, it seems like naked-eye obser-
vation and there is never any difficulty.

This goes to show that by experience it is as easy to interpret
inverted as erect images. This is further illustrated by the printer
who learns to read type without difficulty, although it is a great
puzzle to one who has learned to read the appearances only after the
type has been printed on paper.

§ 357. Physical analysis by the dark-field, the polarizing and the
ultra-violet microscope. — If one looks at objects with the bright-
field microscope only, there may seem to be a complete revelation
of the structure and form. But how inadequate that revelation is

CH. VII] INTERPRETATION OF APPEARANCES 277

will become apparent if one or more of the special microscopes are
used. For the application of these special microscopes, see Chapters
III, IV and VI. Do not fail to get the additional information if the
instruments are available.

§ 358. Summary for proper interpretation. — To summarize this
chapter and leave with the beginning student the result of the
experience of many eminent workers:

(1) Get all the information possible with the unaided eye. See
the whole object and all sides of it, so far as possible.

(2) Examine the preparation with a simple microscope in the
same thorough way for additional detail.

(3) Use a low power of the compound microscope.

(4) Use a higher power.

(5) Make sure that the mirror is in the best position to give the
most favorable light. Vary the aperture by opening and closing the
iris diaphragm to find the aperture which gives the clearest image in
each case.

(6) Shade the top of the stage of the microscope to cut off the
light from above and thus avoid confusion from that source.

(7) Use the highest power available and applicable. In this way
one sees the object as a whole and progressively more and more de-
tails.

(8) If one has the apparatus, it is a good plan to examine speci-
mens with a binocular microscope to gain the best notion possible
of the relative position of parts of the specimen.

(9) Use the dark-ground illuminator (§ 349), the spectroscope,
the polariscope, and the ultra-violet microscope (§§ 312-318, 357).

(10) Try staining the preparations to be studied in various ways
to bring out the structural details; remember also the advantage of
a color picture over a pure refraction image (§ 152) and especially of
a combined color and refraction image. Keep in mind also that the
microscopic image cannot be expected to reveal structural details
that are not in some way clearly differentiated in the specimen.

(n) If artificial light must be used, employ a screen of daylight
glass (§ 76) between the source of illumination and the microscope;
then one can obtain true color effects.

278 INTERPRETATION OF APPEARANCES [Cn. VII

(12) The composite picture derived from all available means of
observation is much more likely to be correct than that obtained by
only one or two means of observation.

(13) According to Wright, p. 46, it is far more difficult to prepare
and illuminate a specimen properly than to get a good image of it
after it is thus prepared and lighted.

COLLATERAL READING FOR CHAPTER VII

For general discussions: Carpenter-Dallinger; A. E. Wright, Principles of Mi-
croscopy, Ch. V; Beale; Spitta, Microscope, Ch. XVIII; Chamot, Chemical
Microscopy.

For pedesis, see Jevons in Quart. Jour. Science, n.s., Vol. VIII (1878), p. 167;
Rutherford, Science, N. S. Vol. XXX, 1909, pp. 289-302. For the original
account of this see Robert Brown, " Botanical appendix to Captain King's
voyage to Australia," Vol. II, p. 534 (1826).

For overcoming pedesis for photography see Gage, The use of a solution of
gelatin to obviate pedesis in photographing milk globules and other minute ob-
jects in water, Transactions Amer. Micr. Soc., Vol. XXIV, 1902, p. 21.

For figures (photo-micrographs, etc.) of the various forms of starch, see Bulle-
tin No. 13 of the Chemical Division of the U. S. Department of Agriculture. For
hair and wool, see Bulletin of the National Association of Wool Growers, 1875,
p. 470; Proc. Amer. Micro. Soc., 1884, pp. 65-68; Herzfeld, translated by Sal-
ter, The technical testing of yarns and textile fabrics, London, 1898.

HAUSMAN, L, A. — A micrological investigation of hair structure of the mo-
notremata. Amer. Jour. Anat., Vol. 27, 1920, pp. 463-488. Many figs.

For different appearances due to the illuminator, see Nelson, in Jour. Roy. Micr.
Soc., 1891, pp. 90-105; and for the illusory appearances due to diffraction phe-
nomena, see Carpenter-Dallinger, p. 434; Mercer, Trans. Amer. Micr. Soc., V.
18 p. 321-396; also, A. E. Wright's Principles of Microscopy, especially the first
five chapters; and chapter IX and the appendix. Conrad Beck. The Theory
of the Microscope. Cantor Lectures before the Royal Society of Arts, Nov. Dec.,
I9°7- 59 pages, London, 1908. See also collateral reading in previous chapters.

CHAPTER VIH

MAGNIFICATION AND MICROMETRY
§§359-398; FIGURES 143-166

WHY A MAGNIFIED IMAGE is NECESSARY

§ 359. The fundamental reason for using a microscope lies in the
structure of the eye and its possibilities of adjustment for objects
at different distances.

The sensory receptors or neuro-epithelium (rods and cones) of the
eye stand in general \\ ith their long axes \* ith the parallel rays of light
entering the eye, hence the image of any external object falls on the
ends of the sensory receptors. Now it is believed that if any image
falls wholly upon one of the receptors, it will appear as a point;
and if the image of two objects close together were to fall on one
receptor, the two objects would appear as one.

§ 360. Robert Hooke (1674), in dealing with the power of the hu-
man eye to distinguish double stars and to see two points or two
details of an object as two, concluded that the two stars or the two
points of any object must be at least far enough apart to rtiake the
visual angle one minute. A few people can distinguish double stars
with a visual angle less than one minute, but for many people the
visual angle must be greater. If the visual angle is too small, then
the two stars or two points appear to fuse and form one. The
visual angle of one minute then does not represent the limit of
visibility, but the limit of resolution, that is, seeing two objects as
two separate things.

Now as the visual angle under which any given object is seen
depends upon its distance from the eye, and the power of accommo-
dation for distance in the eye is limited, if very small objects are
to be seen, or the parts of larger objects are to be distinguished as
separate details, there must be some means of enabling the eye to get
very close to the object.

279

280

MAGNIFICATION AND MICROMETRY

[CH. VIII

The microscope serves to increase the visual angle under which an
object is seen, thus virtually making it possible to get the eye very

FIG. 143. CONSTANT RETINAL IMAGE (R I) AND CONSTANT VISUAL ANGLE
WITH VARYING SIZE OF OBJECT AT DIFFERENT DISTANCES.

RI Retinal image. To keep this of constant size the visual angle must
remain constant.

Object The object varying in size directly as the radius to keep the visual
angle and the retinal image constant.

The radii in this figure are in the proportion of i, 2, 4.

close to the object and still retain the sharpness of the retinal image.
Or to put it in another way, the microscope helps the eye lo produce
a larger retinal image, and makes the details large enough to fall on
more than one of the retinal elements, thus making resolution pos-
sible.

The sensory receptors of the retina — the rods and cones — are
quite close together and over the greater part of the retina are
commingled, there being more rods than cones. In the region of

CH. VIII]

MAGNIFICATION AND MICROMETRY

281

greatest visual acuity (fovea centralis of macula lutea), only cones
are present. In general the rods are 2ju and the cones 6/i in di-
ameter. In the fovea,
however, the cones are
slender, being only
about 2}Ji to 3/i in
diameter. These sizes
give a clue to the size
the retinal image must
have in order that
there be resolution,
that is, that two points
appear as two or two
lines appear as two.

If we assume that
Hooke was correct in
the assumption that
for two points to appear
as two a visual angle

of i minute is neces-
sary, the diameter in

FIG. 144. CONSTANT SIZE OF OBJECT, THE VIS-
UAL ANGLE AND THE RETINAL IMAGE VARYING
WITH THE DISTANCE.

R I The retinal image varying inversely as
the distance of the object.

V A The visual angle varying with the dis-
tance of the object from the eye.

Object The object of constant size but varying
distance from the eye. The distance of the object
is in the ratio of i, 2, 4. The entire circle is
shown at the right, but only a small arc in the
other figures.

millimeters or inches of
the object, or the sep-
aration of the two
points to render them
visible as two, is easily determined as follows.

The nodal point or optic center of the eye is considered to be at
the center of a circle (fig. 143), and the object at the circumference.
No matter how great or how small the visual distance, the object
must subtend one minute of the arc of the circle at whose circum-
ference it is situated, in order that its two extremities shall appear
separate. And so with any two details; they must be far enough
apart to make the visual angle one minute.

To determine the actual length in millimeters required to subtend
one minute of arc in any case, it is necessary to remember only that
the entire circumference is 6.2832 times its radius (2wr), and that
this circumference is divided into 360° or 21,600 minutes.

282 MAGNIFICATION AND MICROMETRY [Cn. VIII

If, now, the radius of the circle, or the distance of the eye from
the object, is i meter, the circumference of the circle will be 6.2832
meters or 6283.2 millimeters. As there are 21,600 minutes in the
entire circumference, the actual length of one minute with a circle
having a radius of one meter is 6283.2 mm. divided by 21,600 equals
0.29088 mm. That is, the eye at one meter distance requires two
points or two lines to be separated a distance of 0.29088 mm. in
order that they may be seen as two and not appear to be fused to-
gether.

It is assumed by workers with the microscope that the distance of
most distinct vision for adults when looking at objects for details of
structure is 254 mm. or 10 inches. This is the standard distance
selected for the determination of magnifying power in microscopy
also.

The question now is, how large a retinal image will be formed by
an object giving a visual angle of i minute at the standard distance
of 254 mm.

First must be found the actual size of the object to give a visual
angle of i minute at 254 mm. distance. It is known from the above
calculation that for one meter or icoo mm. the object must have a
size of 0.29088 mm. Now for 254 mm. the length must be -fi^fa of
this number or 0.07388352 mm., that is, a little more than one-
fourth the size at i meter.

Now to determine the size of the retinal image at 254 mm. image
distance, the distance from the center or nodal point of the eye must
be known as well as the image distance and the size of the object.
The distance of the retinal image from the nodal point is assumed to
be 15 mm. (Ho well, p. 311); then the size of the retinal image will
be: 0.07388352 :x 1:254 : 15 = 0.00436 mm. or 4.36^1, and this size
would make the image fall on at least two of the cones of the fovea,
and therefore there would be resolution and any two points would
appear as two and not as one.

§ 361. The magnification, amplification, or magnifying power of
a simple or compound microscope is the ratio between the apparent
and real size of the object examined. The apparent size is obtained
by measuring the virtual image (figs. 145-146). For determining

CH. VIII]

MAGNIFICATION AND MICROMETRY

magnification the object must be of known length and is designated
a micrometer (§ 366). In practice a virtual image is measured by
the aid of some form of camera ,

lucida (figs. 149, 169), or by double
vision (§ 363). As the length of
the object is known, the magnifica-
tion is easily determined by divid-
ing the size of the image by the
size of the object. For example, if
the virtual image measures 40 mm.
and the object magnified, 2 mm.,
the amplification is 40 -s- 2 = 20,
that is, the apparent size is twenty-
fold greater than the real size.

Magnification is expressed in
diameters or times linear; that is,
but one dimension is considered.
In giving a scale at which a micro-
scopical or histological drawing is
made, the word " magnification "
is frequently indicated by the
sign of multiplication: thus, X4$o
upon a drawing means that the
figure or drawing has the width
or length of every detail 450 times

as great as the object.

§ 362. Magnification of real im-
ages. — In this case the magnifi-
cation is the ratio between the size
of the real image and the size of
the object, and the size of the real
image can be measured directly.
By recalling the work on the
function of an objective, it will be
remembered that it forms a real image on the ground-glass placed
on the top of the tube, and that this real image could be looked at

FIG. 145. SIMPLE MICROSCOPE
WITH THE VIRTUAL IMAGE AT 250
MM. FROM THE EYE.

Axis The principal optic axis of
the microscope and of the eye.

/ The principal focus of the mi-
croscope.

A1B1 The object just above the
focus (/).

B2 A'1 the retinal image; it is in-
verted.

A* B* The virtual image at 250
mm. from the eye; it is erect.

Cr Cornea of the eye.

R Single refracting surface of the
schematic eye.

L The crystalline lens of the eye.

284

MAGNIFICATION AND MICROMETRY

[Cn. VIII

Axis The principal optic axis of the microscope and of the eye.

// Principal focus of the objective, and of the ocular, r im, the real image
formed by the objective just above the principal focus of

the ocular.

cr The cornea of the eye.

rs The single refracting / \j / \ surface of the schematic eye.

/ The crystalline lens of I W 1 the eye.
The retinal image; it | ^JJBU^/. I is erect.

The tube-length of the mi-
limeters, and the image dis-
250 millimeters. For more com-

croscope (fig. 26) is 160 mil-
^% tance of the virtual image,
% rs plete explanation see fig. 18.

FIG. 146. COMPOUND MICROSCOPE SHOWING ALL THE IMAGES.

CH. VIII] MAGNIFICATION AND MICROMETRY 285

with the eye or measured as if it were an actual object. For ex-
ample, suppose the object were three millimeters long and its image
on the ground-glass measured 15 mm., then the magnification is
I5 + 3 = 5> that is, the real image is 5 times as long as the object.
The real images seen in photography are mostly smaller than the
objects, but the magnification is designated in the same way by
dividing the size of the real image measured on the ground-glass by
the size of the object. For example, if the object is 400 millimeters
long and its image on the ground-glass is 25 millimeters long, the ra-
tio is 25 4- 400 = iV,. That is, the image is iV as long as the object
and is not magnified but reduced. In marking negatives, as with
drawings, the sign of multiplication is put before the ratio, and in
the example the designation is X TB . In photography and when us-
ing the magic lantern and the projection microscope, the images are
real, and may be measured on the screen as if real pictures (fig. 147).

§ 363. The magnification of a simple microscope is the ratio be-
tween the virtual image (figs. 6, 145, A3B3) and the object magnified
(A1!}1). To obtain the size of this virtual image, place the tripod
magnifier near the edge of a support or block of such a height that
the distance from the upper surface of the magnifier to the table is
250 millimeters.

As object, place a scale of some kind ruled in millimeters on the
support under the magnifier. Put some white paper on the table
at the base of the support and on the side facing the light.

Close one eye, and hold the head so that the other will be near the
upper surface of the lens. Focus if necessary to make the image
clear. Open the closed eye and the image of the rule will appear as
if on the paper at the base of the support. Hold the head very still,
and with dividers get the distance between any two lines of the
image. This is the so-called method of double vision in which
the microscope image is seen with one eye and the dividers with the
other, the two images appearing to be fused in a single visual field.

§ 364. Measuring the spread of the dividers. — This should be
done on a steel scale divided to millimeters and J mm.

As | mm. cannot be seen plainly by the unaided eye, place one
arm of the dividers at a centimeter line, and with the tripod magni-

286 MAGNIFICATION AND MICROMETRY [Cn. VIII

f.er count the number of spaces on the rule included between the
points of the dividers. The magnifier simply makes it easy to count

a 6

FIG. 147. REAL IMAGE FORMED BY A PROJECTION MICROSCOPE.

(From the Essays of George Adams).

A B Mirror reflecting the parallel rays of the sun upon the condenser (C D.)
abcdef Parallel beams of light.
C D The condenser.

N O The stage of the projection apparatus.
E F The object.
G H The projection objective.

L M The screen upon which the real image is shown.
/ K The real image of the object (R F).

the space on the rule included between the points of the dividers —
it does not, of course, increase the number of spaces or change their
value.

As the distance between the points of the dividers gives the size
of the virtual image (fig. 145), and as the size of the object is known,
the magnification is determined by dividing the size of the image by the
size of the object. Thus, suppose the distance between the two lines
at the limits of the image is measured by the dividers and found on
the steel scale to be 15 millimeters, and the actual size of the space
between the two lines of the object is 2 millimeters, then the mag-
nification {815-5-2 = 7.5; that is, the image is 7.5 times as long or

CH. VIII]

MAGNIFICATION AND MICROMETRY

287

wide as the object. In this case the image is said to be magnified
7.5 diameters, or 7,5 times linear.

Stage
Micrometer

0.1 mm
0.01mm

FIG. 148. STAGE MICROMETER RULED ON A COVER-GLASS.

The tenths millimeter (o.i mm.) spaces are divided by short lines making the
whole micrometer one with o.i, 0.05, and o.oi millimeters.

The magnification of any simple magnifier may be determined
experimentally in the way described for the tripod magnifier; but
this method is, of course, only possible when the observer has two
good eyes. If he has but one eye, or his eyes are very unlike, then
the magnification can be determined with one eye by using a camera
lucida or the eikonometer (§§ 367, 390).

§ 365. The magnification of a compound microscope is the ratio
between the final or virtual image and the object magnified.

The determination of the magnification of a compound microscope
may be made as with a simple microscope (§ 363), but this is fa-
tiguing and unsatisfactory.

§ 366. Stage or object micrometer. — For determining the mag-
nification of a compound microscope and for the purposes of mi-
crometry, it is necessary to have a finely divided scale or rule on
glass or on metal. Such a finely divided scale is called a micrometer,
and for ordinary work one mounted on a glass slide (i X 3 in., 25 X
76 mm.) is most convenient.

The spaces between the lines should be o.i and o.oi mm. (or if in
inches, o.oi and o.ooi in.). Micrometers are sometimes ruled on the
slide, but more satisfactorily on a cover-glass of known thickness,
preferably 0.15-0.18 mm. The covers should be perfectly clean before
ruling, and afterwards simply dusted off with a camel's hair duster,
and then mounted, lines downward over a shellac or other good

288

MAGNIFICATION AND MICROMETRY

[CH. VT;I

cell ( § 525). If one rubs the lines the edges of the furrow
made by the diamond are likely to be rounded and the sharp-

ness of the micrometer is
lost. If the lines are on the
slide and uncovered one
cannot use the micrometer
with an oil immersion, as the
oil obliterates the lines.
Cleaning the slide makes
the lines less sharp, as
stated. If the lines are
coarse, it is an advantage
to fill them with plumbago
or graphite. This may be
done with some very fine
plumbago on the end of a
soft cork, or by using a
soft lead pencil. Lines
properly filled may be cov-
ered with balsam and a cover-glass as in ordinary balsam mount-

ing (§ 533)-

§ 367. Determination of magnifica-
tion. — This is most readily accom-
plished by the use of some form of
camera lucida, that of Wollaston being

FIG. 149. WOLLASTON'S CAMERA LUCIDA.

most convenient, as it may be used for ET^G'WJ|£ A RING ONTHE

all powers, and the determination of the LINES.

standard distance of 250 millimeters at

which to measure the images is readily accomplished (fig. 149).

Employ the 16 mm. (lox) objective and a 5x ocular with a stage
micrometer as object. For this power the o.i mm. spaces of the
micrometer should be used as object. Focus sharply.

It is somewhat difficult to find the micrometer lines. To avoid
this it is well to have a small ring enclosing some of the micrometer
lines (fig. 150). The light must also be carefully regulated. If too
much light is used, i.e., too large an aperture, the lines will be

Cn. VIII] MAGNIFICATION AND MICROMETRY 289

drowned in the light. In focusing with the high powers be very
careful. Remember the micrometers are expensive and one cannot
afford to break them. As suggested above, focus on the edge of the
cement ring enclosing the lines; then, in focusing down to find the
lines, move the preparation very slightly, back and forth. This will
bring the lines into the field and the shadow made by them will
indicate their presence, and one can then focus until they are
sharp.

After the lines are sharply focused, and the slide clamped in
position, make the tube of the microscope horizontal by bending the
flexible pillar, being careful not to bring any strain upon the fine
adjustment (fig. 26).

Put a Wollaston camera lucida (fig. 149) in position, and turn the
ocular around if necessary so that the broad flat surface may face
directly upward, as shown in the figure. Elevate the microscope by
putting a block under the base, so that the perpendicular distance
from the upper surface of the camera lucida to the table is 250 mm.
(§ 37o)- Place some white paper on the work table beneath the
camera lucida.

Close one eye, and hold the head so that the other may be very
close to the camera, lucida. Look directly down. The image will
appear to be on the table. It may be necessary to readjust the focus
after the camera lucida is in position. If there is difficulty in seeing
both dividers and image, consult § 408. Measure the image with
dividers and obtain the power exactly as above (§ 364).

Thus: If two of the o.i mm. spaces are taken as object and the
image is measured by the dividers, and the spread of the dividers is
found on the steel rule to be 9.4 millimeters, the magnification
(which is the ratio between size of image and object) is 9.4 -5- 0.2 =
47. That is, the magnification is 47 diameters, or 47 times
linear.

Put the IQX ocular in place of the 5x, and then put the camera
lucida in position. Measure the size of the image with dividers and
a rule as before. The power will be considerably greater than when
the low ocular was used. That is, the virtual image (fig. 146) seen
with the high ocular is larger than the one seen with the low one.

2QO

MAGNIFICATION AND MICROMETRY

[Cn. VIII

F. Image 10

FIG. 151. DIAGRAM TO SHOW THE
SIZES OF THE IMAGES WHEN THE OBJECT
Is AT DIFFERENT DISTANCES FROM THE
PRINCIPAL Focus.

The farther the object from the
principal focus ' L. F.) the nearer
the image to the lens (F. Object, N.
Image), and the nearer the object to
the focus the farther will be the
image from the lens (N. Object, F.
Image). The sizes of the images
will vary directly as their distances
from the upper or conjugate focus
(U.F.).

Lengthen the tube of the
microscope 50-60 mm. by
pulling out the draw-tube.
Remove the camera lucida and
focus; then replace the camera
and obtain the magnification.
It is greater than with the
shorter tube. That is, the real
image (fig. 151) is formed
farther from the objective
when the tube is lengthened,
and the objective must be
brought nearer the object.
The law is: the magnification
varies directly with the rela-
tive distance of the image and
object from the center of the
lens (fig. 152).

§ 368. Varying the magni-
fication of a microscope. —
There are five ways of varying
the power of a compound mi-
croscope:

(1) By using a higher or
lower objective.

(2) By using a higher or
lower ocular.

CH. VIII]

MAGNIFICATION AND MICROMETRY

291

Image

(3) By lengthening or shortening the tube
of the microscope.

(4) By increasing or diminishing the dis-
tance at which the virtual image is projected

(fig- 153).

(5) By changing the relative position of
the combinations in an adjustable objective
(§§ 29, 149) or by the use of an amplifier

(§ 369).

§ 369. Amplifier. — In addition to the
methods of varying the magnification given
in § 368, the magnification is sometimes in-
creased by the use of an amplifier, that
is, a diverging lens or combination placed
between the objective and ocular and
serving to give the image-forming rays
from the objective an increased divergence.
An effective form of this accessory was
made by Tolles, who made it as a small
achromatic concavo-convex lens to be screwed
into the lower end of the draw-tube (fig. 26)
and thus but a short distance above the ob-
jective. The divergence given to the rays
usually increases the size of the real image
about twofold. Object

§ 370. Standard distance at which the FIG. 152. To SHOW
virtual image is measured. - For obtaining ^^J^D^E™
the magnification of both the simple and the UPON ITS RELATIVE
compound microscope the directions were to CENTE^
measure the virtual image at a distance of JECTIVE.

250 millimeters. That is, some standard obJect . x The .object
,. A , , , „., A ,.-. ^ one unit of distance

distance must be chosen so that different from the center of the

workers can compare their results. The lens (££)•

'£. *.- u i- £ j A. i *. Image i, 2, 3, 4 The

magnification could be found at almost any image four units of dis-

distance, and in getting the magnification of *ance from the lens and
. , . ?. . , hence four times as long

drawings the image distance is rarely ex- as the object

OF^HE

MAGNIFICATION AND MICROMETRY

[On. VIII

actly 250 millimeters. Whenever the magnification of the micro-
scope as a whole or of the objective or the ocular is mentioned, how-
ever, it is always understood that this magnification is at the
standard distance of 250 mm. The necessity for the adoption of
some common standard will be seen at a glance in fig. 153, where is
represented graphically the fact that the size of the virtual image

depends directly on
the distance at which
it is projected, and
this size is directly
proportional to the
vertical distance from
the apex of the tri-
angle of which it
forms a base. The
distance of 250 milli-
meters has been
chosen on the sup-
position that it is the
distance of most dis-
tinct vision for nor-
mal adults when
examining details.

In preparing draw-
ings it is often of
great convenience to make them at a distance less or greater than
the standard. In that case the magnification must be determined
for the image distance actually used,

§ 371. Magnification and relation of the object to the principal
focus. — As shown by figures 154 and 155, independent of the equiv-
alent focus of the simple microscope or the objective, the real image
or the virtual image, as the case may be, will be larger the nearer
the object is to the principal focal point.

In figure 156 it is shown also that if the object or the real image is
in the plane of the principal focus, the rays emerging from the simple
microscope or the ocular will be in parallel bundles, and when pro-

FIG. 153. DIAGRAM TO SHOW THAT THE SIZE OF
THE VIRTUAL IMAGE DEPENDS UPON THE PROJECTION
DISTANCE,

a Size of image at a projection distance of 25 cm.

b Image at 3 <; cm.

The sizes are directly as the projection distances.

C The camera lucida and under it a spectacle lens
to aid the eye in focusing the pencil point; this is
only needed by those with defective eyes.

CH. VIII]

MAGNIFICATION AND MICROMETRY

293

FTG. 154. DIAGRAMS TO SHOW THAT THE SIZE OF THE REAL IMAGE OF A LENS
DEPENDS UPON THE DISTANCE OF THE OBJECT FROM THE PRINCIPAL Focus.

Axis The principal optic axis extended above and below. A B, B A The
object and the inverted real image. /, / The principal focus above and below
each lens. Lc The lens.

The object is the same size in the two cases, but the images differ, depending
upon the distance of the object from the principal focus, being longer the nearer
the object is to the focus.

FIG. 155. DIAGRAMS TO SHOW THAT THE SIZE OF THE VIRTUAL IMAGE OF A LENS
DEPENDS UPON THE DISTANCE OF THE OBJECT FROM THE PRINCIPAL Focus.

A B, A B The object and the virtual image. // The principal focus. L The
lens, ep The eyepoint. c The single, ideal refracting plane.

As with real images, the size of virtual image in a given lens depends upon
the nearness of the object to the principal focus,

294

MAGNIFICATION AND MICROMETRY

[Cn. VIII

jected by the eye must also be in parallel bundles. It is further
shown in such a case that the rays emanating from any point in the
object or real image will not in that case form a virtual point

Ocular

///Object \\

/// ! \\\

I:!

// '

250

FIG. 156. DIAGRAMS OF SIMPLE AND COMPOUND MICROSCOPES WITH PARALLEL
BEAMS EMERGING ABOVE AND PROJECTED BELOW.

Axis The principal optic axis.

Object The object.

Objective The objective of the compound microscope.

r i The real image formed by the objective.

Ocular- Magnifier The ocular and magnifier for the real image in the com-
pound microscope, and for the object in the simple microscope.

Eyepoint The most favorable position for the eye of the observer.

Below, at 250 mm., the usual position of the projected image, no image is
formed with parallel rays. These only seem to come from a point at a dis-
tance where their separation is less than one minute of arc (§ 359-360).

focus at the standard distance of 250 mm., as shown in fig. 145, but
will remain parallel. At that distance, then, the image on the retina
would be a diffusion circle. In order that there be the appearance
of a point focus the distance must be great enough so that the
parallel rays from a point will be separated less than one minute

§ 372. Table of magnification and of the valuations of the ocular

CH. VIII]

MAGNIFICATION AND MICROMETRY

295

micrometer. — The table should be filled out by each student. In
using it for micrometry and drawing it is necessary to keep clearly in
mind the exact conditions under which the determinations were
made, and also the ways in which variations in magnification and the
valuation of the ocular micrometer may be produced.

OCULAR OCULAR

5x lox

OBJECTIVE

TUBE

TUBE
OUT

— MM.

TUBE

TUBE
OUT

— MM.

OCULAR MICROMETER
VALUATION
TUBE IN. OUT — MM.

SIMPLE MICROSCOPE. x

OCULAR MICROMETER AND ITS VALUATION

§ 373. This, as the name implies, is a micrometer to be used in
connection with an ocular. It consists of rulings of fixed or of
movable lines on a cover-glass.

This form cf micrometer is placed at the level where the real
image is formed, i.e., at the level of the ocular diaphragm of all

296 MAGNIFICATION AND MICROMETRY [Cn. Vllt

oculars. With positive oculars it would therefore be outside the
ocular (figs. 22-23) and with negative or Huygenian. oculars, be-
tween the lenses (figs. 24-25). The image of the object under the
microscope appears to be directly upon or immediately under the
ocular micrometer, and hence the number of spaces on the ocular
micrometer required to measure the real image may be read off
directly. This, however, is measuring the size of the real image,
and the actual size of the object can be determined only by finding
the ratio between the size of the real image and that of the object.
In other words, it is necessary to get the valuation of the ocular
micrometer in terms of a stage micrometer.

§ 374. Valuation of the ocular micrometer. — This is the value of
the divisions of the ocular micrometer for the purposes of microm-
etry, and is entirely relative, depending on the magnification of
the real image formed by the objective; consequently it changes
with every change in the magnification of the real image, and must
be especially determined for every change modifying the real image
of the microscope (§ 368).

It will be seen when the ocular micrometer valuation is found for
different objectives, that the greater the magnification of the objec-
tive, the less will be the ocular micrometer valuation; and con-
versely, the less the magnification of the objective, the greater will
be the ocular micrometer valuation.

§ 375. Obtaining the ocular micrometer valuation for an ocular
micrometer with fixed lines. — If the ocular micrometer is on a
cover-glass, place it on the diaphragm of the $x or lox ocular after
removing the eyelens. Screw the eyelens back in place, and put the
ocular in the tube of the microscope. Put a 16 mm. (lox) objective
in place. Use the stage micrometer as object. Light the field well
and look into the microscope. The lines of the ocular micrometer
should be very sharply defined. If they are not, raise or lower the
eyelens to make them so; that is, focus as with the simple magnifier.

When the lines of the ocular micrometer are distinct, focus the
microscope (§ 367) for the stage micrometer. The image of the
stage micrometer appears to be directly under or upon the ocular
micrometer.

CH. VIII]

MAGNIFICATION AND MICROMETRY

297

Make the lines of the two micrometers parallel by rotating the
ocular or changing the position of the stage micrometer or both if
necessary, and then make any two lines of the stage micrometer
coincide with any two on the ocular micrometer (fig. 157). To do
this it may be necessary to pull out the draw-tube a greater or lesser
distance. See how many spaces are included in each of the microm-
eters (figs. 157, 165).

Divide the value of the included
space or spaces on the stage mi-
crometer by the number of divi-
sions on the ocular micrometer
required to include them, and the
quotient so obtained will give the
valuation of the ocular microme-
ter. For example, suppose the
millimeter is taken as the unit for
the stage micrometer and this unit
is divided into spaces of o.i and o.oi
millimeters. If with a given optical
combination and tube-length it re-
quires 10 spaces on the ocular microm-
eter to include the real image of o.i
millimeter on the stage micrometer,
obviously one space on the ocular
micrometer includes only one-tenth as
much, or o.i mm. -7-10 = o.oi mm.
That is, each space on the ocular micrometer includes o.oi of a milli-
meter on the stage micrometer, or o.oi millimeter of the length of any
object under the microscope, the conditions remaining the same.
Or, in other words, it requires 100 spaces on the ocular micrometer
to include i millimeter on the stage micrometer; then, as before,
i space of the ocular micrometer would have a valuation of o.oi
millimeter for the purposes of micrometry. The size of any minute
object may be determined by multiplying this valuation of one space
by the number of spaces required to include it. For example, sup-
pose the fly's wing or some part of it covered 8 spaces on the ocular

Fip. 157. THE IMAGES OF THE
OCULAR AND OF THE STAGE
MICROMETER, SHOWING HOW TO
ARRANGE THE LINES.

o.m Ocular, s.m Stage mi-
crometer lines.

A Lines of the ocular mi-
crometer opposite the middle of
the lines of the stage micrometer.

B Lines of the ocular mi-
crometer at the right side of the
lines of the stage micrometer
(compare fig. 165.)

2Q8

MAGNIFICATION AND MICROMETRY

[CH. VIII

micrometer; it would be known that the real size of the part meas-
ured o.oi mm. X 8 = 0.08 mm. or 8o/x (§§ 380-382).

Proceed in exactly the same manner to get the ocular micrometer
valuation when using any objective, whether it is of higher or lower
power than the one in this section.

Any Huygenian ocular may be used as a micrometer ocular by
placing the ocular micrometer at the level of the ocular diaphragm
where the real image is formed. If there is a slit in the side of the
ocular and the ocular micrometer is mounted properly, it may be
introduced through the opening in the side. This was a common
method with the older microscopes. When there is no side opening,
the eyelens may be unscrewed and the ocular micrometer on a
cover-glass laid upon the ocular diaphragm.

OCULAR MICROMETER WITH MOVABLE SCALE

§ 376. Ocular micrometer with movable scale. — The form here
shown is a Huygenian ocular with a micrometer scale on the diaphragm

of the ocular. The
eyelens is adjustable
up and down for fo-
cusing the scale, and a
drum with 100 divi-
sions is attached to the
screw which moves the
scale from side to side.
Each interval on the
drum represents o.oith
of a complete interval
on the scale, thus en-
abling one to measure
an object o.oith the
size of one requiring a
whole scale-interval.
This ocular micrometer

combines the advantages of the ocular micrometer with fixed lines and
the filar micrometer. To complete the measurement of an object not

FIG. 158. OCULAR MICROMETER WITH MOVABLE
SCALE AND RECORDING DRUM.

(From the Catalogue of the Spencer Lens Co.)

CH. VIII]

MAGNIFICATION AND MICROMKTRY

290

included exactly between any two lines of the scale, the drum need
be revolved only partly around.

§ 377. Valuation of the movable scale ocular micrometer (fig,
158). — Use a 4 mm. Uox) objective and proceed exactly as for the
micrometer with fixed lines, ex-
cept that a partial stage mi-
crometer space can be measured
by rotating the drum until the
ocular micrometer exactly coin-
cides with the stage micrometer.
Make sure that the lines of the
two micrometers are correctly
related, as shown in figs. 157
and 165. One can then count
up the number of spaces on the
ocular micrometer required to
measure one or more spaces of
the stage micrometer. To this
is then added the ifo spaces on
the drum. For example, sup-
pose that three o.oi mm. spaces
of the stage micrometer are
taken as object, and that it re-
quires seven complete spaces
of

FIG. 150. FIELD OF THE MICROSCOPE
SHOWING THE MOVABLE SCALE OF
THE HUYGENTAN MICROMETER OCU-
LAR (Fio. 158).

The arrow indicates that the scale may
be moved in both directions.

o, 5, io, 15, 20 These figures indicate
the 20 spaces in groups of 5. Each
space represents a total revolution of
the screw (screw with \ mm. pitch).
Each of the 100 divisions on the drum
(fig. QI) represents then ^fa mm.

Object The circular object in the field
measures 5 intervals on the ocular mi-
crometer and 45 intervals on the drum,
hence the entire diameter of the object is
5.45 intervals on the ocular micrometer.

the ocular micrometer and
on the drum to include the
three spaces on the stage mi-
crometer; then each space on
the ocular micrometer would be
equal to 0.03 mm. divided by
7.50 = 0.004 mm. or 4/z. One of the spaces on the drum which
represents one hundredth of an interval on the ocular micrometer
would have a valuation under these conditions of w divided by
100 = 0.04 microns. This gives a notion of the minuteness of the ob-
ject which can be measured, and of the smallness of the error in
measuring large objects, even if the observation erred in getting the
obiect one or more of the drum divisions too large or too small.

300 MAGNIFICATION AND MICROMETRV [CH. vm

For an actual measurement with this ocular micrometer, sec

§ 387.
One would proceed exactly as above for getting the valuation with

any other objective.

FILAR OCULAR MICROMETER

§ 378. This form of ocular micrometer usually consists of a
Ramsden ocular with fixed cross lines and a movable line (fig. 161).

For obtaining
the valuation of
this ocular microm-
eter proceed as
follows: Employ a
4 mm. Uox) ob-
jective. Carefully
focus the-i^ir mm.
lines. The lines of
the ocular microm-
eter should also
FIG. 160. FILAR MICROMETER OCULAR. be sharp; if they

(From the i6th ed. of the Catalogue of the Bausch & are not, focus them

Lomb Optical Co.). , . ,

This is a Ramsden ocular, and the recording drum is by moving the OCU-
divided into TOO equal divisions, and as the pitch of the lar up or down in
screw is 0.5 mm., each division on the drum represents f, cliHino- tiihp
an actual movement of 0.005 mm. of the movable line. L1JC ""uiug LUUC.

Make the vertical

lines of the ocular micrometer parallel with the lines of the stage mi-
crometer (figs. 157, 165). Note the position of the graduated drum
and the teeth of the recording comb, and then rotate the wheel until the
movable line traverses one space on the stage micrometer. Each tooth
of the recording comb indicates a total revolution of the wheel, and by
noting the number of teeth required and the graduations on the
wheel, the revolutions and part of a revolution required to measure
the o.oi mm. of the stage micrometer can be easily noted. Measure
in like manner 4 or 5 spaces and get the average. Suppose this
average is ij revolutions or 123 graduations on the wheel, to meas-
ure the o.oi mm. or io/i (see §§ 380-382), then one of the gradua-

CH. VIII]

MAGNIFICATION AND MICROMETRY

301

ations on the wheel would measure lo/j divided by 125 = 0.08/4.
In using this valuation for actual measurement, the tube of the
microscope and the objective must be exactly as when obtaining the
valuation (§§ 368-377).

The valuation of the filar micrometer can be obtained for any
objective by proceeding exactly
as above. (See § 388 for
measurement.)

Micrometry is the determina-
tion of the size of objects by
the aid of a microscope.

MICROMETRY WITH THE SIMPLE
MICROSCOPE

§ 379. With a simple micro-
scope (i), the easiest and best
way is to use dividers and then

with the simple microscope de-

, ,, . . f ,i FIG. 161. FIELD or THE MICROSCOPE

termme when the points of the SHOWING THK LlNES AND THE RECORD-

dwiders exactly include the ob- ING COMB OF THE FILAR MICROM-

ject. The spread of the dividers ET*R (FlG* l6o)'
above

is then obtained as
(§§ 363-364). This amount will
be the actual size of the object,
as the microscope was used only
in helping to see when the di-
vider points exactly enclosed
the object.

The recording comb. Each tooth
represents a complete revolution of the

fW mflv mir the nhiWt

Une may put me oDject

cross lines.

'ml', nil The movable line.

The arrow shows that the movable
line can be moved in both directions.

0 Object, the full movable line (ml)
shows it at one edge of the object and
the broken line shows it at the other
edge of the object. The intervening
teeth

the comb show that the screw

___ned two whole revolutions and

under the simple microscope and the recording drum showed 90 divisions,

rhpn a* in rterprrmnintr the making two and nine tenths revolutions
tnen, as in determining me Q£ the drum to carry the movable line

power (§ 363), measure the from one edge of the object to the other,
image at the standard distance.

If the size of the image so measured is divided by the magnification
of the simple microscope, the quotient gives the actual size of the
object. One might use the eikonometer also (§ 391).

302 MAGNIFICATION AND MICROMETRY [Cn. VIII

Use a fly's wing or some other object of about that size and try
to determine the width in the two ways described above. If all the
work is done accurately, the results will agree.

MICROMETRY WITH THE COMPOUND MICROSCOPE

There are several ways of varying excellence for obtaining the size
of objects with the corr pound microscope, the method with the
ccular micrometer (§ 373) being most accurate.

§ 330. Unit of measure in micrometry. — Most of the objects measured with
the compound microscope, and many of those in physics and chemistry are smal-
ler, often much smaller, than any of the originally named divisions of the meter.
To express these very small dimensions in common or in decimal fractions of a
meter or millimeter is not only cumbersome, but likely to give rise to errors;
consequently workers in microscopy, in physics and in chemistry have sought to
avoid the difficulties by selecting and naming as units such small divisions of the
meter that the minute dimensions can be expressed as whole numbers.

The Micron unit (pt) has been generally adopted in microscopy, and is widely
used for minute sizes in all branches of science. Harting recommended it for mi-
croscopy in 1859, but he named it micro-millimeter, or milli-millimeter, and gave
as a symbol mwm. Since the definite meaning for micro, as one millionth of the
unit before which it is placed, has been decided on by metrologists, micro-milli-
meter should mean one millionth of a millimeter, not one thousandth. Harting's
Milli-millimeter is correct, but awkward. Occasionally one meets the symbol ju/i
for millimicron (mju). ;u/i should stand for the millionth, not for the thousandth,
of a micron.

Up to the present three such special units have been designated and have re-
ceived the sanction and use of the highest authorities. They are:

§ 380a. 1. The Micron (symbol JJL). This is the one millionth of a meter
(o.oooooi, m.); one thousandth of a millimeter (o.ooi mm.); one thousand
millimicrons (1000 mju); ten thousand Angstrom units (10,000 A).

§ 381. 2. The Millimicron (mju). This is the one billionth of a meter (o.opo
ooo ooi m.); the one thousandth of a micron (o.ooi ju); ten Angstrom units
(10 A.).

§ 3*2. 3. The Angstrom Unit (A.) or Tenthmeter (To~10 m.). It is the one
ten billionth of a meter (o.ooo ooo ooo i m); the ten thousandth of a micron
(0,000 i /x); the one tenth of a millimicron (o.i m/x).

See Jour. Roy. Micr. Jour. Soc., 1888, p. 502. Nature, Vol. XXXVII, p. 388;
Bit. Bureau Standards, Vol. VIII, p. 540.

§ 383. Micrometry by the use of a stage micrometer on which to
mount the object. — In this method the object is mounted on a mi-
crometer and then put under the microscope, and the number of
spaces covered by the object is read off directly. It is exactly like
putting any large object on a rule and seeing how many spaces of
the rule it covers. The defect in the method is that it is impossible

CH. VIII] MAGNIFICATION AND MICROMETRY 303

to arrange objects properly on the micrometer. Unless the objects
are circular in outline they are likely to be oblique in position, and in
every case the end or edges of the object may be in the middle of a
space instead of against one of the lines; consequently the size must
be estimated or guessed at rather than really measured.

§ 384. Micrometry by dividing the size of the image by the mag-
nification of the microscope. — For example, employ the 4 mm.
(4ox) objective, and $x or lox ocular. For measurement use a prep-
aration of the blood corpuscles of the frog, necturus, or other
animal with large oval corpuscles. Obtain the size of the image of
the long and short axes of three corpuscles with the camera lu-
cida and dividers, exactly as in obtaining the magnification of the
microscope (§ 367). Divide the
size of the image in each case by
the magnification, and the result
gives the actual size of the blood
corpuscles. Thus, suppose the

image of the long axis of the cor- FIG. 162. BLOOD PREPARATION

i.0 J j.1- •£. WITH A RING AROUND A GROUP

puscle is 18 mm. and the magnifica- OF CORPUSCLES.

tion of the microscope 400

diameters (§361), then the actual length of this long axis of the

corpuscle is 18 mm. -5- 400 = 0.045 mm. or 45/4 (§ 364).

As the same three blood corpuscles are to be measured in three
ways, it is an advantage to put a delicate ring around a group of
three or more corpuscles, and make a sketch of the whole enclosed
group, marking on the sketch the corpuscles measured (fig. 162).
The different corpuscles vary considerably in size, so that accurate
comparison of different methods of measurement can be made only
when the same corpuscles are measured in each of the ways.

§ 385. Micrometry by the use of a stage micrometer and a
camera lucida. — Employ the same object, objective and ocular as
before. Put the camera lucida in position, and with a lead pencil
make dots on the paper at the limits of the image of the blood cor-
puscles. Measure the same three that were measured in § 384.

Remove the object, place the stage micrometer under the micro-
scope, focus well, and draw the lines of the stage micrometer so as to

304 MAGNIFICATION AND MICROMETRY [Cn. VIII

include the dots representing the limits of the part of the image to be
measured. As the value of the spaces on the stage micrometer is
known, the size of the object is determined by the number of spaces
of the micrometer required to include it.

This simply enables one to put the image of a fine rule on the
image of a microscopic object. It is theoretically an excellent
method, and nearly the same as measuring the spread of the dividers
with a simple microscope (§ 364).

§ 386. Micrometry with the ocular micrometer with fixed lines. —
Use the 4 mm. (4ox) objective, and the ocular with the ocular microm-
eter. For object use the same corpuscles as in §§ 384-385. Make
sure that all the conditions are exactly as when the valuation was
determined; then put the preparation under the microscope and
find the same three red corpuscles that were measured in the other
ways (§ 384).

Count the divisions on the ocular micrometer required to enclose
or measure the long and the short axis of each of the corpuscles,
multiply the number of spaces in both cases by the valuation of the
ocular micrometer, and the results will represent the actual length of
the axes of the corpuscles in each case.

The same corpuscle is, of course, of the same actual size, when
measured in each of the three ways, so that if the methods are
correct and the work carefully enough done, the same results should
be obtained by each method.

§ 387. Micrometry with the movable scale ocular micrometer. —
Use the same preparation and objective as before. Arrange the
micrometer ocular so that the long axis of the corpuscle will coincide
with the cross line in the micrometer scale (figs. 158-159). Get one
end of the corpuscle exactly level with one division of the microm-
eter scale. Note the position of the drum, and then rotate it until
the other end of the corpuscle is exactly against the nearest line of
the micrometer. Count up the entire intervals required and the
partial interval on the drum. Suppose it requires 5 entire and 0.60
intervals (see explanation of fig. 159); then the whole corpuscle
must be 5.60 intervals multiplied by 4/1 (§ 37?)> tne value °* one
interval; 5.6 X 4 = 22.4ju.

CH. VIII] MAGNIFICATION AND MICROMETRY 305

§ 388. Micrometry with the filar micrometer. — Use the same
preparation and objective as before, but use a filar micrometer.
Note how many graduations on the recording comb and drum (fig.
1 60) are required to measure each dimension of the corpuscle, and
multiply by the valuation as in the other cases.

The advantage of the filar micrometer is that the evaluation of
one graduation is so small that even the smallest object to be meas-
ured would require several graduations to measure it. In ocular
micrometers with fixed lines, small objects like bacteria might not
fill even one space; therefore estimations, not measurements, must
be made. For large objects, like most of the tissue elements, the
ocular micrometers with fixed lines answer very well, for the part
which must be estimated is relatively small and the chance of error
is correspondingly small (§ 389).

§ 389. There are three ways of using the ocular micrometer, or of
arriving at the size of the objects measured with it:

(1) By finding the value of a division of the ocular micrometer for
each optical combination and tube-length used, and employing this
valuation as a multiplier. This is the method given in the text, and
the one most frequently employed. Thus, suppose with a given
optical combination and tube-length it required five divisions on the
ocular micrometer to include the image of 0.2 millimeter of the stage
micrometer, then obviously one space on the ocular micrometer
would include f or 0.2 or 0.04 mm.; the size of any unknown object
under the microscope would be obtained by multiplying the number
of the divisions on the ocular micrometer required to include its
image by the value of one space, or in this case 0.04 mm. Suppose
some object, as the fly's wing, required 15 spaces of the ocular
micrometer to include some part of it, then the actual size of this
part of the wing would be 15 x 0.04 = 0.6 mm.

(2) By finding the number of divisions on the ocular micrometer
required to include the image of an entire millimeter of the stage
micrometer, and using this number as a divisor. This number is
also sometimes called the ocular micrometer ratio. Taking the same
case as in (i), suppose five divisions of the ocular micrometer are
required to include the image of 0.2 mm., on the stage micrometer,

306 MAGNIFICATION AND MICROMETRY [Cn. VIII

then evidently it would require 5 -r 0.2 = 25 divisions on the ocular
micrometer to include a whole millimeter on the stage micrometer,
and the number of divisions of the ocular micrometer required to
measure an object divided by 25 would give the actual size of the
object in millimeters or in a fraction of a millimeter. Thus, suppose
it required 15 divisions of the ocular micrometer to include the image
of some part of the fly's wing, the actual size of the part included
would be 15 -r 25 = | or 0.6 mm. This method is really exactly like
the one in (i), for dividing by 25 is the same as multiplying by -^
or 0.04.

(3) By having the ocular micrometer ruled in millimeters and
divisions of a millimeter, and then getting the size of the real image
in millimeters! In employing this method a stage micrometer is
used as object and the size of the image of one or more divisions is
measured by the ocular micrometer, thus: Suppose the stage microm-
eter is ruled o.i and o.oi mm. and the ocular micrometer is ruled
in millimeters and o.i mm. Taking 0.2 mm. on the stage microm-
eter as object, as in the other cases, suppose it requires 10 of the o.i
mm. spaces of i mm. to measure the real image, then the real image
must be magnified i.o -f- 0.2 = 5 diameters, that is, the real image is five
times as great in length as the object, and the size of an object may
be determined by putting it under the microscope and getting the
size of the real image in millimeters with the ocular micrometer and
dividing it by the magnification of the real image, which in this case
is 5 diameters.

Use the fly's wing as object, as in the other cases, and measure
the image of the same part. Suppose that it required 30 of the o.i
mm. divisions = 3 mm. to include the image of the part measured,
then evidently the actual size of the part measured is 3 mm. 4- 5
= f mm., or 0.6 mm., the same result as in the other cases. See
also § 390 on the eikonometer.

In comparing these methods it will be seen that in the first two
the ocular micrometer may be simply ruled with equidistant lines
without regard to the absolute size in millimeters or inches of the
spaces. In the last method the ocular micrometer must have its
spaces some known division of a millimeter or inch. In the first two

CH. VIII]

MAGNIFICATION AND MICROMETRY

307

methods only one standard of measure is required, viz., the stage
micrometer; in the last method two standards must be used, viz.,
a stage micrometer and an ocular micrometer.

§ 390. Eikonometer for magnification and
micrometry. — The eikonometer is something
like an eye. It has a converging lens serving
in place of the crystalline lens to focus the
rays from the eyepiece of the compound micro-
scope, or from the simple microscope upon a
micrometer scale, the scale taking the place of
the retina in the eye (figs. 145-146). This
scale is ruled in o.i mm. Above the scale is a
Ramsden ocular of 25 mm. equivalent focus,
giving a magnification of 10. The eikonometer
scale, therefore, is a millimeter scale when seen at
the distance of 250 mm. in the visual field of the
normal human eye, and it enables one to put a
millimeter scale on the image of any object
studied.

To use it for magnification a stage microm-
eter is put under the microscope and carefully
focused. Then the eikonometer is put in place
the ocular. The microscopic image of the

over

stage micrometer and the scale of the eikono-
meter will then appear in the same field as
with the ordinary ocular micrometer (§ 375).
The two sets of lines should be made parallel
(§§ 374~3?6). See how many divisions of the
eikonometer millimeter scale are required to
measure one or more of the divisions of the
image of the stage micrometer. Suppose it
requires 6 intervals or millimeters of the
eikonometer scale to measure the image of 0.03
mm. on the stage micrometer. The size of the
object is then 0.03 mm., and of its image 6 mm.
tion is therefore (§ 361) 6 •*- 0.03 = 200.

U«o

FIG. 163. WRIGHT'S

EIKONOMETER.

(From Sir A. E.
Wright's Principles
of Microscopy).

o Object.

vi Virtual image.

ob Objective.

Microscope Ocu-
lar, the objective,
tube and ocular of
the microscope.

Eikonometer The
Ramsden ocular (Ro)
magnifying 10 diam-
eters, and field lens
(fi) above the ocular
of the microscope.

es The real image
formed at the dia-
phragm of the eikon-
ometer.

The magnifica-

308 MAGNIFICATION AND MICROMETRY [Cn. VIII

For determining the magnification of a simple microscope the
eikonometer is placed over the simple microscope as it was over the
ocular above. With this instrument, as with the camera lucida, only
one eye is used (figs. 149, 169).

§ 391. Micrometry with the eikonometer. — In the first place the
magnification of the microscope must be determined as described
in the preceding section; and one must keep in mind the factors
which will vary the magnification (§ 368). The object to be meas-
ured is put under the microscope and focused and the eikonometer
put in position. The virtual image is then measured in millimeters
by the scale of the instrument. The size of this virtual image is
then divided by the magnification and the result will be the actual
size of the object as in § 384.

For example, suppose the long axis of a necturus' red blood cor-
puscle measures 9 mm. on the eikonometer scale. If the magnifica-
tion of the microscope is 200, as found above, then the actual length
of the corpuscle is 9 mm. -*• 200 = 0.045 mm., or 45/z.

§ 392. Micrometry by the aid of the condenser image of a scale.
— Probably every one is all too familiar with the cross bars of the
window "in the field of the microscope. This is, as well known, a real
image of the window produced by the condenser at the level of the
object. The possibility of projecting a real image at the level of the
object is taken advantage of for purposes of micrometry as follows:
A lantern slide is made of net lines (fig. 164) or of any parallel,
equidistant lines. The lantern slide is then set up exactly 10 cm. or
some other exact distance in front of the microscope. A good light
from the window or from one of the daylight lanterns (figs. 46, 53)
must traverse the lantern slide. This light is reflected up through
the condenser by the plane mirror. The condenser will form a real
image of the network or parallel lines at about the level where the
object is placed on the slide. If now one focuses a 16 mm. (lox) or
other objective upon this real image, it will appear very clearly in
the field of the microscope. In order to utilize the image for
micrometry the valuation of the spaces must be determined by the
use of a stage micrometer as with the ocular micrometer (§ 375).
Place a stage micrometer under the microscope and focus the lines

CH. VIII]

MAGNIFICATION AND MICROMETRY

sharply. Then with the screw or rack of the substage condenser
focus the condenser up and down until the image of the lines or net

FIG. 164. NET SCALE FOR USE IN MICROMETRY WITH THE CONDENSER

IMAGE.

on the lantern slide is also sharp. Arrange the stage micrometer
so that the lines are parallel with the lines of the condenser image.
Make any two of the lines coincide. Count the number of spaces in
the condenser image included between any two of the lines of the
stage micrometer, and divide the value of the space in the stage
micrometer by the number of spaces of the condenser image in-
cluded. The quotient will represent the valuation of the spaces of
the condenser image in millimeters. For example, suppose the stage
micrometer is ruled in o.i mm. and that 12 spaces of the condenser
image are included in 9 spaces of the stage micrometer; then each
space of the condenser image has a valuation of 0.9 mm. -r 12 =
0.075 mm-

As the size of the image varies with the distance of the object
from the center of the condenser (§ 362), if the object (lantern
slide of the lines) is always placed exactly the same distance in front
of the microscope, the real image formed by the condenser will be of
the same size, and hence have the same valuation for micrometry
regardless of the power of the objective or the length of tube

310 MAGNIFICATION AND MICROMETRY [Cat. VIII

used. It is a very convenient method of micrometry for all coarser
objects, but not exact enough for the finer objects* A movable scale
or filar ocular micrometer should be used for the most exact work.

Example of an actual measurement by means of the condenser
image: The long axis of a red corpuscle of necturus measured 0.61
of a space of the condenser image. As each space represents 0.075
mm. the length of the corpuscle is: 0.61 X 0.075 = o-°4575 mm- or
45.75/x. (See Chamot, pp. 155-15?.)

§ 393. Remarks on micrometry. — In using adjustable objectives
(§§ 29, 149) the magnification of the objectives varies with the posi-
tion of the adjusting collar, being greater when the adjustment is
closed, as for thick cover-glasses, than when open, as for thin ones.
This variation in the magnification of the objective produces a corre-
sponding change in the magnification of the entire microscope and
the ocular micrometer valuation; therefore it is necessary to
determine the magnification and ocular micrometer valuation for
each position of the adjusting collar.

While the principles of micrometry are simple, it is very difficult
to get the exact size of microscopic objects. This is due to the lack
of perfection and uniformity of micrometers and the difficulty of
determining the exact limits of the object to be measured. Hence,
all microscopic measurements are only approximately correct, the
error lessening with the increasing perfection of the apparatus and
the skill of the observer.

A difficulty when one is using high powers is the width of the lines
of the micrometer. If the micrometer is perfectly accurate, half the
width of each line belongs to the contiguous spaces, hence one should
measure the image of the space from the centers of the lines border-
ing the space, or, as this is somewhat difficult in using the ocular
micrometer, one may measure from the inside of one bordering line
and from the outside of the other, that is, from the right side of all
the lines, or from the left side of all. If the lines are of equal width
this is as accurate as measuring from the center of the lines. Evi-
dently it would not be right to measure from either the inside or the
outside of both lines (figs. 157, 165).

It is also necessary in micrometry to use an objective of sufficient

CH. VIII]

MAGNIFICATION AND MICROMETRY

power to enable one to see all the details of an object with great dis-
tinctness. The necessity of using sufficient amplification in microm-
etry has been especially remarked upon by Richardson, Monthly
Micr. Jour., 1874, 1875; Rogers, Proc. Amer. Soc. Microscopists,
1882, p. 239; Ewell, North Amer. Pract., 1890, pp. 97, 173.

Correct

Incorrect

FIG. 165. CORRECT AND INCORRECT ARRANGEMENT OF THE OCULAR AND OF
THE STAGE MICROMETER LINES.

(From Chamot).

The fine lines are those of the ocular micrometer and the coarse ones of the
stage micrometer (compare fig. 157).

As to the limit of accuracy in micrometry, one who has justly
earned the right to speak with authority expresses himself as fol-
lows: " I assume that o.2ju is the limit of precision in microscopic
measures beyond which it is impossible to go with certainty." W.
A. Rogers, Proc. Amer. Soc. Micr., 1883, p. 198.

In comparing the methods of micrometry with the compound
microscope given above (§§ 383-390), the one given in § 383 is im-
practicable; that given in §§ 388-390 is open to the objection that
two standards are required — the stage micrometer and the steel
rule; it is open to the further objection that several different
operations are necessary, each operation adding to the probability of
error. Theoretically the method given in § 385 is good, but it is
open to the very serious objection in practice, that it requires so
many operations which are especially likely to introduce errors.
The method that experience has found most safe and expeditious,
and applicable to all objects, is the method with the ocular microm-
eter. If the valuation of the ocular micrometer has been accu-
rately determined, then the only difficulty is in deciding on the
exact limits of the object to be measured and so arranging the ocular
micrometer that these limits are enclosed by some divisions of the
micrometer. Where the object is not exactly included by whole

312 MAGNIFICATION AND MICROMETRY [Cn. VIII

spaces on the ocular micrometer, the chance of error comes in, in
estimating just how far into a space the object reaches on the side not
in contact with one of the micrometer lines. If the ocular microm-
eter has some quite narrow spaces, and others considerably larger,
one can nearly always manage to exactly include the object by some
two lines. The ocular screw micrometers (figs. 158, 160) obviate
this entirely, as the cross hair or lines traverse the object or its real
image, and whether this distance be great or small it can be read off
on the graduated wheel, and no estimation or guess work is neces-
sary.

INDEPENDENT MAGNIFICATION OF OBJECTIVES AND OCULARS

§ 394. Independent magnification of an objective. — The inde-
pendent magnification of an objective is like that of a projection
microscope when the objective alone is used (figs. 147, 166). As
pointed out in § 370 it is necessary to select some standard distance
for the projection of the real or of the virtual image, for the size of
the image varies directly as its distance from the center of the lens
(fig. 152 for real and 145 for virtual images; in the latter the pro-
jection distance is from the nodal point in the eye to the image).
The image distance for magnification most commonly employed is
250 mm. (§ 370).

While the magnification distance in microscopy has been fixed as
250 millimeters by general agreement, in actual use with a short or
160 mm. tube, the magnification of the objective is less than that
which would be found by getting the magnification at the standard
distance of 250 mm.

Now that the actual magnification produced by the objective on
the short tube is used in designating it and this magnification number
is correct no matter what kind of an ocular is used, it is worth while
to know how it is obtained. In section eighteen the method
is given. Briefly it is as follows: A stage micrometer is put upon the
stage, and the objective to be used is put in place. A Huygenian
ocular is inserted in the tube which has been set for a tube-length of
exactly 160 millimeters (figs. 18, 26). The stage micrometer is then
focused as sharply as possible. The Huygenian ocular is removed

CH. VIII] MAGNIFICATION AND MICROMETRY 313

and a Ramsden micrometer ocular put in its place. Without fo-
cusing the microscope the least bit, the micrometer ocular is moved
in the tube, or the tube is lengthened or shortened as necessary to
give again a sharp image of the stage micrometer. The lines of the
two micrometers are made parallel and the image of one or more
spaces of the stage micrometer measured. Suppose the image of o.io
mm. on the stage micrometer measures i mm., then the magnifica-
tion of that objective with the short tube is 10, and this number is
the one now marked on objectives of 16 mm. (lox) equivalent focus.

§ 395* Magnification due to the ocular. — The final magnifica-
tion of the microscope (fig. 18) is due to the magnification of the
objective multiplied by the magnification of the ocular. That is, the
objective gives a real, magnified image, and the ocular as a whole
gives a magnified image of the real image formed by the objective.
The image formed by the ocular is measured at 250 millimeters dis-
tance, not at 1 60 millimeters as with the objective.

One of the best ways to determine the magnifying action of the
ocular is to determine the magnification of the whole microscope
(§§ 365, 367). Knowing the entire magnification, and knowing the
magnification due to the objective, the part played by the ocular is
the entire magnification divided by the objective magnification.
For example, if the objective gives a magnification of 10, and the
entire magnification of the microscope is 100 then W - *o> that is,
the ocular must also have magnified 10.

If the ocular's magnification is 10 for a 250 mm. image distance,
its equivalent focus must be W = 25, and the designation of this
ocular would be lox, or 25 e.f . and have an equivalent focus of 25 mm.
It would magnify 10 diameters with any objective.

It may be puzzling to see how an objective with a magnification of
10, for example, could give the same final image with positive ocu-
lars, as with the negative oculars (figs. 24-25). The field lens serves
to make the real image of the objective smaller (figs. 24-25) while
the real image of the objective is formed below all the lenses of the
positive oculars (figs. 22-23) and they all unite in acting as a
magnifier.

The difficulty is overcome in this way: The curvature of the

MAGNIFICATION AND MICIICMETRY [Cu. VIII

Ocular

Objective

FIG. 166-167. MICROSCOPE MAGNIFICATION BY PROJECTION.

e I Eyelens of the ocular serving to project a real image to the screen.

// Field lens, the lower lens in a negative ocular; it reduces the size of the
real image formed by the objective.

r i Real image formed by the objective and field lens.

rf i' Position and size of the real image if no field lens were present.

The screen distance of 250 millimeters is measured along the axis from the
eyepoint, not from the eyelens.

eyelens or combination as the case may be, is made enough greater
to compensate for the reducing action of the field lens, and thus the
ocular as a whole gives the desired increase in magnification, and its
total action may be indicated with the same definiteness as with
positive oculars, hence the magnification number on a Huyge-
nian ocular while it gives no clue to the action of the individual
lenses composing ity does indicate its final effect in producing the
magnification of the microscope.

§ 396. Nelson's projection method of determining the magnifica-
tion of the entire microscope. — This method which has been
rigidly tested by several observers and by myself side by side with
the camera lucida method, gives such uniformly accurate results
that it is recommended for general adoption. It is illustrated clearly
by fig. 1 66. As used by the writer, the work was done by night or
in a dimly lighted room.

The microscope is made horizontal and fastened to a block which slides on
an optical bench (fig. 179). A dark-field lamp (figs. 79-80) is placed in line or
at right angles to the microscope opposite the mirror (fig. 182).

A vertical white screen or a piece of finely ground glass is set up on a movable
block beyond the ocular. The microscope is moderately lighted and the microm-
eter lines focused with extreme sharpness, then by means of a white card or piece
of ground glass the position of the eyepoint of the ocular is determined, and the
white vertical screen placed exactly 250 millimeters from the eyepoint. This is
important. If the distance were measured from the top of the ocular, it would

CH. VIII] MAGNIFICATION AND MICROMETRY 315

not give the correct result, and the error would be greater the higher the eye-
point, as with the "telaugic" oculars (§§ 41, 145). The light in the microscope
is now made as brilliant as possible, and the lines of the micrometer made as
sharp as possible on the \\ hite screen by a slight turn of the fine adjustment.

With bow-dividers or other fine dividers the image of one or more spaces near
the middle of the field is measured, and the spread of the dividers determined
?s in § 364. The total magnification can then be found by dividing the size of
the image by the actual size of the micrometer space measured by the dividers
(§ 367)- (E- M. Nelson, Jour. Quekett Micr. Club, vol. xii, 1913, pp. 374~379-)

§ 397. Phelps Gage's method of obtaining the magnification for ocular and
objective, and for the whole microscope. —

(1) A stage micrometer is used as object and fcci sed sharply on the scale
of an ocular micrometer in a positive ocular, Loth rricron eters bdrg in fractions
of a millimeter.

(2) Make the microscope horizontal, light brilliantly \\ith the io8-watt,
6-volt or other lamp. Determine the position of the eye-point of the ocular
(§ 99). Put a vertical \\hite screen 250 rr,m. from the e>e~pcint and focus the
stage micrometer on it. If the ccular micron: eter is not also in focus on the
ecreen, raise or lower the positive ocular con hination until the lines are sharp.
Refocus the stage micrometer if necessary.

(3) With bow or other fine dividers measure one or more spaces of each
micrometer image.

(a) The image of the stage micrometer will represent the magnification of the
entire microscope, objective and ocular.

(6) The image of the ocular micrometer v\ill show the separate* magnification
<f the positive ocular.

(t) The magnification of the whole microscope divided by the ocular magni-
fication will represent the magnification of the objective on the 160 mm. tube.

If one knows the magnification of the objective, the magnification
of any ocular, positive or negative, can be found by dividing the
magnification of the whole microscope obtained as in 30 by that
of the objective.

As the field lens of the negative ocular reduces the magnification of
the objective, one can find the amount of the reduction by getting
first the magnification of the whole microscope with the field lens in
place and then with the field lens removed. As the eye-point is
higher when the field lens is removed, one must readjust the micro-
scope or the image screen to make the screen distance 250 mm. from
the eye-point when getting the magnification.

§ 398. Magnification of drawings. — In determining the magnifi-
cation of a drawing made with a camera lucida or with projection-
apparatus, by far the best method is to remove the specimen and
put in its place a stage micrometer and project the image of the
micrometer upon the drawing paper. Make a few lines of the
micrometer image and indicate the value of the spaces (fig. 172)

3i6 MAGNIFICATION AND MICROMETRY [Cn. VIII

then at any time one can determine exactly what the magnification
is (§ 409)-

COLLATERAL READING FOR CHAPTER VII

Sir A. E. Wright's Principles of Microscopy. Chamot, Chemical Microscopy,
Chamot & Mason.

For those especially interested in micrometry in its relation to medical juris-
prudence the following are recommended. They treat the subject in a practical
as well as in a scientific spirit. The papers of Prof. Wm. A. Rogers on microm-
eters and micrometry, in the Amer. Quar. Micr. Jour., Vol. I. pp. 97, 208;
Proceedings Amer. Soc. Microscopists, 1882, 1883, 1887. Dr. M. D. Eweli,
Proc. Amer. Soc. Micr., 1890; The Microscope, 1889, pp. 43~4S; North Amer.
Pract. 1890, pp. 97, 173. Dr. J. J. Woodward, Amer. Jour, of the Med, Sci.,
1875. M. C. White, Article " Blood Stains," Ref. Hand-Book Med. Sciences,
1885. Medico-Legal Journal, Vol. XII. For the change in magnification due to
a change in the adjustment of adjustable objectives, see Jour. Roy. Micr. Soc.
1880, p. 702; Amer. Monthly Micr. Jour., 1880, p. 67. Carpenter-Dallinger,
p. 270 and end of § 196.

If one consults the medico-legal journals, the microscopical journals, the Index
Medicus, the Index Catalog of the library of the Surgeon General's Office, and
The Quarterly Cumulative Index Medicus under Micrometry, Blood, and Juris-
prudence, he can get on track of the main work which has been and is being
done in legal medicine.

Optic Projection, S. H. & H. P. Gage.

Microscopy, E. J. Spitta.

The Microscope and its Revelations, Carpenter-Dallinger.

Journal of the Royal Microscopical Society.

Transactions of the American Microscopical Society, especially the address of
Hon. J. D. Cox, 1884, pp. 5-39 on Aperture, and 1893, pp. 1-16, and A. C.
Mercer, 1896, pp. 321-396.

John C. Shedd, The Index of Refraction. School Science and Mathematics,
Vol. VI, 1906, pp. 678-680.

(This article gives a brief history of the discovery of the law of refraction;
it also discusses the ratio of velocities in different media, and shows that the
coefficient of retardation of velocity in a transparent medium is the reciprocal
of the index of refraction.)

According to Nelson, "Par-focal" oculars have been made by Powell since
1839.

NELSON, E. M. — Eyepieces for the Microscope. Jour. Roy. Micr. Soc., 1908,
p. 149. See also for other discussions of oculars by Nelson, same journal, 1907,

BECK, CONRAD. — The Microscope; Theory and Practice, London, 1938, pp. 45-
47, 225-226.

CHAPTER IX

DRAWING WITH THE MICROSCOPE AND WITH PROJECTION

APPARATUS; CLASS DEMONSTRATIONS

§§399-450; FIGURES 168-199

§ 399. Methods of drawing. — There are five principal methods
for obtaining drawings in general, and all the methods are applicable
to the production of drawings of microscopic objects:

(i) Free-hand drawings. This is the simplest method if one has
natural ability and adequate training, for one needs only an object,
pencil, pen and paper.

(2) Camera lucida drawings. By this method the outlines and
proportions can be accurately traced (§§ 401-408).

(3) Camera obscura drawings. By this method the real image
obtained in a photographic camera can be traced (§ 410).

(4) Projection drawings. In this method real images like those
of the magic lantern and projection microscope can be traced di-
rectly upon the drawing paper (§ 418).

(5) Line drawings on blue prints and on the back of photo-
graphs (§§ 413-414).

In many laboratories all the methods are used, sometimes sepa-
rately, but more often combined.

§ 400. Free-hand drawings. — Microscopic objects may be drawn
free-hand directly from the microscope, but in this way a picture
giving only the general appearance and relations of parts is obtained.
For pictures which shall have all the parts of the object in true pro-
portions and relations, it is necessary to obtain an exact outline of
the image of the object, and to locate in this outline all the princi-
pal details of structure. It is then possible to complete the picture
free-hand from the appearance of the object under the microscope.

§ 401. Camera lucida. — This is an optical apparatus for enabling
one to see objects in greatly different situations as if in one field of
vision, and with the same eye. In other words, it is an optical de-
vice for superimposing or combining two fields of view in one eye.

DRAWINGS AND DEMONSTRATIONS

[CH. IX

As applied to the microscope, it causes the magnified virtual
image of the object under the microscope to appear as if projected
upon the table or drawing board, where it is visible with the draw-
ing paper, pencil, dividers, etc., by the same eye, and in the same
field of vision. The microscopic image appears like a picture on the
drawing paper (§ 4O4a). This is accomplished in two distinct
ways:

(i) By a camera lucida reflecting the rays from the microscope
~*^ so that their direction when

they reach the eye coincides
with that of the rays from
the drawing paper, pencil,
etc. In some of the camera
lucidas from this group
(Wollaston's, fig. 168), the
rays are reflected twice, and
the image appears as when
looking directly into the
microscope. In others the
rays are reflected but once,
and the image has the in-
version produced by a plane
mirror. For drawing pur-
FIG. 168. WOLLASTON'S CAMERA LUCIDA. poses this inversion is a
Axis The optic axis of the microscope, r,*™* ^i^ti™ « '4-
Ocidar The upper end of the ocular. 8reat objection, as it IS

A, B Two rays outside the axis to show necessary to invert similarly
that they cross twice and hence have the Oii ft,_ A^n\\« A A A t
same relative position as when they emerge a11 the details added free-
from the ocular. hand.

Camera lucida The quadrangular piece of /-\ T>, i • i

glass giving the double internal reflection to W By a Camera lucida
change the direction of the axkl ray 90°. reflecting the rays of light

CD. AB The virtual image, drawing pacer r ,1 j

and pencil partly overlapping. \Vhere they f r°m the drawing paper, etc.,
overlap the appearance is that of one field. so that their direction when

they reach the eye coincides

with the direction of the rays from the microscope (fig. 169).
In all of the camera lucidas of this group, the rays from the paper
are twice reflected and no inversion appears.

CH. IX] DRAWINGS AND DEMONSTRATIONS 319

The better forms of camera lucidas (Wollaston's, Grunow's,
Abbe's, etc.) may be used for drawing both with low and with high
powers. Some require the microscope to be inclined (fig. 168) while
others are designed to be used on the microscope in a vertical posi-
tion. As in biological work it is often necessary to have the micro-
scope vertical, the form for a vertical microscope is to be preferred
(fig. 169).

§ 402. Avoidance of distortion. — In order that the picture drawn
by the aid of a camera lucida may not be distorted, it is neces-
sary that the axial ray from the image on the drawing surface shall
be at right angles to the drawing surface (figs. 168, 170).

§ 403. Wollaston's camera lucida. — This is a quadrangular
prism of glass put in the path of the rays from the microscope, and
it serves to change the direction of the axial ray 90 degrees. In
using it the microscope is made horizontal, and the rays from the
microscope enter one-half of the pupil, while rays from the drawing
surface enter the other half of the pupil. As seen in fig. 168, the
fields partly overlap, and where they do so overlap, pencil or dividers
and microscopic image can be seen together.

In drawing or using the dividers with the Wollaston camera lucida
it is necessary to have the field of the microscope and the drawing
surface about equally lighted. If the drawing surface is too bril-
liantly lighted, the pencil or dividers may be seen very clearly, but
the microscopic image will be obscure. On the other hand, if the
field of the microscope has too much light, the microscopic image
will be very definite, but the pencil or dividers will not be clearly
visible. It is necessary, as with the Abbe camera lucida (§ 404), to
have the Wollaston prism properly arranged with reference to the
axis of the microscope and the eyepoint. If it is not, one will be
unable to see the image well, and may be entirely unable to see the
pencil and the image at the same time. Again, as rays from the
microscope and from the drawing surface must enter independent
parts of the pupil of the same eye, one must hold the eye so that
the pupil is partly over the camera lucida and partly over the
drawing surface. One can tell the proper position by trial. This is
not a very satisfactory camera to draw with, but it is a very good

320

DRAWINGS AND DEMONSTRATIONS

[CH. IX

form to measure the vertical distance of 250 mm. at which the draw-
ing surface should be placed when determining magnification (fig.

153).

§ 404. Abbe camera lucida. — This consists of a cube of glass
cut into two triangular prisms and silvered on the cut surface of the
upper one. A small oval hole is then cut out of the center of the

FIG. 169. DIAGRAM OF ABBE'S CAMERA LUCIDA WITH A VERTICAL MICROSCOPE.

Axis, Axis The axial ray of the microscope and from the field of the drawing
surface.

Ocular The upper part of the microscope ocular.

Mirror The mirror of the camera lucida reflecting the rays from the drawing
surface at right angles to the axis.

P, P The drawing pencil in the field, and the prism of the camera lucida.

Q The quadrant attached to the mirror to give the angle.

G Smoked glass.

a b The silvered surface in the prism with a hole made in the center for the
light to pass upward from the microscope. The silvered part reflects the rays
from the drawing surface.

The geometrical figure at the left gives the angles when a 45° mirror is used.

CH. IX] DRAWINGS AND DEMONSTRATIONS 321

silvered surface and the two prisms are cemented together in the
form of the original cube with a perforated 45 degree mirror within
it (figs. 169-170). The upper surface of the cube is covered by a per-
forated metal plate. This cube is placed over the ocular in such a
way that the light from the microscope passes through the hole in
the silvered face and thence directly to the eye. Light from the
drawing surface is reflected by the mirror to the silvered surface of
the prism and reflected by this surface to the eye in company with
the rays from the microscope, so that the two fields appear as one,
and the image is seen as if on the drawing surface (figs. 168-171,
§ 4°4a).

§ 404a. For some persons the image and the drawing surface, pencil, etc., do
not appear on the drawing board as stated above, but under the microscope, ac-
cording to the general principle that "objects appear in space where they could
be touched along a perpendicular to the retinal surface stimulated," — that is, in
the line of rays entering the eye. This is always the case with the Wollaston
camera lucida. The explanation of the apparent location of the image, etc., on
the drawing board with the Abbe camera lucida is that the attention is concen-
trated upon the drawing surface rather than upon the object under the micro-
scope. With some observers it is possible to make the image appear under the
microscope or on the drawing surface at will by concentrating the attention of
one position or the other. (Dr. W. B. Pillsbury).

§ 405. Arrangement of the camera lucida prism. — In placing
this camera lucida over the ocular for drawing or for the determi-
nation of magnification, the center of the hole in the silvered surface
is placed in the optic axis of the microscope. This is done by prop-
erly arranging the centering screws that clamp the camera to the
microscope tube or ocular. The prism must not only be centered
to the axis of the microscope, but it must be at the right level, or
more or less of the field will be cut off. In all the good modern
forms of this camera lucida it is fastened to the tube of the micro-
scope by a clamp which enables one to raise or lower it so that it
may be at the right position with reference to the eyepoint of the
ocular being used (§99).

One can determine when the camera is in a proper position by
looking into the microscope through it. If the field of the micro-
scope appears as a circle and of about the same size as without the
camera lucida, then the prism is in a proper position. If one side of
the field is dark, then the prism is to one side of the center; if the

322

DRAWINGS AND DEMONSTRATIONS

[CH. IX

field is considerably smaller than when the prism is turned off the
ocular, it indicates that it is not at the correct level, i.e., it is above

or too far below the
eyepoint.

§ 406. Arrangement
of the mirror and the
drawing surface. —
The Abbe camera
lucida was designed
for use with a vertical
microscope (fig. 169).
On a vertical micro-
scope if the mirror is
set at an angle of 45°,
the axial ray is at
right angles with the
table top or drawing
board which is horiz-

FIG. 170. DIAGRAM OF THE ABBE CAMERA LU-
CIDA WITH THE DRAWING SURFACE ELEVATED TO

MAKE THE Axis PERPENDICULAR WITH DEPRESSED ontal, and a drawing

MIRROR

A, Axis, Axis The axial ray from the microscope
and from the drawing surface.

made under these con-
ditions is in true pro-

Ocular The upper part of the microscopic ocuhr. p0rtion and not dis-

\A ' iwnv TV»£» mirrrvr nf t\\& r^amArn liifirlo* if 10 HA_ *

torted. The stage of
most microscopes, how-
ever, extends out so far
at the sides that with
a 45° mirror the image
appears in part on the
stage of the micro-
scope. In order to
avoid this, the mirror
may be depressed to

some point below 45°, say at 40° or 35° (fig. 170). But as the
axial ray from the mirror to the prism must still be reflected hori-
zontally, it follows that the axial ray no longer forms an angle of
90° with the drawing surface, but a greater angle. If the mirror is

Mirror The mirror of the camera lucida; it is de-
pressed from 45° to 35° to make the axis from the
drawing surface perpendicular to the axis of the mi-
croscope.

A — B The drawing surface elevated 20°; that is,
twice as many as the mirror is depressed belo,v 45°.

W Wedge under the drawing board.

P, P The drawing pencil and the prism of the
camera lucida.

Q Quadrant of the mirror.

B Geometrical figure to show why the drawing
board must be raised twice as many degrees as the
mirror is depressed to keep the axial ray perpendicu-
lar to the drawing surface.

CH. IX] DRAWINGS AND DEMONSTRATIONS 323

depressed to 35°, then the axial ray makes an angle of 110° with
a horizontal drawing surface (fig 170 B). To make the angle 90°
again, so that there shall be no distortion, the drawing board must
be raised toward the microscope 20°. The general rule is to raise
the drawing board twice as many degrees toward the microscope as
the mirror is depressed below 45°. Practically, the field for drawing
can always be made free of the stage of the microscope, at 45°, at
40°, or at 35°. In the first case (45° mirror) the drawing surface
should be horizontal, in the second case (40° mirror) the drawing
face should be elevated 10°, and in the third case (35° mirror) the
drawing board should be elevated 20° toward the microscope.
Furthermore, it is necessary in using an elevated drawing board to
have the mirror bar of the camera lucida project directly laterally
so that the edges of the mirror are in planes parallel with the edges
of the drawing board; otherwise there will be front to back dis-
tortion, although the elevation of the drawing board avoids right to
left distortion. If one has a micrometer ruled in squares (net
micrometer) (figs. 131, 164), the distortion produced by not having
the axial ray at right angles with the drawing surface may be very
strikingly shown. For example, set the mirror at 35° and use a
horizontal drawing board. With a pencil make dots at the corners
of some of the squares, and then with a straight edge connect the
dots. The figures will be considerably longer from right to left
than from front to back. Circles in the object appear as ellipses in
the drawings, the major axis being from right to left.

The angle of the mirror may be determined with a protractor,
but that is troublesome. It is much more satisfactory to have a
quadrant attached to the mirror and an indicator on the projecting
arm of the mirror. If the quadrant is graduated throughout its
entire extent, or preferably at three points, 45°, 40° and 35°, one can
set the mirror at a known angle in a moment; then the drawing
board can be hinged and the elevation of 10° and 20° determined
with a protractor. The drawing board is very conveniently held
up by a broad wedge. By marking the position of the wedge for 10°
and 20° the protractor need be used but once; then the wedge may
be put into position at any time for the proper elevation.

324

DRAWINGS AND DEMONSTRATIONS

[CH, IX

§ 407. Abbe camera and inclined microscope. — It is very fati-
guing to draw continuously with a vertical microscope, and many
mounted objects admit of an inclination of the microscope, when one
can sit and work in a more comfortable position. The Abbe camera
is as perfectly adapted to use with an inclined as with a vertical micro-
scope. All that is requisite is to be sure that the fundamental law
is observed regarding the axial ray of the image and the drawing

FIG. 171. BERNHARD'S DRAWING BOARD FOR THE ABBE CAMERA LUCIDA.

(From the Catalogue of Zeiss).

This drawing board can be elevated and tipped; it can also be inclined, carrying
the microscope with it.

surface, viz.. that they should be at right angles. This is very easily
accomplished as follows: The drawing board is raised toward the
microscope twice as many degrees as the mirror is depressed below
45° (§ 4°6) ; then it is raised exactly as many degrees as the micro-
scope is inclined, and in the same direction, that is, so that the end
of the drawing board shall be in a plane parallel with the stage of

CH. IX] DRAWINGS AND DEMONSTRATIONS 325

the microscope. The mirror must have its edges in planes parallel
with the edges of the drawing board also (fig. 171).

§ 408. Drawing with the Abbe camera lucida. — (i) The light
from the microscope and from the drawing surface should be of
nearly equal intensity, so that the image and the drawing pencil can
be seen with about equal distinctness. This may be accomplished
with very low powers (16 mm. (rox) and lower objectives) by cover-
ing the mirror of the microscope with white paper when transparent
objects are to be drawn. For high powers it is best to use a substage
condenser. Often the light may be balanced by using a larger or
smaller opening in the diaphragm. One can tell which field is
excessively illuminated, for it is the one in which objects are most
distinctly seen. If it is the microscopic, then the image of the
microscopic object is very distinct and the pencil is invisible or very
indistinct. If the drawing surface is too brilliantly lighted, the
pencil can be seen clearly, but the microscopic image is obscure.

When opaque objects, that is, objects which must be lighted with
reflected light (figs. 19, 43), like dark colored insects, etc., are to
be drawn, the light must usually be concentrated upon the object in
some way. The microscope may be placed in a very strong light
and the drawing board shaded, or the light may be concentrated
upon the object by means of a concave mirror, or a bull's-eye con-
denser or the small arc lamp may be used.

If the drawing surface is too brilliantly illuminated, it may be
shaded by placing a book or a ground-glass screen between it and
the window, also by putting one or more smoked glasses in the path
of the rays from the mirror (fig. 169). If the light in the microscope
is too intense, it may be lessened by using white paper over the
mirror, or by a ground-glass screen between the microscope mirror
and the source of light (Piersol, American Monthly Microscopical
Journal, 1888, p. 103). It is also an excellent plan to blacken the
end of the drawing pencil with carbon ink. Sometimes it is easier to
draw on a black surface, using a white pencil or style. The carbon
paper used in manifolding letters, etc., may be used, or ordinary
black paper may be lightly rubbed on one side with a moderately
soft lead pencil. Place the black paper over white paper and trace

326 DRAWINGS AND DEMONSTRATIONS [CH. IX

the outlines with a pointed style of ivory or bone. A corresponding
dark line will appear on the white paper beneath (Jour. Roy. Micr.
Soc., 1883, p. 423).

(i) It is desirable to have the drawing paper fastened with thumb
tacks, or in some other way. (2) The lines made while using the
camera lucida should be very light, as they are likely to be irregular.
(3) Only outlines are drawn and parts located with a camera lucida.
Details are put in free-hand. (4) It is sometimes desirable to draw
the outline of an object with a moderate power and add the details
with a higher power-. If this is done, it should always be clearly
stated. It is advisable to do this only with objects in which the
same structure is many times duplicated, as in a nerve or a muscle.
In such an object all the different structures can be shown, and by
omitting some of the fibers the others may be made plainer with-
out undesirable enlargement of the entire figure. (5) If a drawing
of a given size is desired and it cannot be obtained by any com-
bination of oculars, objectives, and lengths of the tube of the mi-
croscope, the distance between the camera lucida and the table
may be increased or diminished until the image is of the desired
size. This distance is easily changed by the use of a book or a block,
but more conveniently if one has a drawing board with adjustable
drawing surface like that shown in fig. 171. (6) It is of advantage
to have the camera lucida hinged so that the prism may be turned
off the ocular for a moment's glance at the preparation, and then
returned without the necessity of loosening screws and readjusting
the camera. This form is now made by several opticians, and many
of them add graduations so that the angle of the mirror is readily seen.

§ 409. Scale of drawings. — The scale should be given for every
drawing (fig. 172). Sometimes the drawing is larger than the object,
as with microscopic specimens, and sometimes it is of the same size
or much smaller, as in drawing large objects.

In getting the scale at which an object is drawn with the micro-
scope or projection microscope, the object is removed and a microm-
eter in half millimeters for low powers and one in tenths and
hundredths of a millimeter (fig. 148) for high powers is put in
place of the specimen. The image of the micrometer lines and

CH. IX] DRAWINGS AND DEMONSTRATIONS 327

spaces will be of the same enlargement as the drawing, provided
nothing has been changed except the micrometer for the object. If
now a few of the lines of the micrometer image (figs. 148, 172) are
traced at one corner of the drawing paper and their actual value
given, the enlargement can be determined accurately as follows:
Suppose the micrometer spaces are tenth millimeters, and the image
of the spaces measures 2 millimeters. The enlargement must be the
size of the image divided by the size of the object or 2 ~ o.i = 20;
that is, the image is 20 times the size of the object.

In using the photographic camera for negatives or for tracing,
if the metric scale (fig. 173) is put with the object, its image will
appear with the image in the negative or in the tracing, and the en-
largement or reduction can be found as above. Suppose the image
of .the 10 cm. scale on the negative or in the tracing is 2 cm. long.
Obviously the picture must be 2 cm. •*• 10 = i\ or |, that is, the
picture is only one-fifth the size of the object.

For any form of projection apparatus (figs. 178-183), the magic
lantern or projection microscope, after the image is traced, the ob-
ject is removed and a micrometer in half millimeters for the magic
lantern and low powers of the microscope is put in place of the ob-
ject and the image of the scale projected
upon the drawing paper. Suppose the image
of one of the micrometer half millimeter
spaces measures 15 millimeters, then the

scale of the drawing must be 30 (i.e., 15 -r \ _/IG- J72- MAGNIFIED

6 ° ^ ' ° * MICROMETER SPACES TO

= 30) . SHOW THE METHOD c F

If one is drawing from the projected INDICATING THE SCALE

° . , . . AT WHICH DRAWING

image of a negative or lantern slide it is neces- WAS MADE.

sary to know the scale at which the negative

or slide was made as well as the scale at which the drawing from
the projected negative or slide is being made. For example, if the
scale of the negative is 50 times the size of the object, and the draw-
ing is 10 times the size of the negative, the final drawing must be
10 x 50 = 500 times the size of the original object.

If, on the other hand, the negative is -rV the size of the original
object and the drawing is 5 times the size of the negative, the final

328

DRAWINGS AND DEMONSTRATIONS

[CH.

drawing will be the size of the negative (TV the original) multiplied
by the magnification (in this case 5) which is i\ x 5 =* A or %.
That is, the drawing is one-half the size of the original object.

For the projection microscope with objectives of 40 to 16 mm.
(4x to IQX) a micrometer in | mm. is good. For objectives above
16 mm. (IQX) it is better to use a micrometer in o.i mm. and o.oi
mm. (fig. 148).

After the drawing has been made, remove the specimen and put
the micrometer under the microscope and draw a few spaces of the
micrometer image (fig. 172) giving the actual value of the spaces;
then one can compute the enlargement of the drawing by measuring
the image spaces and dividing by the actual value. For example,
suppose the image of one of the o.i mm. spaces measures on the
drawing 4 cm. or 40 mm. The scale of the drawing or its magnifica-
tion is 40 + o.i = 400.

§ 409a. For diagrams and other large objects a very serviceable micrometer
can be made by using the 10 cm. metric rule (fig. 173) as object and making a
negative of it on a lantern slide exactly natural size or half natural size.

TO CENTIMETER RULE

The upper edge is in millimeters, the lower in centimeters
The metric system

The most commonly used divisions and multiples.
Centimeter (cm.) O.OE Meter; Millimeter (mm.), o.ooi
Meter; Micron (ju), o.ooi Millimeter; the Micron is the
unit in Micrometry.
Kilometer, 1000 Meters; used in measuring roads and other

• long distances.
Milligram (mg.), o.ooi Gram.

Kilogram, 1000 Grams, used for ordinary masses, like groceries,
etc.

Cubic Centimeter, (cc.), O.OOT Liter. This is more common

than the correct form, Milliliter.

Units are indicated by the Latin prefixes; deci. o.i; centi,
o.oi; ntittij o.ooi; micro, one millionth (O.OOOOOT) of any unit.
^ Multiples are designated by the Greek prefixes; deka, 10 times; hecto, 100
times; kilo, 1000 times; myria, 10,000 times; mega, one million (1,000,000)
times any unit.

UNITS.

THE METER FOR
LENGTH

THE GRAM FOR
WEIGHT

THE LITER FOR
CAPACITY,

Divisions of the'

FIG. 173. METRIC SCALE AND SUMMARY OF THE METRIC SYSTEM.

CH. IX] DRAWINGS AND DEMONSTRATIONS 329

DRAWINGS BY THE AlD OF THE PHOTOGRAPHIC CAMERA AND
THE MAGIC LANTERN

§ 410. Drawings by the aid of a photographic camera. — The

photographic camera (camera obscura) gives help for getting pictures
of objects in three ways:

(1) By producing real images which can be traced (§ 411).

(2) By producing negatives which can be projected upon the
drawing paper and traced, or the drawing can be done directly on
the print, and all but the drawing removed from the print; or the
drawing can be made on the back of the print (§§ 413-414).

(3) By producing large prints for retouching (§ 416).

411. Real images by the camera. — For drawing with a photo-
graphic camera it is a great help to have a frame with a piece of
clear glass to use instead of the ordinary ground-glass focusing
screen. The tracing paper is stretched over the glass. The object
is arranged as desired and placed in a strong light. The camera is
then arranged to give the desired view, and the bellows pulled out,
and the whole camera moved toward or away from the object until
the desired size is obtained. This tracing is transferred to the draw-
ing paper in the usual manner and inked in. A camera like that
shown in fig. 174 answers well; also a copying camera.

While inking in, and indeed whenever free-hand and optical
methods of getting drawings are combined, the object should be
available for constant observation so that accuracy may be obtained.

§ 412. Negatives by the camera. — The object is arranged as
desired and placed in a good light. A photographic camera is then
used and a negative on glass made in the usual manner. If the nega-
tive is to be used for prints on which to trace and draw with ink or
pencil, the negative is made the size of the desired finished picture.
On the other hand if the negative is to be used for projection, it
should be of about the size of a lantern slide (§ 416).

§ 413. Drawings upon blue prints. — This is especially available
for objects with definite outlines and clear details like the wing veins
of insects (Comstock) or apparatus, furniture, etc.

A negative of the object is made of the desired size and a blue

330

DRAWINGS AND DEMONSTRATIONS

[CH. IX

print made. Then with waterproof India ink all the lines are gone
over, and all the points indicated which are to be shown in the
finished cut.
Bleach out the blue by soaking the print in a solution of 10%

FIG. 174. VERTICAL PHOTOGRAPHIC CAMERA ON A Low TABLE.

T Table about =50 cm. high and 50 cm. by 70 cm. on the top.

d d Drawer with combination lock.

Base The heavy base of the vertical camera support.

p Pillar in which the graduated rod (vgr) rotates.

ss Set screw to fix the graduated rod in any position.

c s, c s Set screws to enable the operator to set the camera bellows at any
desired extension.

wr Magnification rod with its set screw rs. When any desired magnification
is arranged, the rod set screw is tightened; then by loosening the camera set
screws (cs) the bellows can be moved up and down on the graduated rod to get
the focus.

Fs Focusing stand; this is a microscope stand with coarse and fine adjust-
ment (cf) and two stages (st st) for supporting the object or the dish containing
it (sp c).

Ob Photographic objective in the lower end of the camera.

VC Vertical camera bellows.
fg Focusing glass.

CH. IX] DRAWINGS AND DEMONSTRATIONS 331

neutral oxalate of potash. Wash in water and dry on gauze. Only
the ink lines will show in the finished print. This line drawing can
then be lettered in any desired way, and the engraver can make a
line cut for the printing press.

Ordinarily it is best to make the picture two or three times the
size of the final engraving. Defects are minimized in the reduction.
Always have the object in view in finishing the drawing.

§ 414. Drawings on the back of photographic prints. — The
easiest way to obtain line cuts of many objects is to make a photo-
graph of them and then draw the outlines on the back of the photo-
graph. This is an application of the old method of tracing the veins
of leaves and the details of other objects by holding against a well
lighted window, and making the tracing on a sheet of paper over the
object.

A negative should be made of the size desired or a small negative
is made and a large print obtained by projection (§ 484). Prints of
both sides of the negative should be made. A print from the
front or film side will give an erect image like the object. One from
the glass side or back of the negative will give a reversed image.
The tracing on the back of the reversed or inverted image will give
an erect image like the object.

If one prints by projection (§ 484), the erect image is made by
making the negative, film side, face the printing paper; the inverted
image is obtained by having the glass side of the negative face the
paper.

If the original negative is of the desired size, one print is made by
putting the sensitive paper in contact with the front or film side of
the negative. For the inverted or reversed print the glass side is
placed up in the printing frame, and the sensitive paper put on the
glass. This will make the print slightly out of focus, but by print-
ing this image with the plate holder a meter or more distant, and
directly under the printing light, a moderately sharp print can be
made except for very thick glass negatives. There will be no
trouble with films.

§ 415. Making the tracings. — When the enlarged prints are
ready, proceed as follows: Work in a dark or dimly lighted place.

332

DRAWINGS AND DEMONSTRATIONS

CH. IX] DRAWINGS AND DEMONSTRATIONS 333

Use a drawing shelf containing a glass window (fig. 180), or use a
table with a heavy glass set in a window in the top. Have a 100-
watt lamp in a reflector underneath to illuminate the print.

Place the inverted print, face down, on the glass. The light
shining through the print will make it appear almost as if the face
were up.

Trace all the outlines with a lead pencil, using a triangle or T-
square for the straight lines. In doing the tracing it is advantageous
to have the erect print to look at, and occasionally one should hold
the tracing in a good light to see that all the lines are present.

After getting the outlines with a pencil, the lines are inked in.
For this one should work in a well-lighted place, and have the
actual object in view and the erect print to serve as guides. Some
additions may be put in free-hand. One may also wish to add
accessory apparatus, or enlargements of some of the details. This
was done with figures 80 and 180.

The paper used for photographic prints is excellent both for the
draughtsman and for the photo-engraver. There is some advantage
in using double-thick paper for the tracings, as the prints are flatter.
The single thickness of paper shows the details of the print some-
what more clearly.

Of course, one could make tracings on the back of blue prints,
and then no bleaching would be necessary, but the details are not
so sharp and definite in blue prints as in silver prints. One can
draw on the face of silver prints and remove the silver print with
chemicals, but that is not so satisfactory as drawing on the back of a
reversed print as described above.

Many of the line drawings in this book were made by tracing
them on the back of inverted photographic prints. Much has also
been made of the method for all sorts of objects during the last ten
years, and its usefulness is increasingly appreciated. The amateur
artist has the advantage of correct proportions and perspective
without the trouble of many measurements; he is also perfectly
free to add artistic touches, and to combine free-hand sketches.
The line cuts have the great advantage of definiteness, and can be
printed on any good book paper. For lettering the drawings, see

334 DRAWINGS AND DEMONSTRATIONS [Cn. IX

§ 427. Do not make the lettering so prominent that the drawing
itself is submerged.

§ 416. Retouching photographs for halftone reproduction. — For
pictures of animals, organs, and dissections to be reproduced by the
halftone process, very successful drawings can be made as follows:
Arrange the object as it is to appear in the finished drawing; light it
to bring out clearly the features desired; then use a long focus pho-
tographic objective and get a small, sharp picture. The negative
should be about the size of a lantern slide. Make a large print on
thick developing paper exactly as described in section 484. This
print should not be dark, but two or three shades lighter than the
usual print to give opportunity for the added shading. The picture
should be erect.

When the print is dry, put it on a drawing board and with a car-
bon drawing crayon, pen, India ink, and an air brush, if it is avail-
able, the picture can be made almost perfect with a minimum of labor.

In case the negative shows parts not needed or if the background
is not as desired, the superfluous parts can be eliminated and the
background made perfectly white by painting on the glass surface
of the negative Gihon's or other opaquing medium. In the print
there will be pure white where the opaque is painted on the glass.
Use a fine brush and put on a layer which does not allow any light
to pass. The opaque is put on the glass surface so that it can be
removed easily if desired. In case some parts are not light enough
or white points are to be added, use some of the white recommended
by the photo-engravers (Blanc d Argent, etc.).

As in all drawing, the actual object should be before the artist
when retouching the photograph, so that accuracy may be secured.

§ 417. Tracing pictures natural size on drawing paper. — It
frequently happens in preparing the drawings for a book or for a
scientific paper that figures from another book or scientific
paper are needed. If there is to be no modification in the figure, the
simplest method is to borrow an electrotype. If this cannot be
done and the picture is not available to put in the hands of the pho-
to-engraver for a new cut, or if one wants to make minor changes, it
is very easy to get a tracing on any good drawing paper as follows;

CH. IX]

DRAWINGS AND DEMONSTRATIONS

335

Put the picture on the glass of the drawing shelf (fig. 180) and place
over it some good drawing paper like Whatman's hot-pressed
drawing paper or Reynolds' bristol board. Turn on the light, and
even through the thick drawing paper the outlines of the picture are
so clear that the tracing can be made with ease. After the outlines
have been traced, the finishing can be done on a drawing board with
the original picture for reference.

§ 418. Diagrams by projection. — For light use an arc lamp or a
stereopticon mazda lamp ; use a negative which is not too dense or a
lantern slide. It is placed in the lantern-slide holder and by means
of an ordinary projection objective, or better by a photographic

FIG. 178. MAGTC LANTERN WITH PROJECTED IMAGE.
(From Optic Projection).

A small arc lamp connected with the house lighting system is used for light in
this case.

W, So, S — p Electric wires, lamp socket with key switch (s) and a separable
attachment plug.

R Rheostat.

Condenser, W A three lens condenser with a water cell to absorb radiant heat.

LS Lantern slide.

Axis, Objective The principal optic axis of the condenser and of the objective
in one line. The cone of light crosses within the objective at (c).

Screen Image The real image projected upon the screen.

objective, the image is projected upon the drawing paper (fig. 178).
For the proper size either the projection apparatus or the drawing
surface must be movable.

When the size is correct, and the image sharply focused, one can
trace directly on the drawing paper with a pencil all the lines and
details which it is desired to represent. Then the drawing can be
inked in at leisure, remembering always to have the object for con-
stant reference and thus insure accuracy.

336 DRAWINGS AND DEMONSTRATIONS [Cn. IX

In projection it is very easy to make the picture as large as desired
provided the projection apparatus or the drawing surface is movable.
The projection method has the advantage of being applicable to all
forms of objects, gross and microscopic. The only precaution is to
make the negative rather thin, not dense; then the details come out
clearly in the projected image.

PROJECTION MICROSCOPE FOR DRAWING

§ 419. This is the most satisfactory method of drawing small
objects. With it one can draw large diagrams or small figures
directly from the objects; and if the apparatus is properly con-
structed one may make diagrams from objects 60 to 70 mm. in di-
ameter down to those of half a millimeter or less. This method was
much in vogue and was highly commended by the older microscopists
who used the solar microscope (Baker, Adams, and Goring). Since
the general introduction of electric lighting, drawing with the pro-
jection microscope has become once more common and is the most
satisfactory method known, especially for the numerous drawings
necessary for the preparation of models in wax or blotting
paper.

§ 420. Drawings with low powers. — For objectives of 30 to
100 mm. focus, the best method is to use a projection outfit with
a three lens condenser as shown in fig. 179.

For a radiant, a large or a small arc lamp is best (figs. 179, 181),
but a 250- or 400-watt concentrated filament, stereopticon mazda
lamp filled with nitrogen also works fairly well. The mazda lamp
has the advantage that it can be attached to any lighting circuit,
and when once centered and properly arranged, requires no attention
except to turn the switch on and off. A dark room is desirable, but
one can draw in any room at night.

Arrange the object, the lamp, and the condenser so that the object
is fully lighted; then focus the objective and place the drawing sur-
face and objective at a distance apart to give the desired size of
drawing. Focus sharply and trace with a pencil the outlines and
details which it is desired to show. Finally, with the object where it

CH. IX]

DRAWINGS AND DEMONSTRATIONS

337

can be examined at any time, ink in the lines and details. (For
erect images see § 435).

FIG 179. PROJECTION MICROSCOPE.
(From Optic Projection).

+w The positive wire going to the upper carbon (He), and -w, wire to the
lower or vertical carbon (Vc) of the large arc lamp with direct current.

Axis, Axis, Axis The principal optic axis from the source of light (L) through
the condenser, the microscope and to the screen.

W Water cell to absorb radiant heat.

Stage The separate stage of the microscope with its water ceil for cooling
the specimen by conduction.

Microscope In this case the microscope has an objective ( nly; compare fig.
1 80, where an ocular is present also.

Each element, lamp, condenser, stage, and microscope is on a separate movable
block (block i, 2, 3, 4) which slides independently along the optic bench or base
board.

§ 421. Use of a 45° mirror or a prism. — While one can draw on
a vertical surface, it is far easier to draw on a horizontal surface.
This is available for all powers by using a plane mirror at 45° or a
drawing prism. The mirror may be at a distance from the objec-
tive, when it must be large (fig. 181); or it may be close to the ob-
jective, when it may be small (figs. 180, 183). The drawing surface
must be movable to vary the size of the drawing and the magnifica-
tion. Figures 179-181 show the two principal methods of varying
the distance between the objective and the drawing surface, and
consequently the scale of the drawing. (For erect images see §§ 430-

437-)

§ 422. Drawing with objectives of 25 (5x) to 8mm. (20x) focus. —
For this the best way is to use a three lens condenser, as shown in
figs. 179, 1 80, and for a microscope use either the special one for
projection or the ordinary microscope with large tube. For radiant

338

DRAWINGS AND DEMONSTRATIONS

[CH. IX

use a small or a large arc lamp. Remove the substage condenser
or turn it aside and arrange on the optical bench so that the image

Microscope

FIG. 180. PROJECTION MICROSCOPE, TYBLE, AND ADJUSTABLE DRAWING SHELF.
(Modified from Optic Projection).

DB Drawing board with a 25 x 30 cm. glass plate in the middle for tracing
on the back of photographs. It is placed on the brackets to form the adjustable
shelf (ADS).

Is Leveling screws in the bottom of the table legs.

Rheostat The balance for regulating the electric current of the arc lamp.

c c, ks Electric cable and knife switch.

Table The projection table with drawer (d). This table is 100 cm. high, and
the top 125 cm. long and 50 cm. wide. It is stained by aniline black.

ADS Adjustable shelf with a drawing board having a glass center 25 X 30 cm.

bt Bolts with thumb nuts holding the shelf at any desired height on the legs.

N R Mazda lamp and reflector to throw the light up through the picture which
is being traced.

c Cable with separable cap to attach to the lighting system.

Arc Lamp The right-angled carbon arc lamp for supplying light to the pro-
jection microscope.

Condenser The three lens condenser and water bath (fig. 178).

Microscope The compound microscope with substage condenser and ocular.

m 45° mirror or prism for reflecting the light directly downward upon the
drawing shelf.

Axis, Axis The principal optic axis.

of the light source from the large condenser falls directly on the
specimen. Focus and arrange the drawing surface to give the right

CH. IX]

DRAWINGS AND DEMONSTRATIONS

339

size and magnification, then trace the outlines and the details.
Later, ink in, using the specimen to check up with. (For erect images
see §§ 430-437.)

After one has had
sufficient practice, the
drawing can be partly
or wholly completed
under the projection ap-
paratus. For this, one
must light tHe drawing
surface enough either
by means of a portable
lamp or by some means
of letting in daylight.
At the same time there
must be a screen to cut
off the image where one
is doing the finishing.
By removing the screen
the image appears at

FIG. 181. PROJECTION MICROSCOPE WITH MOV-
ABLE DRAWING TABLE AND 45° MIRROR.

(From Optic Projection).

The projection table has the dimensions given
in fig. 1 80.

The arc lamp is automatic and the rheostat
for current may be adjusted to give from 10 to
20 amperes.

The condenser is of the three lens water cell
type, and the microscope with separate stage;
the microscope has an amplifier in place.

The drawing table (Dr. Table) is of a conven-
ient height for sitting beside. It is 76 cm. high
and the top 100 cm. lon^ and 75 cm. wide.

The 45° plate glass mirror is large (75 cm. long
and 60 cm. wide).

any time and serves to
check the work.

§ 423. Drawing with
high powers, 8 (20x) to
2 mm. (90x) focus. —
For this high power

drawing one should use an ocular as well as an objective, and a
substage condenser in addition to the condenser of the lantern or
small lamp (fig. 182), or light of sufficient aperture will not be
supplied to the microscope. In using the highest powers it is
also well to connect the substage condenser to the slide by
homogeneous liquid, as described in § 124. The large or small arc
light or the concentrated or ribbon filament, io8-watt, 6-volt head-
light lamp is needed for good results.

If one has a drawing room, a large or small arc lamp, and direct
current, the arrangements shown in fig. 180 are best, but if direct

340

DRAWINGS AND DEMONSTRATIONS

[CH. IX

current is not available, excellent results can be obtained by using
the small arc lamp or the io8-watt lamp on the alternating current,

house electric lighting
system, and the micro-
scope, shown in figs.
182-183.

The light supplied
to the substage con-
denser should be ap-
proximately parallel.
This is attained with
the small lamp by put-
ting the arc at the
focus of the condenser.
With the large lamp
one should use a long
focus lens for the con-
denser, as shown in

fig. 184.

In all cases the sub-
stage condenser should
be shifted up and down
slightly until the best
effect is produced.
The substage condenser
should, of course, be
centered carefully before commencing to draw (§ 118).

§ 424. Drawings for publication. — The inexpensive photographic
processes of making cuts for the printing press bring within the
reach of every writer the possibility of appealing to the eye by
means of pictures and diagrams illustrating the facts which are
presented in the text. Artistic ability is, of course, indispensable
for a perfect representation, but any one willing to give the time
and the pains can make simple drawings, especially if one or more
of the helps above described are available.
The various helps for making drawings described in this chapter

FIG. 182. DRAWING MICROSCOPE WITH SMALL
ARC LAMP ON THE HOUSE LIGHTING SYSTEM.

(From Optic Projection).

S, Sp The lamp socket and separable attach-
ment plug.

Rh The rheostat not allowing over 5 amperes
of current to flow.

Lamp The small arc lamp at right angles to
the microscope.

Microscope The microscope on a block (B).

mr, mr The mirror of the microscope, and the
mirror over the ocular to reflect the light directly
downward.

Image The picture of the microscopic object
reflected down upon the drawing paper.

Sh Opaque shield to screen the light from the
drawing surface.

CH. IX]

DRAWINGS AND DEMONSTRATIONS

341

will be found useful to the born artist as well as to the person who
has not great artistic ability, for by means of the optical and me-

Substage
Condenser

FIG. 183. THE MICROSCOPE ARRANGED FOR DRAWING ON A HORIZONTAL

SURFACE.
(From Optic Projection).

The microscope is of the handle type (H) with the fine adjustment (fa) on
the side below the coarse adjustment (c a).

The ocular is of the Huygenian form with the real image at (r i).

Prism, the right-angled prism beyond the ocular to reflect the light directly
downward.

chanical helps the outlines and proportions can be secured with
fidelity by any one. Then the born artist can use the time saved
for making the pictures more artistic, and the plodder can feel con-
fident that his efforts are correct.

Young authors are urged to get the Style Brief furnished by the
Wistar Institute of Philadephia. This is a guide for the preparation
of manuscript and drawings for publication in the scientific journals
published by the Institute. The hints to contributors given on
the second page of the cover in all the journals give in a nutshell
the main points. These journals are: The American Journal of
Anatomy; The Anatomical Record; The Journal of Morphology;
The Journal of Comparative Neurology, and The Journal of Experi-
mental Zoology. The little book: Preparation of Scientific and

342

DRAWINGS AND DEMONSTRATIONS

[CH. IX

Technical Papers by Trelease and Yule, 2d. ed. 1927, gives excellent
advice which is illustrated by abundant examples.

FIG. 184. DIAGRAMS TO SHOW THE POSITION OF THE SUBSTAGE CONDENSER

WHEN NO PARALLELIZING LENS is USED.

(From Optic Projection).

A The substage condenser is within the focus (/) at a point where the long
light cone is of about the same diameter as the substage condenser.

B The substage condenser is beyond the focus (/) of the long focus main con-
denser at a point where the diverging cone is of about the same diameter as the
substage condenser. This is the better position for the substage condenser of the
ordinary microscope.

Arc Supply The right-angled carbons of the arc lamp.

L\ LI The first and the second elements of the main condenser.

Wafer CelL This is to remove the radiant heat.

Axis The principal axis on which all the parts are centered.

/ The principal focus of the second element of the main condenser. In both
cases the focus is long.

Substage Condenser This is the first or lowest element of the substage con-
denser. It is of the achromatic type.

A great many good hints can be found by studying the illustra-
tions in well-printed books and in scientific journals, especially
those dealing with the subject in which one is interested.

§ 425. Size of drawings. — For most draughtsmen it is wise to
make the drawings two or three times the size of the final cut for
publication. It is easier to make the details clear, and then little

CH. IX] DRAWINGS AND DEMONSTRATIONS 343

defects are minimized by the reduction, The photo-engraver can
make the cut any desired reduction, but one should remember that
the lines should be heavy enough for the reduction desired, otherwise
the finest details are likely to be lost.

§ 426. Reduction. — - There is some confusion as to the meaning
of reduction in the minds of authors. For the engraver this term
has a perfectly definite significance. It is linear measure, and never
area or solid measure, that he considers. For example, if the en-
graver is directed to make the cut half the size of the drawing, he
will make every line half the length of the corresponding line in the
drawing. The area will then be one-fourth that of the drawing.
If the cut is to be reduced to one-fourth the drawing, each line will
be only one-fourth the length of the original, and the area will be
one-sixteenth that of the drawing (figs. 185-186).

§ 427. Lettering drawings. — After the drawings are finished the
details must be indicated in some way. This may be by having the
full name of the part, an easily intelligible abbreviation, or a letter
or a numeral upon or near it (fig. 188).

The lettering should be done with discrimination in two ways:

(1) The letters, words, etc., should be artistically arranged and
then put on straight. For this one may need to use a T-square and
straight edge. Most persons cannot letter neatly enough to letter
with a pen. Printed words and letters can be pasted upon the draw-
ing. In the final cut the appearance is as if words, letters, or nu-
merals were printed on the picture (fig. 26.)

If the letters, abbreviations, etc., are not upon the parts they are
meant to indicate, then " leaders," that is, full or broken lines
should be drawn from the part to its designating letter, numeral,
abbreviation, or word (figs. 18, 26).

(2) The size of type to be used should correspond to the size of
the picture and the amount of reduction. The letters should not be
the most prominent thing about a picture, neither should they be so
small that one needs a microscope to read them. By consulting
figs. 185-186 one can get a clear notion of the appearance of various
sizes of letters when reduced. If one has a camera (fig. 174), it is a
good plan to put letters of different sizes upon the drawing and

344 DRAWINGS AND DEMONSTRATIONS [Cn. IX

then, having the bellows set to give the reduction desired, look at
the image of the drawing and lettering and see how they wit
look in the final picture.
For photo-engraving, Gothic type gives the best results (fig. 185).

24 Point Type A a
123456789 10

18 Point Type A R S 2 3 4

12 Point Type ABCabc 1234
10 Point Type ABC abc 12345

8 Point Type ABCD abed 12345
6 Point Type ABCDabed12345 I II III IV

ABCD a b o d 123456789 10 I II III IV V VI

FIG. 185. GOTHIC TYPE FOR LETTERING DRAWINGS.
(From Optic Projection).

§428. Fastening the letters to the drawing. — The letters, etc.,
should be printed on thin, smooth, very white paper, and they
should be black, not gray. Tissue paper is often used, but that is
not so easy to handle as a paper about like the so-called " Bible
paper."

The words, letters, and numerals for a drawing are cut out and
arranged on the drawing to get the best effect. Then using a T--
square and straight edge, each letter or word is stuck to the drawing

CH. IX]

DRAWINGS AND DEMONSTRATIONS

34$

in the proper position as follows: Some fresh starch paste is made
by placing in a small tin or aluminum dish 5 grams of laundry
starch and adding 50 cc. of cold water. Stir with a spoon and then
heat gradually with constant stirring on a stove or over a gas flame

24 Point Type A a

123456789 10

18 Point Type A R S 2 3 4

12 Point Type ABCabc 1234

10 Point Type ABC a b c 12345

SPolnlTyp. ABCO • 0 0 d 12345

24 Point Type A a

12345678910

18 Point Type A R S 2 3 4

12 Point TyM A • C • k c 1 1 1 4

JFio. 186. THE GOTHIC TYPE IN FIG. 185 REDUCED TO ONE-HALF AND TO
ONE-FOURTH NATURAL SIZE.

(From Optic Projection).

until the paste is formed. Mucilage and paste which has been made
for some time are not good for pasting the letters. Mucilage turns
the paper yellow and the old paste is lumpy. Any good library paste
will answer, also stainless rubber cement.

Use a fine brush to put the paste on the letters, and then use fine
forceps (fig. 138) to pick up the letters and transfer them to the

346 DRAWINGS AND DEMONSTRATIONS [Cn. IX

proper position. Press down with the finger covered with tissue
paper or very fine cloth or with fine blotting paper. Press directly
downward or the letter is likely to be displaced or distorted by a
lateral thrust.

§ 429. White letters for black background. — The white letters,
words, or numerals are most easily procured by photography. The
letters, words, etc., are printed on tissue paper. This is used as a
negative by placing it face down on a glass plate and in a printing
frame. Use some developing paper, of the contrast variety. Print
as for any negative and develop with a contrast developer so that
the whites and blacks will be perfect. The white letters, etc., are
then cut out and pasted on the drawing as described above. This
photographic paper is rather thick and will show a white edge
where it is cut. Blacken the white edges of the letters or words
with carbon ink after the letters are stuck in place (fig. 188).

AVOIDANCE or INVERSION

§ 430. It is desirable to make drawings like the object without
any inversion whatsoever, provided the object has rights and lefts,
etc. For structural detail like cells, etc., it makes no difference
whether the image is erect or not, but with symmetrical organs and
animals it is very confusing to have the parts inverted in the draw-
ing. For example, it is unsatisfactory to have the liver shown as if
on the left side and the heart on the right side.

In order to avoid inversions, it is necessary to know what inver-
sions are produced by the different optical appliances used to assist
in drawing. Then one can so arrange the object that the image
will be exactly like the object. It is believed that the following
directions will enable the worker to arrange his specimen and the
apparatus so that erect images may be produced without undue
effort.

The simplest of all ways to get the image without inversion is to
arrange the slide on a piece of white paper so that the object is erect
and then to write with a very fine pen the letters a, k, on the cover-
glass of the specimen to be drawn (fig. 187). Now with the low

CH. IX]

DRAWINGS AND DEMONSTRATIONS

347

power (16 to 60 mm. [lox to 2.66x]) objective, project the image
of the specimen and letters upon the drawing paper. One can then
continue to rearrange the slide until the letters are erect; the speci-
men will then also be erect.

§ 431. Images to be traced in the photographic camera. — These
images are wrong side up and the rights and lefts are reversed. This
can be corrected by drawing the picture on the tracing paper in th6
inverted position and then inverting the tracing after it is finished;
or the specimen can be put in the inverted position, then the image
will be erect.

FIG. 187. SLIDE OF SERIAL SECTIONS, SHOWING THE DEVELOPMENT OF THE
EYE WITH THE LETTERS a k, TO AID IN GETTING ERECT IMAGES IN DRAWING
WITH PROJECTION APPARATUS.

(From Optic Projection).

This slide is also to show how to mask preparations which are to be used in class

demonstration.

Demonstrate this by putting the metric card in position and
tracing some of the larger letters or figures on the tracing paper.
Then turn the drawing paper around 180° and the letters or figures
will appear erect.

Put the metric card wrong edge up to start with; then the letters
or figures will appear right side up on the tracing paper.

§ 432. The use of a negative for projection and tracing. — Put
the face of the negative that reads correctly next the source of light
and wrong edge up; then it will appear erect in every way on the
drawing paper. This is the way lantern slides are put in the holder.

348 DRAWINGS AND DEMONSTRATIONS [Cn. IX

§ 433. The Wollaston or Abbe camera lucida. — With these
camera lucidas there are two reflections of the rays (figs. 168-169),
consequently there is no inversion produced by the camera, but the
microscope inverts the image the same as the photographic objec-
tive, and erect images are obtained either by inverting the drawing
after it is made or by putting the object in an inverted posi-
tion under the microscope, just as with the photographic
camera.

Demonstrate that this will. produce erect drawings by using the
letters (fig. 187) and making sketches of their images by the camera
lucida, having the letters right edge up on the stage in one case and
wrong edge up in one.

ERECT IMAGES WITH THE PROJECTION MICROSCOPE

§ 434. Erect images with an objective only or with an objective
and amplifier. — There are two cases: (i) When opaque drawing
paper is used. In this case the object must be put on the stage
with the cover-glass toward the light and the slide toward the ob-
jective, and it must be lower edge up. Only low powers (16 mm.
(IQX) and lower objectives) should be used, for the thick slide intro-
duces aberrations (fig. 64) and is likely to be too thick for the free
working distance (fig. 52 B).

(2) When a translucent drawing paper is used and the drawing
is made on the back. In this case the specimen is put on the stage
lower edge up, but with the cover-glass facing the objective. All
powers can be used. This is similar to the conditions described for
the photographic camera where the tracing paper is used on the
clear glass (§ 431).

Test the correctness of the directions by using a preparation with
the letters a, k, on the cover-glass (§ 430, fig. 187).

§ 435. Erect images with an objective or an objective and an
amplifier and a prism or 45° mirror. — Place the specimen on the
stage lower edge up and with the cover-glass toward the objective.
The image will be erect on the opaque drawing paper. Test with
the lettered specimen (fig. 187).

CH.IX] DRAWINGS AND DEMONSTRATIONS

1 10 CENTIMETER RULE

349

H3i3iMixN3O ot

3JUH H3T3MITVT3O 01

10 GEMJLIiMEJLEB BIIPE

FIG. 1 88. i, 2, 3, 4, ERECT AND INVERTED IMAGES OF THE METRIC SCALE.
(From Optic Projection).

i. Erect image. 2. Inverted image. 3. Mirror image. 4. Inverted mirror image.

§ 436. Erect images with an objective and an ocular. —

(1) Opaque drawing paper. Place the specimen on the stage
right edge up, but with the cover-glass facing the light, the slide
toward the objective.

(2) Translucent drawing paper. If the drawing can be made
on the back of translucent paper the specimen is placed on the stage
right edge up and with the cover-glass facing the objective. Test
with the lettered specimen (fig. 187).

§ 437. Erect images with an objective and ocular and a 46°

350 DRAWINGS AND DEMONSTRATIONS [Cn. IX

mirror or prism. — Place the slide on the stage top edge up, and
with the cover-glass facing the objective. The image will be erect
on an opaque drawing surface. Test with the lettered preparation
(fig. 187).

CLASS DEMONSTRATIONS

§ 438. Demonstration microscopes. — Ever since the microscope
was invented physicians and naturalists have made the greatest use
of it for demonstration purposes. It was a favorite expression of
the older writers that the instrument had created a new world of
the minute. Naturally in the beginning each person used the in-
strument for himself as with the simple microscopes of Roger Bacon.
However, soon after the invention of the compound microscope
Kepler and Scheiner discovered the way to get projection pictures,
and these have been much used for demonstrating to groups of
people the enlarged screen pictures.

Recently the powerful lime and electric lights have made it pos-
sible to carry on these demonstrations to an extent beyond the
hopes of the earlier workers; and have put into the hands of the
teacher facilities which are beyond estimation in value for helping
students. Still for many things and for many persons having charge
of large classes, the individual simple or compound microscope is
still and always will be much used.

DEMONSTRATION MICROSCOPES AND INDICATORS

§ 439. Simple Microscope. — Holding the simple microscope in
one hand and the specimen in the other has always been used for
demonstration, but for class demonstration it is necessary to have
microscope and specimen together or the part to be observed by
the class is frequently missed. Originally blocks of various kinds to
hold both microscope and specimen were devised, but within the
last few years excellent pieces of apparatus have been devised by
several opticians for the purpose.

The tripod magnifier and various pocket magnifiers are excellent

CH. IX]

DRAWINGS AND DEMONSTRATIONS

351

for the purpose (figs. 15-16). Where the microscope and object
should be held in a fixed position, the focusing stand for the simple
microscope is good (fig. 17).

§ 440. Compound demonstration microscope. — This was origi-
nally called a clinical or pocket microscope. It is thus described by
Mayall in his Cantor Lectures on the history of the microscope:
" A small microscope was devised by Tolles for clinical purposes
which seems to me so good in every way that I must ask special
attention for it. The objective is screwed into a sliding tube, and
for roughly focusing the sliding motion suffices; for fine adjustment,
the sheath is made to turn on a fine screw thread on a cylindrical
tube, which serves also as a socket carrier for the stage. The com-

«.

pound microscope is here reduced
to the simplest form I havt met
with to be a really serviceable
instrument for the purpose in
view; and the mechanism is of
thoroughly substantial character.
I commend this model to the
notice of our opticians."

Since its introduction by Tolles
many opticians have produced
excellent demonstration micro-
scopes of this type, but most of
them have not preserved a special
mechanism for fine adjustment.
With it one can demonstrate with
an objective of 6 mm. (3ox) sat-
isfactorily. It has a lock, so that
once the specimen is in the right
position and the instrument
focused it may be passed around

the class. For observation it is necessary for each student only
to point the microscope toward a window or a lamp.

A modification of this clinical microscope was made by Zent-
mayer, in which the microscope was mounted on a board, and a

FIG. 189 A, B. POINTER OCULAR
AND FIELD WITH POINTER.

A. POINTER OR INDICATOR OCULAR
WITH A CAMEL'S HAIR (P) STUCK
TO THE OCULAR DIAPHRAGM AND
EXTENDING OUT INTO THE OPEN

SPACE WHERE THE REAL IMAGE IS

FORMED.

B. THE MICROSCOPIC FIELD OF A
BLOOD PREPARATION WITH THE
POINTER (P) DIRECTED TOWARD A
LEUCOCYTE.

352

DRAWINGS AND DEMONSTRATIONS

[CH. IX

lamp for illuminating the object was placed at the right posi-
tion.

§ 441. Traveling Microscope. — Many years ago the French op-
ticians produced most excellent traveling microscopes. Now the
opticians of America and other countries make serviceable instru-
ments. For the needs of the pathologist and sanitary inspector a
microscope must possess compactness and also the qualities which
render it usable for nearly all the purposes required in a laboratory.
This instrument is a type of much apparatus which has grown up
with the needs of advancing knowledge.

§ 442. Indicator or pointer ocular. — This is an ocular in which
a delicate pointer of some kind is placed at the level where the real
image of the microscope is produced. It is placed at the same level
as the ocular micrometer, and the pointer, like the micrometer, is
magnified with the real image and appears as a part of the projected
image (fig. 189 B). By rotating the ocular or the pointer any part
of the real image may be pointed out as one uses a pointer on a
wall or blackboard diagram. By means of the indicator eyepiece one

999

9 ®

©• 9

999

FIG. 190, RING AROUND ONE OF THE SECTIONS OF A SERIES FOR DEMONSTRAT-
ING SOME ORGAN ESPECIALLY WELL.

FIG. 191. A MICROSCOPIC PREPARATION WITH A RING AROUND A SMALL PART
TO SHOW THE POSITION OF SOME STRUCTURAL FEATURE.

can be certain that the student sees the desired object, and is not
confused by the multitude of other things present in the field. This
device has been invented many times. It illustrates well the adage:
" Necessity is the mother of invention/' for what teacher has not
been in despair many times when trying to make a student see a
definite object and neglect the numerous other objects in the field?
So far as the writer has been able to learn, Quekett was the first to
introduce an indicator ocular with a metal pointer which was ad-

CH. IX] DRAWINGS AND DEMONSTRATIONS 353

justable and could be turned to any part of the field or wholly out
of the field.

It is not known who adopted the simple device of putting a fine
hair on the diaphragm of the ocular, as shown in fig. 189. This may
be done with any ocular, positive or negative. One may use a little
mucilage, Canada balsam, or any other cement to stick the hair on
the upper face of the diaphragm so that it projects about halfway
across the opening. When the eye-lens of the Huygenian ocular is
screwed back in place, the hair should be in focus. If it is not,
screw the eye-lens out a little and look again. If it is not now sharp,
the hair is a little too high and should be depressed a little. If it is
less distinct on screwing out the ocular, it is too low and should be
elevated. One can soon get it in exact focus. Of course it may
be removed at any time. Ordinary hair is too coarse. The tip of
one of the hairs in a camel's hair brush is excellent.

THE PROJECTION MICROSCOPE

§ 443. Projection Microscope. — One of the most useful and
satisfactory means at the disposal of the teacher of microscopic
anatomy and embryology for class demonstrations is the projection
microscope. With it he can show hundreds of students as well as
one, the objects which come within the range of the instrument.

It is far more satisfactory than microscopic demonstrations, for
with the projection microscope the teacher can point out on the
screen the structural features and organs which he wishes to demon-
strate, and he can thus be certain that the students know exactly
what is to be studied. Unless one employs a pointer ocular (fig.
189), there is no certainty that the student selects from the multi-
tude of things in the microscopic field the one which is meant by
the teacher. Like all other means, however, the projection micro-
scope is limited. With it one can show organs both adult and
embryonic, and the general morphology. For the accurate demon-
stration of cells and cell structure the microscope itself must be
used. As a general statement concerning the use of the projection
microscope for demonstration purposes, it may be said that it is

3S4

DRAWINGS AND DEMONSTRATIONS

[CH.IX

entirely satisfactory for objects and details which show under the
microscope with objectives up to 16 mm. (lox) equivalent focus,
For objects and details requiring objectives higher than 16 mm.
(IQX) focus in ordinary microscopic observations, the projection
microscope is unsatisfactory with large classes.

With small classes (10 or 15) where the screen distance can be
reduced to about one meter, demonstrations with oil immersion
objectives are satisfactory. However, when the finest details of

FIG. 192. DIAGRAM OF ADAMS* SOLAR MICROSCOPE. THIS ILLUSTRATES WELL
THE ADVANTAGE OF SOME FORM OF PROJECTION MICROSCOPE FOR DEMONSTRA-
TION PURPOSES.

structure are to be seen most successfully under high powers, each
individual must look into a microscope for himself and attend to all
the finer adjustment and lighting.

§ 444. Euscope for testing laboratories and for demonstration. —
In 1924, Dr. W. G. Exton described in the Jour. Amer. Med.
Assoc., Vol. 82, pp. 1838-1840, a device to enable the observers
in testing laboratories to look at the microscopic image with
both eyes. It is a small self-contained projection microscope.
The microscope is vertical and over it is fitted a pyramidal box with
a screen at the far end to receive and reflect the image. At the

CH. IX] DRAWINGS AND DEMONSTRATIONS 355

top of the tube receiving the tube of the microscope is a totally
reflecting prism which projects the originally vertical beam hori-
zontally to the screen. For demonstrations to a small group the
opaque screen is removed and an extension put in its place. At the
far end of the pyramidal extension is a screen of finely ground
glass. The illumination is by a small arc light or by one of the 108-
watt, 6-volt lamps (figs. 78-80).

For individual use, the observer looks into a kind of hood which
makes it possible to use the instrument in a light room. Although
not an essential part of the apparatus, there is a special magnifying
lens at the eye end of the instrument into which the observer looks.
This instrument in the hands of the author has served an excellent
purpose for demonstration to small groups. It is more satisfactory
with low than with high powers. The Euscope is manufactured
by the Bausch & Lomb Optical Co., and is fully described and
illustrated in their catalogue, pp. 304-305.

CONDUCT OF A DEMONSTRATION WITH THE PROJECTION
MICROSCOPE

§ 445. Preparedness. — From the great difficulty in making
really good projection demonstrations with the microscope the
preparation should be thorough. The following are some of the
most important things to look after:

(1) If any of the objectives used are of the photographic type
and have an iris diaphragm, that should be opened to the fullest
possible extent.

(2) The microscopic slides to be used should be in order so that
they can be grasped easily.

(3) If the slides have many sections upon them, as in a series,
then the slide should be masked by putting some orange paper over
the cover-glass with openings for the sections to be shown; then
these can be found quickly and with certainty (fig. 193).

(4) Indicate in some way which edge of the slide should be
up. This will save time, and add to the respect for the exhibi-
tion.

356 DRAWINGS AND DEMONSTRATIONS [Cn. IX

(5) It is often a great help to have stated on the preparation the
objectives best adapted to bring out the special feature desired.

FIG. 193. SLIDE TRAY WITH MASKED PREPARATIONS TO BE USED IN A DEMON-
STRATION.

(From Optic Projection).

(6) For holding the specimens, a slide tray may be used (fig. 193)
or one of the slide boxes. In any case they must be so that the
slides can be grasped easily.

(7) It is for many lecturers easier to manipulate the projection
microscope themselves and to use a pointer held out in the cone of
light. The pointer appears as sharply as when put on the screen.

(8) For all but the highest powers a substage condenser is not
needed; and one can light objects up to 50 or 60 mm. in diameter if
the object is placed in the right position in the cone of light (fig.
194).

(9) For objectives of higher power than 4 mm. (4ox) a substage
condenser should be used, and if an ocular is used as well as an ob-

CH. IX]

DRAWINGS AND DEMONSTRATIONS

357

jective then the substage condenser is advantageous for powers
above 8 mm. equivalent focus. For lighting see §§ 119, 122, 477.

FIG. 194. ILLUMINATING OBJECTS OF VARIOUS SIZES IN MICRO-PROJECTION
WITH THE MAIN CONDENSER ONLY.

(From Optic Projection).

The object must be put in the cone of light at a point where it will be fully
illuminated.

For high powers it will be at or very near the focus (/). For larger objects and
low powers at 2 or 3, or even closer to the condenser face.

Arc Supply The right-angled carbons of the arc lamp.

L\ £2 The first and second elements of the triple condenser.

Water Cell The water cell for absorbing radiant heat (§ 446). It is in
the parallel beam between the first and second elements of the condenser.

Axis The principal optic axis on which all the parts are centered.

(10) One of the most important points is to have a very white
screen. A cloth or wall screen painted with artist's scenic white
gives a very perfect screen which does not yellow with age, and
it is restored by an occasional coat of fresh white. Semi-mirror
screens are successful only in narrow rooms.

For short screen distances (i or 2 meter screen distances) white
cardboard or a sheet of very white bristol board gives excellent
results.

The apparatus, in contrast to the screen, should be dull black.

§ 446. Heat-absorbing glass and water cell; objectives, amplifiers
and oculars. — As the cone of light from the condenser (fig. 194)
must be focused on the object, the object is likely to be overheated
and spoiled by the longer waves of radiant energy in the light or
accompanying it. These longer waves represent about 90 per cent
of the radiant energy from an artificial light source like the arc
lamp, and the visible waves only about 10 per cent.

358

DRAWINGS AND DEMONSTRATIONS

[Cn. IX

If in some way the invisible 90 per cent of energy could be ab-
sorbed, a larger amount of energy represented by light could be used
and the pictures made more brilliant without injuring delicate
specimens or living objects. This possibility has been realized by
the combination of heat-absorbing glass and a water cell by Dr.
H. P. Gage of the Corning Glass Works. (See Trans. Soc. Mov. Pict.
Eng. May, 1924, pp. 38, 42).

With this combination, osmic acid preparation, Golgi and Weigert
stained sections and living infusoria have been projected from half

FIG. 195. PROJECTION APPARATUS SHOWING THE PARTS AND THE WIRING FOR

AN ARC LAMP.

(From Optic Projection).

The Objective, Condenser, and Arc Lamp are on separate blocks which move
independently along the optical bench.

c Center of the objectives where the rays from the condenser should cross.

i, 2 The first and second elements of the three lens condenser with a water
cell for absorbing radiant heat between the lenses (§446).

V The ventilating hood of the lamp house.

LA, VA The mechanism for fine adjustment of the arc lamp to the sides and
vertically. These are a necessity for projecting with the microscope, otherwise
the crater cannot be kept centered.

FS The fine adjustments for the two carbons.

PWR Separable attachment for the wires from the outlet box to the table
switch.

Wi "Wire from the table switch to the upper carbon.

Wz Wire from the table switch to the rheostat.

Wi Wire from the rheostat to the lower carbon.

CH. IX] DRAWINGS AND DEMONSTRATIONS • 359

a minute to 10 minutes and longer without injury when a 10 ampere,
direct current was used for the arc light.

For individual use, objectives and oculars of all powers can be
employed for projection. For classes it is not satisfactory to use
objectives higher than 8 mm. (2ox) to 4 mm. (4ox); and with the
larger classes it is better to use the large condenser (fig. 194) without
a substage condenser, and to use the objective or an objective and
amplifier for projection. A much larger and more brilliant field can
be shown in this way than when an ocular is used, especially when
the narrow tube for the ocular is removed and a wide (5 cm.) tube
is present on the projection outfit.

§ 447. Centering the optical parts on one axis. — This is one of
the most important procedures of all and no good projection can be
accomplished without it. The easiest way is first to arrange the
crater of the arc lamp, the central point of the large condenser, and
the microscope objective all at the same height from the baseboard
(fig. 179). If then the lamp is turned on and the objective placed
in the focus of the main condenser cone, the image of the crater of
the arc lamp should be formed on the end of the objective, the
brightest part on the front lens. If the image is to one side, above
or below, then the microscope should be raised, or lowered. After
being once carefully centered, the centering will vary slightly with
the burning of the carbons. To compensate for this there must be
fine adjustments to raise and lower the carbons and to move them
from side to side. No good projection can take place unless the full
cone of light shines upon the end of the objective.

To get the very best effect in the easiest way, there should be a
dull black shield over the end of the objective (fig. 196) so that the
image of the crater can be seen without hurting the eyes. When the
crater is focused on the end of the objective, the specimen is moved
up until it is in focus, the .objective not being moved. Of course
this means that the stage must be separately movable (fig. 179).
See also § 422.

§ 448. Demonstrations with a vertical projection microscope. —
Many specimens must be mounted in liquids and cannot be set in a
vertical position; therefore the microscope must be vertical and the

360 DRAWINGS AND DEMONSTRATIONS [Cn. IX

object remain horizontal. In such a case project the light from
the large condenser (fig. 179) or from the small arc lamp (fig. 78)
upon the mirror of the microscope and reflect it directly upward,
and then use a mirror or prism to change the direction from vertical
to horizontal. (See figs. 180, 183, to recall how the beam is changed
in direction 90°).

FIG. 196. METAL HOOD OVER THE OBJECTIVE TO AID IN CENTERING THE

LIGHT.

(From Optic Projection).

A Longitudinal section of the objective to show the metal hood.

B End view of the objective with the crater of the arc lamp directly in the
center, at the left, and to one side of the center at the right. The adjustments,
VAj LA in fig. 195 are to enable one to center the light easily.

A most striking preparation is one of the hay infusion (§350)
projected upon the screen. A water immersion objective of 2 to 3
mm. equivalent focus is excellent for projecting such preparations.
It is especially necessary to have a sheet of heat-absorbing glass
somewhere in the light beam before it reaches the living organisms
(§ 446).

DEMONSTRATION LANTERN AND TABLE FOR ARTIFICIAL DAYLIGHT

§ 449. Special microscopic demonstrations. — As stated above,
if one is to see the finest details of structure, there is no satisfactory
way but to look into the microscope direct. There is also in every
laboratory for microscopic work considerable waste space if depend-
ence is put upon daylight. If artificial light is used regularly, the
method here given is also applicable.

The main points for this kind of demonstration were worked out
by Dr. B. F. Kingsbury for his laboratory of histology and em-
bryology.

CH. IX]

DRAWINGS AND DEMONSTRATIONS

A round top demonstration table of a size for 8 microscopes is
made and in the middle a single mazda lamp of 200 or 250 watts is

FIG. 197. KINGSBURY'S DEMONSTRATION TABLE WITH ARTIFICIAL DAYLIGHT.
(ABOUT 2*5 NATURAL SIZE).

(From the Anatomical Record, June, 1916).

T Top of the metal tube and the separable attachment plug. This tube
reaches about 2 meters above the floor so that the supply cable will be out of the
way.

N S The single 2 50- watt mazda lamp with its metal support.

i, 2, 3, 4, 5, 6, 7, 8 The shields (677; with a disc of daylight glass (a) in each
at the level of the microscope mirror.

installed (fig. 197). Around this lamp are 8 shields, each containing
a piece of daylight glass.

With this arrangement 8 microscopes can be used at once (fig.
197). and the light is sufficient to enable the student to use all
powers of the microscope up to the highest oil immersion. This
method of demonstration has already been in use during the college
years of 1915-1931 and has proved successful beyond expectation.

§ 450. Demonstrations with the polarizing and the ultra-violet
microscopes. — For the best effects with polarized light a dark or
at least a dimly lighted room is most successful. That is, no light
should get to the eye that has not passed through the microscope.

DRAWINGS AND DEMONSTRATIONS

[Cn. IX

FIG. 198. Two MICROSCOPES AND A CHALET LAMP ON A LABORATORY TABLK.
(Above one-ninth natural size).

The Chalet microscope lamp with two windows (fig. 46) serves well for two
observers on opposite side of the same table, or two tables may be placed side by
side and the lamp rested partly on each.

FIG. 199. CHALET MICROSCOPE DAYLIGHT LAMP WITH FOUR WINDOWS.

(From the catalogue of the Spencer Lens Co.).

This lamp serves well for demonstrations with four microscopes. It is also good
for use by four students if on a square-top table.

CH. IX] DRAWINGS AND DEMONSTRATIONS 363

For the ultra-violet microscope the fluorescent light is relatively
so faint that it is almost necessary to work in a darkened room.
The eyes are then adjusted to twilight vision, and the delicate
radiance may be seen with much satisfaction. See also the dis-
cussions in Chapters V and VI.

COLLATERAL READING FOR CHAPTER IX

ATWELL, W. J. — On the conversion of a photograph into a line drawing. Anat.
Record, Vol. 10, pp. 39-41. The lines are made on the face of the photo-
graph, then the photographic image is bleached out by means of hypo and
cyanide.

COMSTOCK, J. H. — The Wings of Insects, 1918. Blue prints were made and the
India ink lines made on their face. The blue was then bleached with po-
tassium oxalate, etc.

GAGE, S. H. AND H. P., Optic Projection, Ch. X.

HARDESTY AND LEE. Laboratory drawing.

FRELEASE, S. AND YULE, E. S. — Preparation of Scientific and Technical Tapers,
2d ed., 1927.

CHAPTER X

PHOTOGRAPHING EMBRYOS AND SMALL ANIMALS;
PHOTOGRAPHIC ENLARGEMENTS; PHOTOGRAPHING

WITH THE MICROSCOPE

§§451-507; FIGURES 200-214

PHOTOGRAPHY

§ 461. From the beginning of the art of photography scientific
men have used it to paint for them the forms in nature and the com-
plex structures found in the physical and the biological world; and
it has been so good a servant that it is more and more called into
requisition to delineate all the phenomena as well as the forms of
nature and art. This is especially true now that successful methods
of color photography have become available.

§ 452. Photography with a horizontal camera. — The most con-
venient position for the camera obscura is the horizontal one,
and for most of the photography actually done it is very easy to
arrange the objects to be photographed in a vertical position, but
for much of the photography of science it is very convenient to use
a vertical camera, leaving the objects in a horizontal position. With
objects in liquids this is a practical necessity.

§ 453. Photography with a vertical camera. — The object can be
left horizontal as well as the camera by the use of a mirror or
totally reflecting prism, but this gives the inversion of a plane
mirror, and as shown in § 435 it will render the image erect on the
film side of the negative, but when the negative is printed the image
will be inverted. To meet all the difficulties the object may be left
in a horizontal position and the camera made vertical (fig, 200).

Since 1879 such a camera has been in use in the Anatomical De-
partment of Cornell University for photographing all kinds of
specimens; among these, fresh brains and hardened brains have
been photographed without the slightest injury to them. Further-
more, as many specimens are so delicate that they will not support

364

CH. X] PHOTOGRAPHY 365

their own weight, they may be photographed under alcohol or water
with a vertical camera and the result will be satisfactory as a
photograph and harmless to the specimen.

A great field is also open for obtaining lifelike portraits of water
animals. Chloretoned or etherized animals are put into a vessel of
water with a contrasting background and arranged as desired, then
photographed. Fins have something of their natural appearance
and gills of branchiate salamanders float out in the water in a
natural way. In case the fish tends to float in the water a little
mercury injected into the abdomen or intestine will serve as ballast.
The photographs obtainable in water are almost if not quite as
sharp as those made in air. Even the corrugations on the scales of
such fishes as the sucker (Catostomus teres) show with great clear-
ness.

While the use of photography diminishes the labor of artists about
one-half, it increases that of the preparator; and herein lies one of
its chief merits. The photographs being exact images of the prep-
arations, the tendency will be to make them with greater care and
delicacy, and the result will be less imagination and more reality in
published scientific figures. The objects prepared with such care
are more likely to be preserved for future reference.

In the use of photography for figures several considerations arise:
(i) the avoidance of distortion; (2) the adjustment of the camera
to obtain an image of the desired size; (3) focusing; (4) lighting
and arranging the object.

(i) While the camera delineates rapidly, the image is liable to
distortion. I believe opticians are agreed that, in order to obtain
correct photographic images, the objective must be properly made,
and the plane of the object must be parallel to the plane of the
ground-glass. Furthermore, as most of the objects in natural his-
tory have not plane surfaces, but are situated in several planes at
different levels, the whole object may be made distinct by using a
long focus objective and a small diaphragm.

§ 454. Scale of photographs. — It is desirable to make all photo-
graphs at some definite scale. To do this without much waste of
time the camera should be calibrated for each objective that is to be

366 PHOTOGRAPHY [Cn. X

used. This is accomplished easily by using a metric scale like that
shown in fig. 173. By lengthening and shortening the bellows of
the camera so that the image distance is greater and less, one can
get the exact position for a group of magnifications and reductions.
If the length of the bellows is noted for each size, and the distance
of the objective from the object when the focus is good is also noted,
one can arrange the camera very quickly for any special size which
may be desired. The sizes found very useful by the author are:
i; f 5 i; J; i; 2; 2-5; 4; 5- For magnifications above 5 it is
better to make a negative natural size and then make an enlarge-
ment of this, as explained in § 484.

The vertical camera shown in fig. 200 has the supporting rod
graduated in centimeters and half centimeters. After the extension
of the camera for any size has been once determined, it is easily
made the same at some future time.

§ 465. Magnification rod for the camera. — Objects vary so much
in thickness that the focusing range of the camera should be con-
siderable. With the ordinary camera there is usually no provision
for moving the camera as a whole for focusing. -With the vertical
camera shown in fig. 200, where both ends of the camera must be
clamped, it is difficult to focus over a large range and keep the
length of camera needed for the desired magnification or reduction.
For this reason the same device was applied to it as to the original
vertical camera of 1879, viz., a rod passing from end to end of the
camera, fixed at one end and clamped at the other. When the
camera is extended the exact amount required for the size in a given
case, the clamp is fixed so that the length of the camera cannot
be changed; then the whole camera may be moved for focusing
without any danger of varying the magnification. This device saves
a great deal of time. In the original camera of 1879, the rod was
graduated in centimeters. This, of course, helps to give the proper
extension with the* least outlay of trouble. In fig. 200 the vertical
supporting rod is graduated in centimeters and half centimeters.

§ 456. Lighting for the vertical camera. — The object should be
so arranged that all the details come out with the greatest distinct-
ness. As the light must be largely from the side, it is often neces-

CH. X]

PHOTOGRAPHY

367

sary to put a piece of white blotting paper or cardboard on the side
of the specimen opposite the window. Occasionally for lighting up
deep cavities it is a great advantage to use a mirror and reflect sun-
light or lamplight into the cavities for a part of the exposure.

Great care must be taken in selecting a
suitable background so that the specimen
will stand out clearly and not be merged into
the background.

When a white background is used, the
shadow of the specimen is often very trouble-
some, and to distinguish the outline of the
object W. E. Rumsey (Canadian Entomolo-
gist, 1896, p. 84) hit upon the plan of plac-
ing the object on a glass plate and putting
the background on a stage below (fig. 200).
A background on the lower stage does
away with the confusion. If daylight is not
available, excellent photographs can be
obtained with mazda lamps with metallic
or white reflectors to direct the light. It is
usually better to employ two portable lamps
and arrange them so that the shadows will
not be too prominent.

§ 457. — Photographing embryos, small
animals, and organs. — The camera shown
in fig. 200 is admirably adapted for this, as
the objects, many of them, must be photo-
graphed under water, alcohol or other
liquids.

If one has a good place to do the work in,
the light can usually be arranged satisfac-
torily with the object in a vessel with a
proper background in the bottom. If not, a
double stage must be used, as shown in fig.
200.

If white embryos or other light objects are to be photographed a

FIG. 200. VERTICAL
PHOTOGRAPHIC CAMERA.

T Low table 50 cm.
high, 50 cm. wide, and 70
cm. long.

Fs Focusing stand
with vessel for holding
embryos and small ani-
mals to be photographed
under liquid.

VC Vertical camera
with an objective (ob.) in
the lower end and a fo-
cusing glass (/#) above.
(See fig. 174 for fuller de-
scription.)

368 PHOTOGRAPHY [Cn. X

black background is best. This is produced by using black glass
on the bottom of the dish. If black glass is not available, a good
background can be produced by smooth white paper blackened with
water-proof carbon ink.

With a proper background make sure that the lighting is such as
to bring out the desired details. Turn the object in various posi-
tions till the desired one is found which shows clearly the points that
are to be emphasized.

§ 458. Focusing stand for the vertical camera. — To hold the
specimen and to provide for the finest focusing, and also some of the
coarse focusing, a modified microscope stand is convenient. It has
no tube, but two stages are attached to the support usually carrying
the tube. This then can be raised and lowered by the coarse and
by the fine adjustment, as in focusing the microscope, except that
here the stages move, the photographic objective remaining station-
ary (fig. 200). With the rod to hold the camera at a fixed ex-
tension, most of the focusing can be accomplished by sliding the
whole camera up and down the vertical graduated support (fig.
200).

§ 459. Focusing glass. — There are two ways of using this:
i. A clear screen is used instead of a ground-glass. On this is a
diamond scratch in the middle. The focusing glass is carefully
focused on the central scratch, which must be in the exact plane
where the sensitized photographic surface will be during the ex-
posure. If now an object is brought to an accurate focus at this
plane, it will also be in focus on the sensitized surface of the dry
plate. Except for aid in arranging the object and for general focus-
ing, the frosted glass can be entirely omitted, and a focusing glass
giving about 8 to 10 diameters magnification is set in a board which
takes the place of the ordinary frosted glass screen. This is put at
the level to bring the focus exactly at the plane where the sensitive
surface of the negative is to be.

The position of the focusing glass is determined as follows:
The plate holder with a clear glass plate or a thin negative is in
the holder. And on the film side is a diamond scratch or an India
ink mark near the middle of the face usually occupied by the sensi-

CH. X]

PHOTOGRAPHY

369

live film. It is very important that the mark should be on the >side
occupied by the film.

The scratch or ink mark is a guide for getting the focus at the
right level. Now with a tripod or
other magnifier, preferably with the
magnifier to be used later, get the
image focused of the metric scale,
and its explanation or other sharp
print exactly on the surface where the
diamond or ink mark is. To make
sure that there is no better focus ob-
tainable, it is worth while to make a
negative of the printed matter used
for focusing. On the excellence of
the focus determined depends the
excellence of all future pictures which
will be made. This method has the
further advantage that the focus
level is determined for the plate
holder and not for a focusing screen.
It is, in fact, an excellent way to

check up the similarity of level of the sam- ™s rendfrs
F J surface transparent.

ordinary focusing screen and the

plate holder. Frequently they do

not agree closely enough for the

more exacting work, especially in

photo-micrography. If the focus is found to be exact, proceed to

set the focusing glass in a board as follows:

Have a board of about 15 mm. thickness in a frame like that used
for the ordinary focusing screen. Bore a hole in the center in which
the focusing glass holder will fit snugly. Now put the frame on the
focused camera and slowly twist the focusing glass into the hole
until the focus seen through it is perfect. If nothing has changed in
the camera, then this focus should give perfect results for any future
setting of the camera, for the focus will be at the exact level oc-
cupied by the sensitive surface of the plate. If it is found perfect

FIG. 201. GROUND-GLASS Fo-
ci SING SCREEN UITH CLEAR CEN-
TER FOR FINE FOCUSING.

j The ground or frosted sur-
face of the glass.

2 A cover-glass stuck to the
frosted surface with Canada Bal-
the frosted

x Pencil mark in the center of
the focusing screen on the frosted
surface to serve as guide when
focusing with a magnifier.

370 PHOTOGRAPHY [Cn. X

by trial, it is wise to put some shellac or other varnish around the
mounting to fix it firmly in place in the wood so that there will be
no change in its position. Of course, any change would result in
imperfect, out-of-focus negatives.

This method of focusing has the great ad-
1 vantage of doing away with all obstructing
glass. One focuses the position of the real
image exactly as for a compound microscope
when a positive ocular (figs. 22, 23) is used.
It is an invaluable way for focusing in photo-
micrography.
§ 460. Objectives and magnification for

FIG. 202. TRIPOD embryos. — It is a good plan to have one
MAGNIFIER TO SERVE
AS A FOCUSING GLASS, picture of natural size in each case, and then,

if the embryos or other objects are very
small, a picture of 5 or more times natural size. And a picture
should go with the embryo or object throughout its entire career
so that the exact appearance before sectioning or dissection will
be available.

The objectives most convenient for making the photographs have
an equivalent focus of from 50 to 150 mm. They are placed in the
front board of the camera as usual (fig. 200). The larger the object
the longer should be the focus of the objective; then the exagger-
ated perspective of short focused lenses will be avoided.

§ 461. Photographing bacterial cultures. — For the successful
photographing of these cultures dark -ground illumination is em-
ployed on the principle stated in § 171. That is, the preparation is
illuminated with rays so oblique that none can enter the objective
directly. Those striking the culture are reflected into the objective.
The clear gelatin around the growth or colonies does not reflect the
light, and therefore the space between the colonies is dark.

For supporting the Petri dishes a hole is made in a front board for
the camera. This hole is slightly larger than the dish, Over it is
then screwed or nailed a rubber ring slightly smaller than the Petri
dish. This will stretch and receive the dish, and grasp it firmly, so
that it is in no danger of failing out when put in a vertical position.

OK. X] PHOTOGRAPHY 3 7 1

If the camera has two divisions the board with the Petri dish is put
in the front of the camera, and the objective in the middle division
through the side door. Otherwise the board holding the Petri dish
must be on a separate support.

The vertical camera and focusing stand (fig. 200) lend themselves
admirably for this kind of photography. The black background can
be put on the lower stage and the Petri dish or other bacterial cul-
ture can be set on a glass plate or in a perforated board on the
upper stage. The lighting is very easily accomplished by two
portable lamps so arranged that no light can get directly from them
into the objective.

One may use daylight by putting the culture in a support just
outside a window, leaving the camera in the room. The rays from
the sky are so oblique that they do not enter the objective. One
must use a black, non-reflecting background some distance beyond
the dish as in using artificial light (Atkinson).

In photographing bacterial cultures in test-tubes, the lighting is
as in the preceding section, but a great difficulty is found in getting
good results from the refraction and reflections of the curved sur-
faces. To overcome this one applies the principles discussed in
§ 341, and the test-tubes are immersed in a bath of water or water
and glycerin. The bath must have plane surfaces. Behind it is the
black velvet screen, and the light is in front, as for the Petri dishes.
As suggested by Spitta, it is well to employ a bath sufficiently thick
in order that streak cultures may be arranged so that the sloping
surface will all be in focus at once by inclining the test-tube.

§ 462. Recording and storing negatives. — Each negative should
have a record upon it written on the film side with India ink; then
it will never get mixed up. For ease in finding negatives there-
should be a record on the containing envelope also. Finally, it is a
good plan to have a card catalogue of one's negatives. For a form
see § 483.

For storing negatives a good method, where one does not have
too many, is to put them in envelopes and store in boxes or drawers
like book catalogue cards.

372 PHOTOGRAPHY [Cn. X

PHOTOGRAPHING WITH THE MICROSCOPE

§ 463. The first pictures made on white paper and white leather,
sensitized by silver nitrate, were made by the aid of a solar micro-
scope (1802). The pictures were made by Wedgewood and Davy,
and Davy says: " I have found that images of small objects pro-
duced by means of the solar microscope may be copied without diffi-
culty on prepared paper " (§ 463%).

Thus among the very first of the experiments in photography the
microscope was called into requisition. Naturally, plants and mo-
tionless objects were photographed in the beginnings of the art when
the time of exposure required was long.

Although first in the field, photo-micrography has been least
successful of the branches of photography. This is due to several
causes. In the first place, microscope objectives have been con-
structed to give the clearest image to the eye; that is, the visual
image, as it is sometimes called, is for microscopic observation of
prime importance. The actinic or photographic image, on the other
hand, is of prime importance for photography. For the majority
of microscopic objects transmitted light (§ 70) must be used, not
reflected light as in ordinary vision. Finally, from the shortness of
focus and the smallness of the lenses, the proper illumination of the
object is accomplished with some difficulty, and the fact of the lack
of sharpness over the whole field with any but the lower powers has
combined to make photo-micrography less successful than ordinary
macro-photography. So tireless, however, have been the efforts
of those who believed in the ultimate success of photo-micrography,
that now the ordinary achromatic objectives with panchromatic or
isochromatic plates and a color screen give good results, while the
apochromatic objectives with projection oculars give excellent re-
sults, even in hands not especially skilled. The problem of illumi-
nation has also been solved by the construction of achromatic and
apochromatic condensers and by the electric and other powerful
lights now available. There still remains the difficulty of trans-
mitted light and of so preparing the object that structural details

CH. X] PHOTOGRAPHY 373

stand out with sufficient clearness to make a picture which ap-
proaches* in definiteness the drawing of a skilled artist.

The writer would advise all who wish to undertake photo-microg-
raphy seriously to study samples of the best work that has been
produced. Among those who showed the possibilities of photo-
micrographs was CoL Woodward of the U. S. Army Medical
Museum. The photo-micrographs made by him and exhibited at
the Centennial Celebration at Philadelphia in 1876 serve still as
models. According to the writer's observation no photo-micro-
graphs of histologic objects have ever exceeded those made by
Woodward, and most of them are vastly inferior. It is gratifying
to state, however, that at the present time many original papers are
partly or wholly illustrated by photo-micrographs, and no country
has produced works with photo-micrographic illustrations superior
to those in Wilson's " Atlas of Fertilization and Karyokinesis " and
Starr's " Atlas of Nerve Cells," issued by die Columbia University
Press.

Most excellent photo-micrographs appear at frequent intervals in
all the great biological journals. These should be studied by the
young photographer ambitious to excel.

§ 463a. Considerable confusion exists as to the proper nomenclature of
photography with the microscope. On the Continent the term micro-photog-
raphy (micro-photographic) is very common, while in English photo-microg-
raphy and micro-photography mean different things. Thus: A photo-micrograph
is a photograph of a small or microscopic object usually made with a micro-
scope and of sufficient size for observation with the unaided eye; while a micro-
photograph is a small or microscopic photograph of an object, usually a large
object, like a man or woman, and is designed to be looked at with a microscope.

Dr. A. C. Mercer, in an article in the Proc. Amer. Micr. Soc., 1886, p. 131,
says that Mr. George Shadbolt made this distinction. See the Liverpool and
Manchester Photographic Journal (now British Journal of Photography), Aug.
i5> 1858, p. 203; also Sutton's Photographic Notes, Vol. Ill, 1858, pp. 205-208.
On p. 208 of the last, Shadbolt's word "photo-micrography" appears. Dr.
Mercer puts the case very neatly as follows: "A Photo-Micrograph is a macro-
scopic photograph of a microscopic object; a micro-photograph is a microscopic
photograph of a macroscopic object." See also Medical News, Jan. 27, 1894, p.
108.

In a most interesting paper by A. C. Mercer on "The Indebtedness of Photog-
raphy to Microscopy," Photographic Times Almanac, 1887, it is shown that:
"To briefly recapitulate, photography is apparently somewhat indebted to
microscopy for the first fleeting pictures of Wedgewood and Davy [1802], the
first methods of producing permanent paper prints [Reede, 1837-1839], the first
offering of prints for sale, the first plates engraved after photographs for the pur-

374 PHOTOGRAPHY [Cn. X

pose of book illustration [ponne & Foucault, 1845], the photographic use of
collodion [Archer & Diamond, 1851], and finally, wholly indebted for the origin
of the gelatino-brorhide process, greatest achievement of them all" [Dr. R. L.
Maddox, 1871]. See further for the history of Photo-micrography, Neuhauss,
also Bousfield, and Photography, 1839-1937. Museum of Modern Art, N. Y.

§ 464. As the difficulties of photo-micrography are so much
greater than of ordinary photography, the advice is almost uni-
versal that no one should try to learn photography and photo-
micrography at the same time, but that one should learn the
processes of photography by making portraits, landscapes, copying
drawings, etc.; and then when the principles are learned, one can
take up the more difficult subject of photo-micrography with some
hope of success.

The advice of Sternberg is so pertinent and judicious that it is
reproduced: " Those who have had no experience in making photo-
micrographs are apt to expect too much and to underestimate the
technical difficulties. Objects which under the microscope give a
beautiful picture which we desire to reproduce by photography may
be entirely unsuited for the purpose. In photographing with high
powers it is necessary that the objects to be photographed be in a
single plane and not crowded together and overlying each other.
For this reason photographing bacteria in sections presents special
difficulties and satisfactory results can be obtained only when the
sections are extremely thin and the bacteria well stained. Even
with the best preparations of this kind much care must be taken in
selecting a field for photography. It must be remembered that the
expert microscopist, in examining a section with high powers, has
his finger on the fine adjustment screw and focuses up and down to
bring different planes into view. He is in the habit of fixing his at-
tention on the part of the field which is in focus and discarding the
rest. But in a photograph the part of the field not in focus appears
in a prominent way, which mars the beauty of the picture."

APPARATUS FOR PHOTO-MICROGRAPHY

§ 465. Camera. — For the best results with the least expenditure
of time one of the cameras especially designed for photo-microg-

CH. X] PHOTOGRAPHY 375

raphy is desirable, but is not by any means indispensable for doing
good work (fig. 200).

The first thing to do is to test the camera for the coincidence of
the plane occupied by the sensitive plate and the ground-glass or
focusing screen. Cameras even from the best makers are not always
correctly adjusted.

For the method of procedure see above, § 459.

The majority of photo-micrographs do not exceed 8 centimeters
in diameter and are made on plates 8 x n, 10 X 13, or 13 x 18
centimeters (3! X 4! in., 4X5 in., or 5 X 7 in.).

For pictures larger than these it is best to make small, very sharp
negatives of moderate enlargement and then print these at any
desired size by means of projection apparatus. (See under enlarge-
ments, § 484).

§ 466. Workroom. — It is almost self-evident that the camera
must be in some place free from vibration. A basement room where
the camera table may rest directly on the cement floor or on a pier
is excellent. Such a place is almost necessary for the best work with
high powers. For those living in cities, a time must also be chosen
when there are no heavy vehicles moving in the streets. For less
difficult work an ordinary room in a quiet part of the house or
laboratory building will suffice. It helps much to have rubber corks
in the lower ends of the table legs. The legs may also be made to
stand on four thick pads of rubber or of thick felt. Finally the
camera and microscope can be placed on a board platform and
that put into a shallow box nearly filled with sawdust or dry sea
sand.

The photo-engravers have overcome vibrations by suspending
their cameras, or using spring coils as a part of the support. In
case of real need this method would serve the photographer with the
microscope.

§ 467. Arrangement and position of the camera and the micro-
scope. — For much photo-micrography a vertical camera and
microscope are to be preferred. Excellent arrangements were per-
fected long ago, especially by the French. (See Moitessier).

Vertical photo-micrographic cameras are now commonly made,

PHOTOGRAPHY

[CH.X

and by some firms only vertical cameras are produced. They are
exceedingly convenient, and do not require so great a disarrange-

FIG. 203. VERTICAL MICROSCOPE AND CAMERA FOR PHOTO-MICROGRAPHY.
(About ,'„ natural size).

I Low table 50 cm. high, 50 cm. wide, and 70 cm. long with felt pads under
the legs (fp) and a drawer with combination lock (cl.d).

M Microscope.

VC Vertical camera supported by the revolving rod (vgr) which is graduated
in centimeters and half centimeters. The camera may be turned aside as shown
by the dotted lines.

Base The heavy iron base and pillar (p) supporting the revolving rod (vgr),
which in turn supports the camera.

cs Clamping screws to fix the two ends of the camera in any desired position.

mr Magnification rod. This serves to hold the extension of the camera
at the right point for any desired magnification; then the camera as a whole
moves up and down on the graduated rod (vgr).

rs Clamp to fix the camera at any desired extension on the magnification
rod (mr).

fg Focusing glass (§ 459).

le Light excluder (fig. 204-205).

Rl Research lamp with io8-watt bulb, and transformer (Tr). For full descrip-
tion see fig. 80.

CH. X] PHOTOGRAPHY 377

inent of the microscope to make the picture as do the horizontal
ones. The variation in size of the picture in this case is mostly
obtained by the objective and the projection ocular rather than by
length of bellows.

It must not be forgotten, however, that penetration varies in-
versely as the numerical aperture, and inversely also as the square
of the power. There is then an advantage in using a low power with
long bellows if one needs penetration. In many cases the best way
is to use a moderate power and a short bellows, and then to print
the negative as for making enlargements for drawings (§ 484).

For convenience and rapidity of work a microscope with mechani-
cal stage is necessary; and for sections where it is desirable to have
the image in some regular position a revolving stage to the micro-
scope helps greatly in orienting the image on . the plate.

It is also an advantage to have a tube of large diameter so that
the field will not be too greatly restricted (fig. 179). In some micro-
scopes the tube is removable almost to the nose-piece to avoid inter-
fering with the size of the image. The substage condenser should be
movable on a rack and pinion. The microscope should have a
•flexible pillar for work in a horizontal position. While it is desirable
in all cases to have the best and most convenient apparatus that is
made, it is not by any means necessary for the production of excel-
lent work. A simple stand with flexible pillar and good fine adjust-
ment will answer.

§ 468. Objectives and oculars for photo-micrography. — The
belief is almost universal that the apochromatic objectives are most
satisfactory for photography. They are employed for this purpose
with a special projection ocular or compensation oculars. Two low
powers are used without any ocular. Some of the best work that has
ever been done, however, was done with achromatic objectives (work
of Woodward and others). One need not desist from undertaking
photo-micrography if he has good achromatic objectives. From a
somewhat extended series of experiments with the objectives of many
makers the modern fluorite and achromatic objectives have been found
to give excellent results when used without an ocular. Most of them
also gave good results with projection and other oculars.

378 PHOTOGRAPHY [('H. X

Recently negative lens combinations (Homals, Ampliplans) have been devised to
use instead of oculars for photo-micrography and for projection. They are in the
nature of amplifiers (§ 369) and serve to compensate for curvature of the field and
for chromatic differences of magnification of the objectives. (See Bausch & Lomb's
Catalog of Scientific Instruments, 1941, p. 146.)

§ 469. Difference of visual and actinic foci. — Formerly there
was much difficulty experienced in photo-micrographing on account
of the difference in actinic and visual foci. Modern objectives are
less faulty in this respect and the apochromatics are practically free
from it. Since the introduction of orthochromatic or isochromatic
and panchromatic plates, and in many cases the use of color screens,
but little trouble has arisen from differences in the foci. This is
especially true when mono-chromatic light and even when petroleum
light is used. In case an objective has its visual and actinic foci at
markedly different levels, it would be better to discard it for photog-
raphy altogether, for the estimation of the proper position of the
sensitive plate after focusing is only guesswork and the result is
mere chance. If sharp pictures cannot be obtained with an objec-
tive when isochromatic or panchromatic plates are used, the fault
may not rest with the objective, but with the plate holder and
focusing screen. They should be very carefully tested to see if there,
is coincidence in position of the focusing screen and the sensitive
film as described in § 459.

LIGHTING FOR PHOTO-MICROGRAPHY

§ 470. Light. — The best light is sunlight. That has the defect
of not always being available, and of differing greatly in intensity
from hour to hour, day to day, and season to season. Following
the sunlight the electric light is the most intense of. the available
lights. (See also figure 129 A and § 499.)

As natural daylight is not constantly available, the photo-mi-
crographer has now at his disposal the artificial daylight by the use
of a nitrogen-filled mazda lamp and daylight glass. The lantern for
this shown in figs. 80, 203 was found to be excellent and the results
obtained by its use in photographing with powers up to the 1.5 mm.
homdgeneous immersion were good. Of course any light

CH. X] PHOTOGRAPHY 379

filters which are adapted to natural daylight would serve perfectly
with the artificial daylight. In most cases the six-volt lamp requires
a special filter for each specimen. See § 491.

For preparations needing a yellow color screen for daylight, a
petroleum or kerosene lamp gives good results for the majority of
low and moderate power work. And even for 2 mm. (9ox) homo-
geneous immersion objectives, the time of exposure is not excessive
for many specimens (40 seconds to 3 minutes).

A lamp with flat wick about 40 mm. wide has been found most
generally serviceable. For large objects and low powers the flame
may be made large and the face turned toward the mirror. This
will light a large field. For high powers the edge toward the mirror
gives an intense light. The ordinary glass chimney answers well,
especially where a shield is used.

In managing the light for photography with the microscope, follow
the directions in § 198. See below for the use of color screens (§ 491).

§ 471. Objects suitable for photo-micrographs. — While almost
any large object may be photographed well with the ordinary
camera and photographic objective, only a small part of the objects
mounted for microscopic study can be photo-micrographed satis-
factorily. Many objects that can be seen clearly by constant
focusing with the fine adjustment, appear almost without detail on
the screen of the photo-micrographic camera and in the photo-
micrograph.

If one examines a series of photo-micrographs, the chances are
that the greater number will be of diatoms, plant sections, or
preparations of insects. That is, they are of objects having sharp
details and definite outlines, so that contrast and definiteness may
be readily obtained. Stained microbes also furnish favorable ob-
jects when mounted as cover-glass preparations, but these give
color images and require a color screen.

For success with preparations of animal tissue they must approxi-
mate as nearly as possible to the conditions more easily obtained
with vegetable preparations. That is, they must be made so thin
and be so prepared that the cell outlines have something of the
definiteness of vegetable tissue. It is useless to expect to get a clear

380 PHOTOGRAPHY [Cn. X

photograph of a section in which the details are seen with difficulty
when studying it under the microscope in the ordinary way.

Many sections which are unsatisfactory as wholes may neverthe-
less have parts in which the structural details show with satisfactory
clearness. In such a case the part of the section showing details
satisfactorily should be marked in some way. If one's preparations
have been carefully studied and the special points in them indi-
cated, they will be found far more valuable both for ordinary
demonstration and for photography. The amount of time saved by
marking one's specimens can hardly be overestimated.

Formerly many histologic preparations could not be satisfactorily
photographed. Now with improved section cutters, better staining
and mounting methods, and with the color screens and isochromatic
and panchromatic plates (§ 505) almost any preparation which shows
the elements clearly when looking into the microscope can be satis-
factorily photographed. Good photographs cannot, however, be
obtained from poor preparations by any method.

In photo-micrography do not forget the three ways in which de-
tails of structure may be brought out clearly:

(1) By difference of refraction of the object and the mounting
medium (refraction images, § 152).

(2) By differential staining (color images, § 154).

(3) By means of dark-ground illumination,

EXPERIMENTS IN PHOTO-MICROGRAPHY

§ 472. The following experiments are introduced to show practi-
cally just how one would proceed to make photo-micrographs with
various powers, and be reasonably certain of fair success. If one
consults prints or the published figures made directly from photo-
micrographs, it will be seen that, excepting diatoms and bacteria,
the magnification ranges mostly between 10 and 150 diameters.

§ 473. Focusing in photo-micrography. — For rough focusing and
as a guide for the proper arrangement of the object one uses a
ground-glass screen, as in gross photography. Wit/i the ground-glass
screen one can judge of the brilliancy and evenness of the illumi-

CH. X] PHOTOGRAPHY 381

nation more accurately than in any other way. For final and exact
focusing two principal methods are employed:

(a) A focusing glass is used either with a clear screen or in a
board screen, as described above (§ 459). The latter method is like
focusing with the compound microscope and a positive ocular. If
the focusing glass is set properly the focus should be easily and accu-
rately determined.

In whatever way one focuses for photo-micrography a difficulty
often appears. No matter how perfect the focus of the microscope,
the picture may be out of focus. This may be due to either one of
two things: (i) the focusing screen or focusing glass may not be in
the right position to make the image sharp on the sensitive plate;
(2) the microscope may get out of focus while the picture is being
made. The reason for this change may be the gradual settling down
of the tube of the microscope. This may be a fault of the fine or
of the coarse adjustment. It is a good plan to focus the object
carefully and, after 10 or 15 minutes, to see if the focus is still good.
If the microscope will not stay in focus, one cannot get a good pic-
ture. In that case it is necessary to study the apparatus and see
which part of the mechanism is at fault.

§ 474. Photo-micrographs of 20 to 50 diameters. — For pictures
under 10 to 15 diameters it is better to use the camera for embryos
with the objective in the end of the camera, and the special micro-
scope stand for focusing (fig. 200).

For pictures at 25 to 50 diameters one may use the microscope
with a low objective, 20 (8x) to 35 mm. (4x) equivalent focus, and
no ocular (fig. 179). The object is placed on the stage of the micro-
scope and focused as in ordinary observation. If a vertical micro-
scope is used, the light from the petroleum lamp or other artificial
light is reflected upward by the mirror. It may take some time to
get the whole field lighted evenly. In some cases it may be ad-
visable to discard the condenser and use the mirror only. For some
purposes one will get a better light by placing the bulls'-eye or
other condenser between the lamp and the mirror to make the rays
parallel, or even to make a sharp image of the lamp flame on the
mirror. Remember also that in many cases it is necessary to

382

PHOTOGRAPHY

[CH.X

have a color screen between the source of light and the object

(§§ 49I~49S)-

For a horizontal camera it is frequently better to swing the mirror
entirely out of the way and allow the light to enter the condenser
directly. When the light is satisfactory, as seen through an ordi-
nary ocular, remove the ocular.

(a) Photographing without an ocular.
— After the removal of the ocular put
in the end of the tube a lining of black
velvet to avoid reflections. Connect
the microscope with the camera, mak-
ing a light-tight joint, and focus the
image on the focusing screen. One may
make a light-tight connection by the use
of black velveteen or, more conveniently,
by the double metal hood which slips
over the end of the tube of the micro-
scope, and into which fits a metal
cylinder on the lower end of the camera
(figs. 204-205). In figure 205 the con-
nection has been made.

It will be necessary to focus down con-
siderably to make the image clear.

FIG. 204. LIGHT EXCLUDER
FOR CONNECTING THE CAMERA
AND MICROSCOPE (ZKISS FORM)

(About \ natural size).

The front board of the Lengthen or shorten the bellows to
make the image of the desired size,
then focus with the utmost care. In
case the field is too much restricted on
account of the tube of the microscope,
remove the draw-tube. When all is in
readiness, it is well to wait for three to
five minutes and then to see if the
image is still sharply focused. If it

gets out of focus simply by standing, a sharp picture cannot be

obtained. If it does not remain in focus, something is faulty.

When the image remains sharp after focusing, make the exposure.

From 20 to 60 seconds will usually be sufficient time with

camera.

2 Connecting piece to fit
over i and extend down into
J.

3 Piece to fit over the up-
per end of the tube of the
microscope and to receive the
lower end of 2 (compare fig.
205 where the parts are to-
gether as in making an expo-
sure).

CH.X]

PHOTOGRAPHY

383

medium plates and light as described. If a color screen is
used it will require 50-300 seconds, i.e., 2 to 5 times as long, for a
proper exposure (§ 497).

(b) Photographing with an ocular. — If the object is small enough
to be included in the field of a projection or other ocular (fig.
208), use that for making the negative as follows: Swing the camera
around so that it will leave the microscope free (fig. 203). Use an
ordinary ocular, focus and light the object, then insert a projection
ocular in place of the ordinary one, and swing the camera back over
the microscope. It is not necessary to use an ordinary ocular for the
first focusing, but as its field is larger, ,

it is easier to find the part to be photo-
graphed. The first step is then to
focus the diaphragm of the projection
ocular sharply on the focusing screen.
Bring the camera up close to the
microscope and then screw out the eye-
lens of the ocular a short distance.
Observe the circle of light on the
focusing screen to see if its edges are
perfectly sharp. If not, continue to
screw out the eyelens until it is. If
it cannot be made sharp by screwing it
out, reverse the operation. Unless the
edge of the light circle, i.e., the dia-
phragm of the ocular, is sharp, the
resulting picture will not be satis-
factory.

It should be stated that for the 2x

projection ocular the bellows of the camera must be extended about
30 or 40 centimeters or the diaphragm cannot be satisfactorily
focused on the screen. The 4x projection ocular can be focused
with the bellows much shorter. For either projection ocular the
screen distance can be extended almost indefinitely.

When the diaphragm is sharply focused on the screen, the micro-
scope is focused, that is, first with the unaided eye then with the fo-

Draw-
Tube

FIG. 205. LIGHT EXCLUDER
POR PHOTO-MIC ROG R A PHY.
(About 1 natural si/e).
Tn this figure the different
parts of the light excluder are
in position for making an ex-
posure.

1 The front hoard of the
camera.

2 The intermediate part
connecting the camera and
the hollow cylinder on the
upper end of the microscope
draw-tube. (Compare fig.
204).

384 PHOTOGRAPHY [Cn. X

cusing glass. The exposure is made in the same way as though no
ocular were used (§ 474a), although one must have regard to the
greater magnification produced by the projection ocular and increase
the time accordingly; thus, when the 4x ocular is used, the time
should be at least doubled over that when no ocular is employed.
The time will be still further increased if a color screen is used

(§ 501).

Tt is recommended that when the bellows have sufficient length the lower pro-
jection oculars be used, but with short bellows the higher ones.

§ 476. Magnification of a photomicrograph. — This is easily determined by
removing the specimen and putting in its place a stage micrometer in i/ioth
and i/iooth mm. spaces. If the image of the micrometer is focused sharply one
can measure the image of the spaces between the lines. By dividing the size
of the image by the known size of the space the quotient will be the magnifi-
cation. For example, if the micrometer spaces used are i/ioth mm. and the
image of one space measures 12 mm. the magnification is 120 (12 -f- i/ioth
= 120). With this method one must make the determination for every photo-
micrograph taken unless a camera of fixed bellows length is employed.

The following procedure is more satisfactory and much less trouble in the
long run. The extensible bellows of the camera is clamped at some definite
length, then negatives are made of the stage micrometer with trie different
objectives and oculars which are to be used in photographing. The magnifica-
tion for each combination is determined as indicated above and marked on the
negative. When a photographic negative of some microscopic object is made
with the same combination one can see at once the magnification by consulting
the micrometer negative.

This method has a further advantage if one wishes the photomicrographic
print or diagram made from the original negative to be of greater or less magni-
fication than the original. For example, as in the case above where the magni-
fication is 120, suppose the print desired is to be at 350 magnification. The
negative of the micrometer is put in the enlarging apparatus and the distance
found by trial where the image of i/ioth mm. projected on the screen measures
35 mm. (35 -*• i/io = 350). If now one puts the negative of the microscopic
object at a magnification of 120 in place of the micrometer negative, its image
will be 350 diameters on the screen, and if a print or diagram is made at that
screen distance the picture will be at a magnification ot 350. Suppose on the
other hand that a classroom diagram is desired at a magnification of 10,000
diameters of this same negative. As before the negative of the micrometer in
i/ioths and i/iooths mm. is put in the enlarging apparatus and moved to a
distance from the screen so that when in sharp focus the image of i/iooth mm.
measures on the screen 100 mm., the magnification will then be as desired
(100 -T- i/iooth = 10,000), and if the negative of the object at 120 magnifi-
cation is diagrammed at this distance the magnification will be 10,000.

If a reduced image is desired, it is likewise simple; e.g., if a print of exactly
TOO diameters is desired from this same negative it can be obtained by using
the micrometer negative and getting the image of i/ioth mm. to measure
exactly 10 mm. on the screen (10 -f- i/ioth = 100).

§ 476. Photo-micrographs at a magnification of 100 to 150 diameters. — For
this, the simple arrangements given in the preceding section will answer, but the
objectives must be of shorter focus, 8 to 3 mm.

CH.X]

PHOTOGRAPHY

385

Exc

FIG. 206! VIEW OF THE BACK
OF THE OBJECTIVE, SHOWING THE CON-
DENSER OUT OF CENTER AND CENTERED.

Exc The spot of light (D) is to one
side of the center, showing that the optic
axis of the condenser is not in line with
that of the microscope.

C The spot of light (U) is in the cen-
ter, showing that the optic axis of the con-
denser and microscope are in line.

§ 477. Lighting for photo-micrography with moderate and high
powers. (100 to 2500 diameters), — No matter how good one's
apparatus, successful photo-micrographs cannot be made unless the
object to be photographed is
properly illuminated. The be-
ginner should go over with
the greatest care the direc-
tions for centering the con-
denser, for centering the source
of illumination, and the dis-
cussion of the proper cone of
light and lighting the whole
field, as given in §§ 135-137.
Then for each picture he must
take the necessary pains to
light the object properly. An
achromatic condenser is al-
most a necessity (§ 128).
Whether a color screen should be used depends upon judgment

and that can be attained only
by experience. In the begin-
ning one may try without a
screen and with different
screens and compare results.
A plan used by many skilled
workers is to light the objec/
and the field around it well,
and then to place a metal
diaphragm of the proper size
in the camera just under the
plate holder. This will in-
sure a clean, sharp margin to
the picture. This metal di-
aphragm must be removed
while focusing the diaphragm of the projection ocular, as the di-
aphragm opening is smaller than the image of the ocular diaphragm.

c £xc

FIG. 207. FIELD OF THE MICROSCOPE
SHOWING THE LlGHT IN THE CENTER AND
TO ONE SIDE

CFl The light is in the center and
illuminates the object.

Exc Fl The light is at one side of the
center and does not illuminate the object.
(The field is not fully lighted, as a low
power is used to center the object and
the light).

386

PHOTOGRAPHY

[CH. X

If the young photomicrographer will be careful to select for his
first trials objects of which really good photo-micrographs have al-
ready been made, and then persists with each one until fairly good
results are attained, his progress will be far more rapid than as if
poor pictures of many different things were taken. He should, of
course, begin with low magnifications.

§ 478. Adjusting the objective for cover-glass.
— After the object is properly lighted, the ob-
jective, if adjustable, must be corrected for the
thickness of cover. If one knows the exact
thickiiess of the cover and the objective is
marked for different thicknesses, it is easy to
get the adjustment approximately correct
mechanically; then the final corrections depend
on the skill and judgment of the worker. It is
to be noted, too, that if the objective is to be
used without a projection ocular, the tube-length
is extended practically to the focusing screen,
and as the effect of lengthening the tube is the
same as thickening the cover-glass, the adjusting
collar must be turned to a higher number than
the actual thickness of the cover calls for (see

FIG. 208. PRO-
JECTION OCULAR iisr
SECTION.

(About } natural
Size).

1 The upper or
eye end of the oc-
ular; it is composed
of two convex and
one concave lens
and serves to pro-
ject the real image
formed by the ob-
jective and field lens
at d upon the screen
or photographic
plate. It is mov-
able to permit of
focusing at differ-
ent screen distances.

2 Field lens of
the projection oc-
ular.

d Diaphragm
where the real im-
age is formed.

§ 479. Photographing without an ocular. —
Proceed exactly as described for the lower
power, but if the objective is adjustable, make
the proper adjustment for the increased tube-
length (§ 149).

§ 480. Photographing with a projection ocular.
— Proceed as described in § 474-b, only in this
case the objective is not to be adjusted for
the extra length of bellows. If it is corrected
for the ordinary ocular, the projection ocular then projects this
correct image upon the focusing-screen.

§ 481. Photo-micrographs at a magnification of 500 to 2000 di-
ameters. — For this the homogeneous immersion objective is em-

CH. X]

PHOTOGRAPHY

387

ployed, and as it requires a long bel1 nvs to get the higher mag-
nification with the objective alone, it is best to use the projection
oculars. Compensation oculars may also be used.

For this work the directions given in §§ 135-137 must be followed
with great exactness. The edge of the petroleum lamp flame is
sufficient to fill the field in most cases. With many objects the time
required with good lamplight is not excessive; viz., 2 to 3 seconds.
The reason for this is that while the illumination diminishes directly
a,s the square of the magnification, it increases with the increase in
the numerical aperture, so that the illuminating power of the
homogeneous immersion is great in spite of the great magnifica-
tion.

For work with high powers a stronger light than the petroleum
lamp is employed by those doing considerable photo-micrography,
e.g., the arc light or the io8-watt, 6-volt lamp (figs. 80, 128).

It may be well to recall the statement made
in the beginning, that the specimen to be pho-
tographed must be of special excellence for all
powers. No one who undertakes to make photo-
micrographs at a magnification of 500 to 2000
diameters will dc abt the truth of the statement.

If one has a complete outfit with electric arc
light or the ic<4-watt lamp, the time required for
photographing objects is much reduced, i.e.,
ranging from i to 20 seconds even with the
color screen. As the light is so intense with
the arc light it is necessary to soften it greatly
for focusing. Several thicknesses of ground-glass
placed between the lamp and the microscope will
answer. These are removed before taking the
negative. It is well also to have a water bath
on the optical bench to absorb the radiant heat.
This should be in position constantly (figs. 179,
180).

§ 482. Use of oculars in photo-micrography. — There is much
diversity of opinion whether or not the ordinary oculars used for

FIG. 209. ACH-
ROMATIC SUBSTAGE
CONDENSKR FOR
PHOTO-MICROG-
RAPHY.

(From Watson's
Catalogue).

i, 2, 3 The three
optical parts of the
condenser. (Com-
pare figs. 60- 6 1 also
the construction of
objectives in figs.
IQ-II and note that
the condenser is like
an inverted objec-
tive.)

388 PHOTOGRAPHY [Cn, X

observation should be used in photographing. Excellent results
have been obtained with them and also without them.

When an ocular is used, the eyelens serves to project a real image
of the objective, not to act as a magnifier with the eye as an
ordinary observation; therefore for the best results in photography
this eyelens should be a combination which will give a correct
image. For apochromatic objectives the projection or the com-
pensation oculars should be used, not ordinary Huygenian oculars.
The projection and compensation oculars work well with the best
high-angled achromatic objectives also.

§ 483. Negative record in photography. —

Name No. Location

Camera. • • •

Date

Exposure

Obiective

Developer

Ocular

Fixer

Condenser • •

Mag. x

Remarks

Object stained with

plate

PROJECTION APPARATUS FOR PHOTOGRAPHIC ENLARGEMENTS

§ 484. Enlarged prints of small negatives. — There is great
advantage, in making pictures of large objects at a considerable
distance with a long-focus objective, so that the perspective will be
correct and all levels of the object will be in good focus. It is also
advantageous to make pictures of microscopic objects without undue
enlargement; , then there is greater sharpness of the object as a
whole.

If now one wishes a large print, any good negative can be used
and a print obtained of almost any desired enlargement by using a
photographic objective for projecting the image upon the photo-
graphic paper. This is done with projection apparatus in a dark

CH. X3 PHOTOGRAPHY 389

room as follows: The management of the projection apparatus is as
for drawing. The negative is placed in some kind of holder and
put in the cone of light of the main condenser where the part of it
to be enlarged is fully illuminated. An erect image will be printed
on the paper if the film side of the negative faces the sensitive paper
exactly as for contact printing. Of course, if it is desired to reverse
the position, it can be done by turning the film side toward the
source of light.

§ 485. Size of condenser required. — The general law is that the
diameter of the condenser must be equal to or somewhat greater
than the diagonal of the negative or part of the negative to be
enlarged. For example, to enlarge the whole of a lantern slide
negative (85 X 100 mm.), the condenser should have a diameter of
14 cm. For a negative 100 X 125 mm. the condenser should be 18
cm. in diameter; for one 125 x 175 mm. the condenser should be 23
cm. in diameter; and for a negative 200 x 250 mm. the condenser
should be 35 cm. in diameter.

§ 486. Objectives to use for enlarging. — It is necessary to
use an objective which has been corrected for photography. The
ordinary projection objective gives a good visual image, but not a
good photographic image. The iris diaphragm must be wide open

(§ 487).

In preparing for printing, which, of course, is done in a dark
room, put some white paper in a printing frame with a clear glass
in it. Hold it in the path of the beam from the projection appara-
tus, and either by moving a support near the apparatus, or by
moving the projection apparatus, get the desired size of picture.
One can determine the exact magnification by putting a lantern
slide of the metric scale (fig. 173) in place of the negative and
projecting its image upon the white paper in the printing frame.

§ 487. Focusing and printing. — Focus the image of the negative
as sharply as possible. Then put over the end of the objective a
cover of some kind with ruby glass in it. This will allow the light
to pass in part, but it will not affect the photographic paper to be used.

Place in the printing frame some developing paper like vitava
rapid black or velox. Place the printing frame in position. The

3QO PHOTOGRAPHY [Cn. X

image will show clearly on the paper by the red light. When the
frame is in the exact position desired, remove the cap with ruby
glass and make the exposure. With an arc light the time will vary
from about i to 10 seconds, depending on the density of the nega-
tive. Cover the objective, turn off the arc lamp and develop the
print as for contact printed pictures. A mazda lamp may be used
instead of an arc light for enlarging. If the rather large source of
light in the no-volt lamp is used, a diffuser of ground glass is
needed to avoid the shadows between the filaments. When a dif-
fuser is used with the mazda or arc light, the diaphragm of the
objective can be closed as much as desired, but of course it then
takes a much longer exposure. If now one uses a 6-volt mazda
head-light lamp by inserting a transformer in the circuit for the
alternating current, or by using a storage battery for the direct
current, the filament is so concentrated that the source may be
treated like that of an arc light, and no diffuser used. This makes
it possible to use the full opening of the objective. The candle
power of the 6-volt mazda is much less than that of the arc light,
but it has the advantage of requiring no attention after being once
centered (figs. 79-82).

§ 488. Printing the image of an object directly on the paper. —
With the apparatus set up exactly as for drawing or for printing
enlargements, one can expose the developing photographic paper to
the sharply focused image of the specimen. Of course this will give
a negative image, all the lights and shades being reversed, but the
outlines and proportions are perfect. Such pictures serve as useful
a purpose as shade-correct pictures for model making and for keep-
ing a record of one's specimens.

PHOTOGRAPHIC REPRESENTATION OF VISUAL APPEARANCES;
PANCHROMATIC PHOTOGRAPHY WITH COLOR SCREENS

§ 489, Five methods of rendering objects visible. —

(i) The mounting medium and the object must have different
refractive indices, then the outline of the object or of its details are
margined by dark borders (§ 152, refraction images).

CH. X] PHOTOGRAPHY 3^1

(2) The object or its details must have a different color from the
surrounding medium or neighboring objects (color images, § 154).

(3) The object or its details must appear self-luminous, the sur-
rounding field being dark (method of dark-ground illumination or
ultra-violet radiation with resulting fluorescence).

(4) If reflected light is used, some parts of the object must absorb
the light and some parts reflect it; the different parts will then ap-
pear as light and dark.

(5) If transmitted light is used, some parts of the object must be
transparent or translucent and other parts opaque. The opaque
parts will then appear dark, and the transparent or translucent parts
light.

Two, four and five might properly be called absorption images.

§ 490. Photography is admirably adapted to represent the visual
appearances of both naked eye and microscopic objects. There
is only one difficulty which is really serious, and that is in the proper
representation in black and white of the various colors.

This difficulty is inherent in the sensitiveness of the eye to colors
and the unlike sensitiveness of the photographic plate to the same
colors. If both were equally and similarly sensitive, then the photo-
graphic representation of color in shades or tones of black and white
would have the same brightness as the different colors to the eye.
But the eye has its maximum sensitiveness in the green (fig. 210),
while the photographic plate has almost all of its sensitiveness in
the violet-blue end of the spectrum. Indeed it is sensitive to a part
of the ultra violet which is wholly dark to the eye. Hence the
photograph represents the brilliant red-orange-yellow-green image
seen by the eye as dark, while the relatively dark violet-blue to the
eye is rendered white by the photographic plate. The photographic
image of colored objects is then a kind of negative of the same
image to the eye. This has made the use of photography unsatis-
factory where objects have color, and most objects in nature are
colored more or less; and one of the greatest triumphs of micro-
scopic science has been the differentiation of details of structure by
selective staining.

From the earliest history of photography the inability to render

392

PHOTOGRAPHY

[CH. X

the colors properly or in actual colors has been greatly deplored.
To give the proper brightness in tones of black and white to colored
objects, two things had to be attained:

(i) The photographic plates, which were originally sensitive only
to the violet-blue end of the spectrum, had to be rendered sensitive
to the other colors. The first step was in getting plates sensitive to
the spectrum as far as the yellow. These are the so-called isochro-
matic or orthorhromatic plates. The final step was to get plates
sensitive to all the colors of the spectrum, including the orange and
red. These are known as panchromatic or spectrum plates.

I i

:> Violet-Blue

= L_- -^

!/"~\; i

j/+- (\K« 1

B oi IGY! ^s'x">-— — L_

X0.5M

FIG. 210. SENSITIVENESS OF THE EYE TO THE SPECTRUM WITH MODERATE

ILLUMINATION.

(Base Lines * Wave lengths x 250,000 times).

As shown in this curve the normal human eye with moderate illumination
has its maximum sensitiveness at about wave length Xo.55/x, that is, in the
green next the yellow. With very brilliant light the greatest sensitiveness is in
the yellow, while with dim light it moves along well into the green; (See § 288
for designation of wave lengths in microns, etc.).

Ultra-violet Short radiation invisible to the eye. Compare the sensitiveness
of the photographic plate to this radiation (fig. 211-213).

Violet-blue Radiation at the blue end of the spectrum.

Green Radiation in the middle of the spectrum.

Red Radiation at the red end of the spectrum.

Infra-red Long radiation invisible to the eye.

G Y Borderland between green and yellow.

B G Borderland between blue and green.

(2) But as all of these color-sensitive plates are more sensitive to
the violet-blue than to the other colors, it is necessary to use some
means for reducing or blocking out part of the violet-blue light with-
out interfering with the action of the other colors (§ 492). For
gaining contrast effects it was necessary to devise means for blocking
out special parts of the spectrum (§ 493). These selecting media
are known as color screens or ray niters.

CH. X]

PHOTOGRAPHY

393

Green

Red

|GY|

XO.4,1

X0.6M

FIG.

211. NORMAL SPECTRUM SHOWING THE SENSITIVENESS OF ORDINARY

PHOTOGRAPHIC PLATES.
(After Mees; and magnified as in fig. 210).

As shown in this curve, the ordinary photographic plate is sensitive only
in the blue end of the spectrum including the ultra-violet, the maximum sensi-
tiveness being at about wave length Xo.45^. It *is insensitive to all wave lengths
longer than about Xo.52/z. (Compare with fig. 210, 212-213).

FIG. 212. NORMAL SPECTRUM SHOWING THE SENSITIVENESS OF ORTHOCHROMATIC

OR 1SOCHROMATIC PLATES.
(After Mees; magnification as in fig. 210).

These plates have practically the same sensitiveness as the ordinary plates
except that the sensitiveness is continued through the green and yellow. (Com-
pare figs. 210, 2n and 213).

COLOR SCREENS OR RAY FILTERS

§ 491. Color screens or ray filters. — These are transparent,
colored bodies which select the wave lengths of light which they
transmit and absorb the other waves, or they diminish more or less
some of the wave lengths and transmit the others with very slight
loss. The color of such a screen to the eye will be determined by the
light which it transmits in the greatest quantity. For example, if
the violet-blue light is absorbed, the remaining light will appear
yellow, while if green and red are absorbed the transmitted light will
appear blue; if violet-blue and green are absorbed, the light will
appear red; and if violet-blue and red are largely absorbed; the
remaining light will appear green.

394

PHOTOGRAPHY

[CH. X

FIG. 213, NORMAL SPECTRUM SHOWING THE SENSITIVENESS OF PANCHROMATIC

PLATES.

(After Mees; magnification as in fig. 210).

Panchromatic plates have the maximum sensitiveness still in the violet-blue,
but it is extended to include the red. (Compare figs. 210-212).

§ 492. Compensating ray niters. — These are filters or screens
which aid the panchromatic photographic plate in giving a black
and white 'picture of colored objects which shall correspond in
brightness to the different colors as seen by the eye.

As all photographic plates, even the panchromatic ones, are more
sensitive to the violet-blue than to the other colors of the spectrum
(fig. 213), the effect of the violet-blue must be reduced, hence yellow
screens must be used to do this and compensate for the smaller sen-
sitiveness of the plate for the other parts of the spectrum.

Fortunately the great photographic manufacturers have made a
study of the principles of color screens as well as of their plates, and
they supply workers with data showing what wave lengths of light
their different plates are sensitive to, and what wave lengths are
absorbed wholly or in part by their ray filters. They also give
advice from abundant experience as to the proper combination of
plate and color screen to get the best effect in photographing a great
variety of colored objects. By using this information, and profiting by
experience, one can learn to photograph almost any object successfully.

§ 493. Contrast ray filters. — These are filters or screens by the
aid of which strong contrasts in black and white are given to various
colored objects or their details. As given in the general statement
of the basis for visibility of objects and their details, refraction and
opacity are of prime importance for securing sharp outlines. Color
images are also of the greatest advantage in differentiating the
details of microscopic structure; but as color does not appear in the

CH. X] PHOTOGRAPHY 395

ordinary photograph, the differentiation of colored objects must be
secured by producing shades of light and dark up to complete black-
ness in some cases. For example, in some microscopic specimens
important details may be stained violet or blue. To the eye these
violet or blue objects stand out with great clearness. In the
photograph, on the other hand, without special help from a color
screen, they are wholly lost or are so faint that they can hardly be
seen. To make such details stand out in shades of black, a yellow
color screen absorbing violet-blue and allowing the other colors to
pass is used with a plate sensitive to the other colors to be photo-
graphed. A picture is thus obtained which shows the violet-blue
objects in black and the other details in various shades.

A contrast color screen does not, of course, give correct brightness,
but the purpose in using it is to bring out in the most striking man-
ner the form of certain structures. The general law is: For con-
trast effects, use a color screen which absorbs the light transmitted
normally by the colored object, but allows the other colors to pass.

§ 494. Refraction and opacity and color screens. — It should
not be forgotten in using color screens and color-sensitive plates that
refraction and opacity exert their full effect in producing the final
result. The color screen acts only to suppress or lessen certain
definite wave lengths. Refraction and opacity tend to suppress all
wave lengths in certain limited borders or definite areas. Hence any
stain like hematoxylin which tends to make an object more opaque
to all parts of the spectrum will increase the contrast even if no
color screen is used.

§ 495. Lessening contrast. — With some specimens it is necessary
to lessen contrast in order to bring out details of structure. One
of the striking examples frequently referred to is whalebone. A
microscopic section of this has a reddish appearance by transmitted
light. If now a blue screen is used with a panchromatic plate, the
greatest possible contrast is obtained, and the object loses all detail
in the photograph. If, on the other hand, a red screen is used, the
photograph shows good detail and the general appearance is like
that seen by the eye in looking into the microscope.

The general law is: When the contrast is too great, use a color

396 PHOTOGRAPHY [Cn. X

screen of the same color as the object, and, of course, a plate must
be used sensitive to that color.

§ 496. Use of the micro-spectroscope in photo-micrography. — If
one studies his specimens with the micro-spectroscope and makes sure
exactly what light is transmitted by them, it will be possible to
judge with intelligence what plate and what color screen to use to
bring out in the most satisfactory manner their structural appearances.
Fortunately the manufacturers furnish the information concerning
their plates and the color filters, so that labor is spared the indi-
vidual worker. It might be worth while for him to check up the color
screens occasionally to make sure that they have not deteriorated.

§ 497. Time of exposure for photo-micrographs. — This varies from
the fraction of a second to several minutes, depending on four factors:

(1) The nature and intensity of the light.

(2) The magnification of the microscope. The higher the mag-
nification, the longer must be the exposure.

(3) The transparency of the specimen. The more transparent,
the shorter the exposures.

(4) The thicker or deeper the color of the ray filter, the longer
must be the exposure.

(5) Red stains require longer exposure than blue stains.

§ 498. Daylight. — This has served for some of the best photo-micrographs
that have ever been made. Its great defect is that it is not always available.

§ 499. Artificial lights. — Kerosene (petroleum) and now the electric lights
(arc light, incandescent lamp) are most used. The io8-watt, ribbon-filament
lamp is most convenient and suitable (§ 198).

The excess intensity in the red end of the kerosense light, and to a less degree
of the electric illuminants, serves in part at least to compensate for the greater
sensitivity of most photographic media to the violet-blue end of the spectrum.
(See figures 211-213.)

For many purposes the line spectra of the high pressure, capillary mercury
arc are of advantage, especially the blue, green and orange wave-lengths which
with suitable screens or filters can be used separately or in combination, depend-
ing on the character of the object to be photographed (fig. 129 A).

Ultra-violet and infra-red radiations are also coming to have a prominent
place in photo-micrography and in general photography. See especially the
Kodak manuals in the collateral reading.

§ 500. Mutual adaptation of color screen and light. — As the
color screen is for a very definite purpose in absorbing certain parts

CH. X] PHOTOGRAPHY 397

of the light, it follows that the character of the light and that of the
color screen must be mutually adapted. For example, it is self-evi-
dent that the same color screen for a given preparation would not
serve for both daylight and the light from a mazda lamp (see fig.
45). So also the same color screen would not be successful if
used both for the mazda light and for the light of a kerosene flame.

For the most successful use of color screens and different light
sources, one should have curves of the intensity of the light in dif-
ferent parts of the visible spectrum like that for the rnazda lamp and
sunlight (fig. 45). Then one should know the absorption by each
color filter for each kind of light. Knowing these facts and the
absorbing and transmitting qualities of his specimens, and the sensi-
tiveness of the photographic plates used, one could make intelligent
selections and reasonably expect good results.

§ 501. Exposure with color screens. — The color screen naturally
increases the time of exposure. It depends on the color and density
of the screen. In general the exposure is increased from 2 to 5
times. The increase necessary is usually given by the manufacturers,
therefore each individual worker does not have to find out by
experiment. There is plenty of opportunity for the use of his
judgment with the different qualities of his specimens (§ 49?) •

§ 502. Developers. — It is best to use the developers recom-
mended by the manufacturers of the plates used. The experts
employed by the manufacturers have found the best means for
developing the plates, and it is safe to follow their advice. One
usually has a choice of developers; and as a general statement it
should be said that the beginner would be wise to prefer a slow
developer, for it allows a greater latitude than a rapid developer.
In general, a developer containing much bromide works slowly and
gives very strong contrasts. Sometimes this is desirable, but often
it is better to get the soft effects that come with a small amount of
bromide. If one studies the little manuals sent out by the manu-
facturers, there will be found formulae which give the various
effects desired. (See collateral reading suggested at the end of the
chapter.)

§ 503. Light to develop by. — The light which can be used in the

398

PHOTOGRAPHY

[CH. X

dark room depends upon the sensitiveness of the plates or the print-
ing paper used. The more sensitive the plates or paper, the less
light. Furthermore, the sensitiveness to the different wave lengths
is also important to consider. If the plates are sensitive only to the
violet-blue of the spectrum, the dark room can be quite brightly
lighted with red light with entire safety. If isochromatic or ortho-
chromatic plates are used, they are sensitive to the spectrum up to
and including yellow, and hence the dark-room light must exclude
those, or be red only.
For panchromatic plates which are sensitive to all wave lengths

FIG. 214. DARK ROOM TOR PHOTOGRAPHY AND DRAWING IN A LARGE ROOM.
(From Optic Projection).

the only safe method is to develop in total darkness, for any light
will fog the plate if it acts sufficiently upon it. Sometimes very
dark green is used, for the eye is most sensitive to green if the light
is very dim, although for bright light the eye is most sensitive to
yellow. But to be able to see clearly enough to determine the stage
of development by the green light dim enough to be safe, one must
be in the dark room for half an hour or more. The total darkness
method is safest. One learns rather quickly to work in total dark-
ness, and the time during which development goes on can be deter-
mined by counting seconds, or by a signal clock ringing minutes or
by an alarm clock which can be set at the beginning for the esti-
mated time to be used. Or finally, one can develop in a tray
which is covered so that no light can reach the plate; then the
ordinary dark-room light can be turned on from time to time to see
when the estimated period for development has been reached.

CH. X] PHOTOGRAPHY 399

It is far safer to use too little light for developing rather than too
much. For ordinary or for isochromatic plates only a brief glance
occasionally is all that is needed. If one holds the plate in the dark-
room light during the whole development or for a considerable time
there is almost always a thin veil of fog which lessens the crispness
of the picture.

The wisdom of the advice to develop isochromatic or ordinary
plates with as small an exposure to the dark-room light as possible
can be demonstrated by the beginner in the following experiment
which he is advised to try.

Put an isochromatic or orthochromatic plate in the plate holder.
Pull out the dark slide till one or two centimeters of the film is ex-
posed, then leave this for half a minute close to the developing-room
light. Pull out the slide another centimeter or two and expose
again to the dark-room light. Continue till the entire plate has been
exposed. The List segment will have an exposure of half a minute,
next to the last a whole minute, and so on. Now develop the pic-
ture in the ordinary way and the chances are that the plate will
show very marked light effects, and the different segments in pro-
portion to the time they were exposed to the dark-room light.

§ 504. Time development. — Assuming that the correct plate
and color screen are used, careful experiments made in the scientific
laboratories of the large plate manufacturers have shown that the
best method of developing photographic negatives is that of devel-
oping a definite time at a definite temperature of the developer.
The time and temperature must, of course, be determined for the
special plate and composition of developer to be used. The variable
then is the exposure of the plate. A perfectly timed plate will con-
tain all the desired detail in the shadows and just sufficient density
in the high lights so that the print will be sufficiently white. The
deepest shadows in such a negative will be almost perfectly trans-
parent.

A convenient and safe method of developing plates by the time
method without having the room absolutely dark and without expos-
ing the plate to any harmful light, is the following: The dark-room
saf elight is directed away from the developing tray and a shield put

400 PHOTOGRAPHY [Cn. X

in position to further screen it. An alarm or other large-faced clock,
with second hand, is put close to the safelight. This light may then
be very dim and still illuminate the clock face sufficiently. If using
isochromatic or orthochromatic plates, the red safelight is good;
but if panchromatic or spectrum plates are used, the green safelight
is better. The exceedingly minute amount of light reaching the
plate from the safelight as here recommended can cause no damage
(Henry Phelps Gage, Optical Department, Corning Glass Works).

§505. Choice of plates* and color screens. — The hints given
in the little manuals sent out by the manufacturers on request by
their patrons give excellent hints for the selection of plates and color
screens for a wide variety of objects. The beginner cannot do better
than to follow those suggestions faithfully, until his own experience
enables him to supplement those suggestions. Finally, of course,
one wishes to be able to use his own judgment.

In general, if any color is present in the object to be photo-
graphed, one will have better success with isochromatic or ortho-
chromatic plates, which are sensitive to violet-blue, green, and
yellow, than with the ordinary plates, which are sensitive only to
the violet-blue of the spectrum (figs. 211-212). If the colors in-
volved contain orange and red, the isochromatic plates are not
adequate, and one must then use panchromatic or spectrum plates,
sensitive to all wave lengths (fig. 213).

For the color screen to employ, remember that color screens are
not of real use for ordinary plates sensitive only to violet and blue.
For isochromatic plates yellow color screens are very helpful for
reducing the excessive effect of the violet and blue (§492) or for cut-
ting them out altogether in getting contrast effects (§ 493). The
same is true for panchromatic plates, only here a wider range of
color screens can be used to get any desired contrast or compen-
sating effect.

COLOR PHOTOGRAPHY

§ 506. Photographs in natural colors. — This has been the aim
of experts in photography ever since its first invention. Lately
methods have been devised by which surprisingly true color photo-

CH. X] PHOTOGRAPHY 401

graphs have been produced. These color pictures are better adapted
to large objects than to those with fine details such as are observed
with the microscope. Still, many objects are fairly well represented
in photo-micrographs.

The author's experience in color photography has been limited
to the " Autochrome Process " (colored starch grain process). The
directions in the small manual sent out with the plates are very
clear. Any one familiar with the ordinary photographic processes
can succeed in color photography. It may be said in passing that
the pictures taken by this process are transparencies and must be
looked at as such to bring out the colors. Furthermore, as colors
are truly rendered only in daylight or by artificial daylight, these
transparencies must be illuminated by natural or artificial daylight
for a true rendering of the color.

While these pictures cannot be used as negatives to give paper
prints in colors, they can be used as colored pictures to get the
proper negatives for printing by the three-color process, so that with
a good autochrome transparency, colored pictures for books and
magazines can be produced without any hand being taken in the
process by an artist; and for many things the transparency gives a
truth and delicacy in coloring not attainable by the artist's brush.

§ 507. Photography with ultra-violet radiation. — As the finest
details of structure are more clearly brought out by the shorter
wave lengths, it has been hoped for a long time that it would be
finally possible to utilize the ultra-violet rays in photography, if
not in vision.

As shown in the chapter on the ultra-violet microscope, quartz
or other ultra-violet transmitting substance must be used for the
reflector, the condenser and the slip for supporting the specimen.
If one is to make photographs by the shorter ultra-violet wave
lengths the cover-slip, • the objectives and the oculars must also
be of ultra-violet transmitting material like quartz, corex, etc.
These materials are expensive, and it requires a high degree of skill
on the part of the operator to manage the source of radiation and,
indeed, the entire instrument. In spite of the difficulties, the
promise of a fuller understanding of structure has spurred men on,

402 PHOTOGRAPHY [Cn. X

and good results have already been attained. Promise of still
greater results is bringing out new means and methods constantly.
It may be remarked in passing, that with the apochromatic objec-
tives, good photographs may be taken with radiation of wave length
as short as 365 m/z (.0365^) (3650 A).

COLLATERAL READING FOR CHAPTER X
BECK, CONRAD. — The Microscope, 1938 ed , p. 197+ ,

DR. AUGUST KOHLER. — Eine mikrophotographische Einrichtung fur ultravio-
lettes Licht (275 m/i) und damit angestellte Untersuchungen organischer
Gewebe. Physikalische Zeitschrift, 5 Jahrgang, pp. 666-673. Four text
figures of apparatus.

Mikrophotographische Untersuchungen mit ultraviolettem Licht. Zeit-
schrift fur wissenschaftliche Mikroskopie und fur mikroskopische Technik.
Band XXI, 1904, pp. 129-165, und 273-304, six plates.

ERNST, HAROLD C., M. D. AND WOLBACH, S. B., M. D. — Ultra- Violet Photo-
micrography. The Journal of Medical Research, Vol. XIV, (N. S. vol. ix.
No. 3) pp. 463-469, April, 1906, seven plates.

LUCAS, FRANCIS F. (Bell Telephone Laboratories). — The Architecture of Living
Celts. A discussion of recent advances in methods of biological research by
means of optical sectioning with the ultra-violet microscope. Proceedings
of the National Academy of Sciences, Vol. 16, pp. 599-607, Sept. 1930. 6
plates, 5 text figures.

LUCAS AND STARK. — Jour. Morph., vol. 52, 1931, pp. 91-113. Many photo-micro-
graphs by ultra-violet.

MARTIN, L. C. — Some recent developments in Microscopy. Journal of the
Royal Society of Arts, Vol. 79, 1931, pp. 871-885; 887-896. Polarizing and
Ultra- Violet Microscopes.

Optic Projection, by S. H. & H. P. Gage.

The Wratten Booklets on Photographic Plates and Color Filters.

The Photography of Colored Objects, by C. E. Kenneth Mees.

Photo-micrography. Published by the Eastman Kodak Co.

Seed Plates, formulie and directions. Eastman Kodak Co.
Furnished by the G. Cramer Dry Plate Company:

Cramer's Manual on Negative Making and Formulas.

Isochromatic Landscape Photography.

The Photographing of Color Contrasts.

Dry Plates and Filters for Trichromatic Work.

Photo-micrographic and Spectrographic Color Filters.

These brochures are naturally very recent and give the meat of the informa-
tion at present available on the kind of photographic plates available and the
proper color niters to use with them to produce the best effects with different
colored objects in gross photography and in photo-micrography.

For the sensitiveness of the human eye to the different parts of the spectrum
see: Herbert E. Ives, Philosophical Magazine, Vol. XXIV, 6th ser. Dec. 1912,
pp. 853-863; P. G. Nutting, Transaction of the Illuminating Engineering So-
ciety, 1914, pp. 633-642.

Photo- technique. — New York, 1940 -f .

CHAPTER XI

CABINETS; SLIPS AND COVER-GLASSES; MOUNTING; LABELING

AND STORING MICROSCOPIC PREPARATIONS; REAGENTS

§§508-616; FIGURES 216-249

§ 608. Slides, glass slides or slips, microscopic slides or slips. —
These are strips of clear, flat glass quartz or corex upon which
microscopic specimens are usually mounted for preservation and
ready examination. The size that has been almost universally
adopted for ordinary preparations is 25 X 76 millimeters (i X 3
inches). For rock sections, slides 25 x 45 mm. or 32 X 32 mm. are
used; for serial sections, slides 25 x 76 mm., 38 X 76 mm. or
50 X 76 mm. are used.

For the ultra-violet microscope the slips must be transparent to
the ultra-violet radiation. Quartz is best. The Corex 1). glass of
the Corning Glass Works is also good and much less expensive

As these quartz and corex slips look like glass, it seems to the
author that a different size should be used, therefore he has adopted
that of 25 x 65 mm. (i x 2.6 inches). Tt is also desirable to put
the name on one end with a writing diamond (fig. 218).

For special purposes, glass slips of the necessary size are employed
without regard to any conventional standard.

Thick slips are preferred by many to thin ones. They should
correspond in thickness with the working distance of the condenser
with which one works, especially if that is of the achromatic-apla-
natic type. Dr. Chamot recommends that they be of half length for
chemical work. He adds further: " It is a great misfortune tha£ the
colorless glass slips used in America and so excellent for ordinary
microscopic work should be easily attacked by all liquids; even
water extracts a relatively enormous amount of alkalies and alkaline
earths. The slips of greenish glass, while not as neat or desirable
for general microscopy, seem to be decidedly more resistant, and are

403

404

CABINETS; SLIPS AND COVERS; MOUNTING [Cn. XI

therefore preferable." Transparent celluloid slides are recommended
by Behrens for work where hydrofluoric acid and its derivatives

B £

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are to be examined. (Chamot, Jour. Appl. Micr., vol. iii, p. 793.
Chemical Microscopy, pp. 123-124).

CH. XI] CABINETS; SLIPS AND COVERS; MOUNTING

405

§ 509. Thickness of slips for special purposes. — It is very im-
portant to observe strictly the requirements for the thickness of
slip for special purposes. As pointed out in discussing the dark-
ground condenser (§ 194), the slip must be thin enough so that the
focus of the condenser will be just above the upper surface where
the object is mounted. If the slip is too thick, the focus will be
beneath the object and the best light cannot be obtained. So
likewise with the best achromatic condensers, especially when used
as homogeneous immersion condensers (§ 265), if the slip is too
thick, the focus of the condenser will fall below the object and the
best and most critical images cannot be obtained.

It is better to use a slip thinner than the maximum permissible,
and plenty of homogeneous liquid between the slip and the con-
denser, then the condenser can be lowered until its focus is upon
the object. This applies equally with the dark-ground condenser.
For getting the thickness of the slips, use the micrometer calipers or
a cover-glass measurer (figs. 219-220).

Co rex

$
i

I'i

FIG. 218. COREX GLASS SLIP
25 x 65 mm.

These are transparent to ultra-violet, and should be used whenever the
specimens are to be examined under the ultra-violet microscope.

§ 510. Cleaning slips for ordinary use. — Place new slips that
are to be wiped at once in a glass vessel of distilled water contain-
ing 5% ammonia. For wiping the slips use a lintless or a well-
washed linen towel. One may avoid large wash bills by using
absorbent gauze (§ 5ioa).

In handling the slips grasp them by the edges. Cover the ringers
of the right hand with the wiping towel or the gauze and rub both

406 CABINETS; SLIPS AND COVERS; MOUNTING [Cn. XI

faces with it. When the slide is wiped thoroughly dry, place it in a
dry glass jar or for larger numbers use a museum jar (fig. 248).
Soap and water are also recommended for new slips.

Alcohol of 50% to 82% is also excellent for cleaning new slips,
and for those which have been freed from mounting media by boil-
ing (§ 511) after a thorough rinsing in clean water.

§ 610a. Absorbent gauze and lintless towels. — The gauze mentioned is
No. 10, sterilized absorbent gauze. It is sometimes called bleached cheese cloth.
In the author's laboratory it is cut into pieces, i, J, /# °f a yafd. When a piece
is soiled, it is thrown away. There has recently appeared specially prepared
towels for wiping glass, etc,, which are called "lintless," as practically no lint is
left on the wiped object. These are f urnishe 1 by Johnson & Johnson of New
York, and cost about 15 cents each in a size 42 X yo cm.

§ 511. Cleaning used slips. — If only watery substances or gly-
cerin or glycerin jelly have been used, one may soak the slips over-
night in ammonia water, then change the water for fresh and wipe
as described in § 510.

When balsam or other resinous media (§ 564) have been used, it is
best to heat the slips over a Bunsen flame and remove the cover-
glass. Place the covers in cleaning mixture (§ 519). The slip may
also be placed in cleaning mixture or in some hot water containing
10% gold dust or other strong alkaline cleaner. When the metal
basin — preferably an agateware basin — is two-thirds full of the
slips, heat until the water comes to a boil. Then let it cool. Add
fresh water and most of the slips may be wiped clean.

If dichromate cleaning mixture is used, the best method is to have
a museum jar of it and drop the slips in as they are rejected, or a
large number at once, as is most convenient. It may require a week
or more to clean the slips with cleaning mixture. As this is a very
corrosive mixture for metals, use only glass dishes in dipping into
it. When the slips are freed from balsam, etc., pour off the clean-
ing mixture into another glass vessel and allow a stream of water to
flow over the slips until all the cleaning mixture has been washed
away. Then add water and wipe the slips from that. Any slips
still not freed from the balsam should be put back into the cleaning
mixture.

§ 512. Cleaning slips for special uses. — In making blood films,

CH. XI] CABINETS; SLIPS AND COVERS; MOUNTING 407

for micro-chemistry and whenever an even film is desired, every
particle of oily substance must be removed, and every other foreign
substance. In a word the glass must be made thoroughly clean.
To accomplish this end the writer has found a slight modification
of the method of Stitt the most effective and convenient. (See
Stitt, " Practical Bacteriology, Blood Work and Animal Parasi-
tology," yth ed., 1923, p. 299). New slips or those that have been
cleaned as described in § 511, are placed one by one into a glass or
agateware dish containing an emulsion of bon ami. For the emul-
sion 5 grams of the bon ami powder is stirred up with 100 cc. of
water. The slips are stirred around in this emulsion and then taken
out one by one and set up on end on blotting paper or gauze to dry.
When thoroughly dry, they are placed in a box for future use.
Whenever a slip is needed, it is wiped well with a piece of fresh
white gauze or one of the lintless towels. As remarked by Stitt,
this is better than any other single method or all of the others com-
bined.

The gauze mentioned is of the heavier grade, white and ab-
sorbent. It has been used several years in our laboratories, and has
been found satisfactory and economical. For use a square yard
is cut into 16 equal pieces for cleaning and polishing glass slips.
For cover-glasses a square yard is cut into 64 equal pieces. In
taking blood samples one of these small pieces should be used but
once and then discarded.

The best way to tell when slips or cover-glasses are free from a
surface film is to drop some water upon the glass and then incline it
to a sloping position. If the glass surface is clean, the water will run
over the glass and leave a wet track. If a film of oily substance is
present, the water will crawl and form ridges or droplets and will not
leave a smooth wet surface. Sometimes it is almost impossible to
get a slip so that a film of blood or other substance can be spread
evenly upon it. Probably the simplest thing in such a case is to use
such a slip for mounting sections in balsam; but Chamot, pp. 149-
150, says that they may in many cases be made suitable by passing
them slowly through a Bunsen flame.

§ 513. Cover-glasses or covering glasses. — These are circular

408 CABINETS; SLIPS AND COVERS; MOUNTING [Cn. XI

or quadrangular pieces of thin glass used for covering and protecting
microscopic objects. They should be very thin, o.io to 0.25 milli-
meter. It is better never to use a cover-glass over 0.20 mm. thick,
then the preparation may be studied with a 2 mm. oil immersion as
well as with lower objectives. Except for objects wholly unsuited
for high powers, it is a great mistake to use cover-glasses thicker
than the working distance of a homogeneous objective (§ 101).
Indeed, if one wishes to employ high powers, the thicker the section
the thinner should be the cover-glass.

The cover-glass should always be considerably larger than the object
over which it is placed.

§ 514. Cleaning cover-glasses for ordinary use. — Covers may
be cleaned well by placing them in 82% or 95% alcohol containing-
hydrochloric acid one per cent. They may be wiped almost imme-
diately.

Remove a cover from the alcohol, grasping by the edge with the
left thumb and index. Cover the right thumb and index with some
clean gauze or other absorbent cloth; grasp the cover between the
thumb and index and rub the surfaces, keeping the thumb and
index well opposed on directly opposite faces of the cover so that no
strain will come on it, otherwise the cover is likely to be broken.

When a cover is dry hold it up and look through it toward some
dark object. The cover will be seen partly by transmitted and
partly by reflected light, and any cloudiness will be easily detected.
If the cover does not look clear, breathe on the faces and wipe
again. If it is not possible to get a cover clean in this way, it
should be put again into the cleaning mixture or thrown away.

As the covers are wiped put them in a clean shell-vial (fig. 227),
glass box or Petri dish. Handle them by their edges, or use fine
forceps. Do not put the fingers on the faces of the covers, for that
will surely cloud them.

§ 515. Cleaning cover-glasses for special uses. — As with glass
slips, cover-glasses intended for films or other purposes where the
least particles of oily substance or other foreign material must be
removed, are most satisfactorily cleaned by Stitt's bon ami method.
New cover-glasses or cleaned used ones are put into a bon ami

CH. XI] CABINETS; SLIPS AND COVERS; MOUNTING

409

FIG. 219.

BROWN & SHARPENS MICROMETER
CALIPERS.

emulsion, 5 grams to 100 cc. of water, in a shallow dish like a saucer
or plate and moved about somewhat.

They are then taken
out one by one and set
on edge against and
on clean blotting paper
or gauze exactly as
for slips (§512); or,
following Mt. Holyoke
College, a tray is
covered with clean
gauze and the covers
laid one by one upon
it. The tray is inclined to about 40 degrees and when the excess
liquid has run down to the lower edge of the covers, it is blotted

off. When dry, the covers are stored

in a glass box.
When ready to use a cover-glass,

wipe it with one of the small pieces of

gauze. If especially exacting, use a fresh
FIG. 22o; STARRETT'S PAPER- piece of gauze for each cover.

Ordinarily many cover-glasses and

slips are cleaned at one time and
stored for future use. If the preparations are to be mounted in
Canada balsam, this method answers fairly well, but it is not satis-
factory in dark-field microscopy. Experience also shows that even
when stored in glass receptacles, the cleaned covers and slips gradu-
ally accumulate a surface film which renders them unfit for even
balsam mounts unless they are recleaned. The cleaning is so rapid
and thorough by the Stitt bon ami method that even for mounting
series it is not a great burden to wipe the slips and covers as they
are needed.

§ 516. Cleaning large cover-glasses for serial sections. — These
large, quadrangular covers are put one by one in bon ami emulsion
and treated in every way like the glass slips and small cover-glasses.
§ 517. Measuring the thickness of cover-glasses. — It is de-

GAUGE

IPERS.

MICROMETER CAL-

410 CABINETS; SLIPS AND COVERS; MOUNTING [Cn. XI

sirable to know the thickness of the covers: for (a) in study-
ing the preparation one would not try to use objectives of a shorter
working distance than the thickness of the cover (§ 101); (b) in
using adjustable objectives with the collar graduated for different
thicknesses of cover, the collar can be set at a favorable point with-
out loss of time; (c) for unadjustable objectives the thickness of
cover may be selected corresponding to that for which the objective
was corrected (§ 254). Furthermore, if there is a variation from the
standard, one may remedy it, in part at least, by lengthening the
tube if the cover is thinner, and shortening it if the cover is thicker
than the standard (§ 256).

Among the so-called No. i cover-glasses of the dealers in micro-
scopical supplies, the writer has found covers varying from 0,10 mm.
to 0.35 mm. To use cover-glasses of so wide a variation in thickness
without knowing whether one has a thick or thin one is simply to
ignore the fundamental principles by which correct microscopic
images are obtained.

From information supplied by Mr. Edward Pennock the thickness
of various cover-glasses should be within the following limits:

No. i cover-glasses. ...0.12 to o. 18 mm.

No. 2 0.18 to 0.25 mm.

No. 3 o. 25 to o 50 mm.

No. o o. 10 mm. slightly more or less.

In general cover-glasses thinner than the minimum (0.12 mm.) of
No. i, actual measurement, will, as stated above, usually show a
much wider variation.

It is then strongly recommended that every preparation shall be
covered with a cover-glass whose thickness is known, and that this
thickness be indicated in some way on the label (fig. 234).

§ 518. Micrometer calipers for measuring glass slips and cover-
glasses. — The micrometer gauges in figs. 219-220 are satisfactory
for getting the thickness of slips and covers. The paper gauge
(fig. 220) is a little safer for cover-glasses as they are grasped by a
broader surface. These instruments may be had for the inch
standard or for the millimeter standard.

CH. XI] CABINETS; SLIPS AND COVERS; MOUNTING 411

With these measures or gauges one should be certain that the
index stands at zero when at rest. If the index does not stand at
zero, it should be adjusted at that point, otherwise the readings will
not be correct.

As the covers are measured, the different thicknesses should be
put into different glass boxes and properly labeled. Unless one is
striving for the most accurate possible results, cover-glasses varying
not more than 0.06 mm. may be put in the same box. For example,
if one takes 0.15 mm. as a standard, covers varying 0.03 mm. on
each side may be put into the same box. In this case the box would
contain covers of 0.12, 0.13, 0.14, 0.16, 0.16, 0.17, and 0.18 mm.

§ 519. Bichromate cleaning mixture for glass. — The cleaning
mixture used for cleaning slides and cover-glasses is that commonly
used in chemical laboratories: (Dr. G. C. CaldwelPs Laboratory
Guide in Chemistry.)

Dichromate of potash (K2Cr207) 200 grams

Water, distilled or ordinary (H20) 800 cc.

Sulphuric acid (H2SO4) 1200 cc.

As great heat is developed in the reaction on mixing the sulphuric
acid with the watery solution of dichromate, it is necessary to use
heat-resisting vessels. The best so far employed are those made of
pyrex glass. Use ordinary tap water and the commercial dichro-
mate and strong sulphuric acid. Chemically pure ingredients are
not demanded.

Dissolve the dichromate in the water by the aid of heat. Use for
this an agate dish. Now place the pyrex dish in the sink on some
asbestos or a piece of board. Pour the warm solution of dichro-
mate into the pyrex dish, and then add the sulphuric acid, stirring
the liquid with a glass rod. The reaction is so great that the liquid
will boil violently. An abundance of chromic acid crystals will form
as the sulphuric acid is added. Let the pyrex dish remain in the
sink until the cleaning mixture is cool and then pour it into a glass-
stoppered bottle for storage.

If the dichromate is well pulverized, it can be put directly into the
pyrex dish with the requisite amount of water, and the sulphuric
acid added as directed.

412 CABINETS; SLIPS AND COVERS; MOUNTING [Cn. XI

This is an excellent cleaning mixture and is practically odorless.
It is exceedingly corrosive and must be kept in glass vessels. It
may be used more than once, but when the color changes markedly
from that seen in the fresh mixture it should be thrown away. An
indefinite sojourn of the slides and covers in the cleaner does not
seem to injure them,

MOUNTING, AND PERMANENT PREPARATION OF MICROSCOPIC
OBJECTS

§ 620. Mounting a microscopic object is so arranging it upon
some suitable support and in some suitable mounting medium that
it may be satisfactorily studied with the microscope.

The cover-glass on a permanent preparation should always be con-
siderably larger than the object; and where several objects are put under
one cover-glass, as with serial sections, it may be confusing to crowd
them too closely together.

§ 621. Temporary mounting; normal fluids. — In a great many
cases objects do not need to be preserved; they are then mounted
in any way to enable one best to study them, and after the study
the cover-glass is removed, and the slide cleaned for future use.
In the study of living objects, of course only temporary preparations
are possible. With amoebae, white blood corpuscles, and many
other objects, both animal and vegetable, the living phenomena can
best be studied by mounting them in the natural medium. That is,
for amoebae, the water in which they are found; for the white
blood corpuscles, a drop of blood is used and, as the blood soon
coagulates, they are in the serum. Sometimes it is not easy or con-
venient to get the natural medium; then some liquid that has been
found to serve in place of the natural medium is used. For many
things, water with a little common salt (water 1000 cc., common
salt, NaCl, 8 grams) is employed. This is the so-called isotonic or
normal salt or saline solution. For the ciliated cells from frogs and
other amphibia, nothing has been found so good as human spittle.
Whatever is used, the object is put on the middle of the slide and a
drop of the mounting medium added, and then the cover-glass.

CH. XI] CABINETS; SLIPS AND COVERS; MOUNTING 413

The cover is best put on with fine forceps, as shown in fig. 221.
After the cover is in place, 'if the preparation is to be studied for
some time, it is better to avoid currents and evaporation by paint-
ing a ring of castor oil around the cover in such a way that part of
the ring will be on the slide and part on the cover (fig. 235).

It cannot be too strongly emphasized that if one is to study
living or fresh tissues, they must be mounted in a liquid which will
not injure them. The liquid in which they are naturally found is
of course the most nearly normal of any, and should be always used
when possible. Water seems a very bland and harmless liquid, but
it has a very decidedly injurious effect on living tissues which are
normally bathed by the
fluids of the body, for * /% \ \ yiy>e forCiJis

- , . . fi ^O I s 1 ' r 1

they always contain salts
and colloid material.

Distilled water is more ^ ^ FlNE FORCEPS FOR HANDLING COVER-
deleterious than tap water GLASSES AND OTHER DELICATE OBJECTS.
because it contains no

salts. It would be deleterious to water organisms, because all nat-
ural waters contain a greater or lesser quantity of organic and inor-
ganic substances in solution. If the water supply of a city or
town has a filtration plant, the water is likely to be unsuitable
for raising water forms like salamander embryos, and the em-
bryos of the frogs and toads, besides many other water forms.
One must take the trouble to get the water from the natural
breeding places if the embryos are to be successfully raised in a
laboratory. (See also §§ 542-543, 606.)

§ 522. Permanent mounting. — There are three great methods of
making permanent microscopic preparations. Special methods of
procedure are necessary to mount objects successfully in each
of these ways. The best mounting medium and the best method of
mounting in a given case can be determined only by experiment.
In most cases some previous observer has already made the neces-
sary experiments and furnished the desired information.

The three methods are the following:

(i) Dry or in air (§§ 523-526).

41 4 CABINETS; SLIPS AND COVERS; MOUNTING [Cn. XI

(2) In some medium miscible with water, as glycerin or glycerin

jelly (§§ 527-531)-

(3) In some medium like Canada balsam, damar, petrolatum, etc.

§ 623. Mounting dry or in air. — The object should be thor-
oughly dry. If any moisture remains, it is likely to cloud the cover-
glass, and the specimen may deteriorate. As the specimen must be
sealed, it is necessary to prepare a cell slightly deeper than the
object is thick. This is to support the cover-glass, and also to
prevent the running in by capillarity of the sealing mixture.

Order of procedure in mounting objects dry or in air.

1. A cell of some kind is prepared. It should be slightly deeper
than the object is thick (§ 525).

2. The object is thoroughly dried (desiccated) either in dry air or
by the aid of gentle heat.

3. If practicable, the object is mounted on the cover-glass; if
not, it is placed in the bottom of the cell.

4. The slide is warmed till the cement forming the cell wall is
somewhat sticky, or a very thin coat of fresh cement is added; the
cover is warmed and put on the cell and pressed down all around till
a shining ring indicates its adherence.

5. The cover-glass is sealed.

6. The slide is labeled.

7. The preparation is catalogued and safely stored.

§ 624. Example of mounting dry, or in air. — Prepare a shallow
cell and dry it (§ 525). Select a clean cover-glass slightly larger
than the cell. Pour upon the cover a drop of 10% solution of
salicylic acid in 95% alcohol. Let it dry spontaneously. Warm the
slide till the cement ring or cell is somewhat sticky; then warm
the cover gently and put it on the cell, crystals down. Press on the
cover all around the edge, seal, label and catalogue.

A preparation of mammalian red blood corpuscles may be made
satisfactorily by spreading a very thin layer of fresh blood on a
cover with the end of a slide. After it is dry, warm gently to re-

CH. XI] CABINETS; SLIPS AND COVERS; MOUNTING

415

move the last traces of moisture and mount blood side down, pre-
cisely as for the crystals. One can get the blood as directed for the
chylomicrons in dark-field work (§ 212).

§ 526. Preparation of mounting cells. — (A) Thin cells. These
are most conveniently made of some of the cements used in micros-
copy. Shellac is one of the best and most generally applicable. To
prepare a shellac cell place the slide on a turn-table (fig. 222) and
center it, that is, get the center of the slide over the center of the
turn-table. Select a guide ring on the turn-table which is a little
smaller than the cover-glass to be used, take the brush from the
shellac, being sure that there is not enough cement adhering to it

FIG. 222. TURN-TABLE FOR MAKING CELLS AND FOR SEALING COVER-GLASSES.

Hand Rest The metal plate supporting the hand that holds the brush. It can
be raised or lowered by means of the screw underneath (s).

sc Spring clips for holding the slide in place.

gc Guide circles to aid in centering the slide or the mounted object.

me Milled circular disc by which the turn-table is whirled when the ring of
cement is being painted around the cover-glass or the mounting cell.

to drop. Whirl the turn-table and hold the brush lightly on the
slide just over the guide ring selected. An even ring of cement
should result. If it is uneven, the cement is too thick or too thin,
or too much was on the brush. After a ring is thus prepared re-
move the slide and allow the cement to dry spontaneously, or heat
the slide in some way. Before the slide is used for mounting, the
cement should be so dry when it is cold that it does not dent when
the finger nail is applied to it.

A cell of considerable depth may be made with the shellac by
adding successive layers as the previous one dries.

4l6 CABINETS; SLIPS AND COVERS; MOUNTING [Cn. XI

(B) Deep cells are sometimes made by building up cement cells,
but more frequently, paper, wax, glass, hard rubber or some metal
is used for the main part of the cell. Paper rings, block tin or lead
rings are easily cut out with gun punches. These rings are fastened
to the slide by using some cement like the shellac.

§ 626. Sealing the cover-glass for dry objects mounted in cells. —
When the cover is in contact with the wall of cement all around
(§ 523)> ^e slide should be placed on the turn-table and arranged so
that the cover-glass and cell wall will be concentric with the guide
rings of the turn-table. Then the turn-table is whirled and a ring
of fresh cement is painted, half on the cover and half on the cell
wall (fig, 235). If the cover-glass is not in contact with the cell
wall at any point and the cell is shallow, there will be great danger
of the fresh cement running into the cell and injuring or spoiling the
preparation. When the cover-glass is properly sealed, the prepara-
tion is put in a safe place for the drying of the cement. It is
advisable to add a fresh coat of cement occasionally. Seal the
square and oblong covers freehand.

§ 627. Mounting objects in media miscible with water. — Many
objects are so greatly modified by drying that they must
be mounted in some medium other than air. In some cases water
with something in solution is used. Glycerin of various strengths
and glycerin jelly are also much employed. All these media keep
the object moist and therefore in a condition resembling the natural
one. The object is usually and properly treated with gradually
increasing strengths of glycerin or fixed by some fixing agent before
being permanently mounted in strong glycerin or either of the other
media.

In all of these different methods, unless glycerin of increasing
strengths has been used to prepare the tissue, the fixing agent is
w ished away with water before the object is finally and permanently
mounted in either of the media.

§ 628. Order of procedure in mounting objects in glycerin. —

i. A cell must be prepared on the slide if the object is of con-
siderable thickness (§ 525).

CH. XI] CABINETS; SLIPS AND COVERS; MOUNTING

417

2. A suitably prepared object is placed on the center of a clean
slide, and if no cell is required a centering card is used to facilitate
the centering (fig, 223).

FIG. 223. GUIDE CARD TO AID IN MOUNTING OBJECTS NEATLY.

3. A drop of pure glycerin is poured upon the object, or if a cell
is used, enough to fill the cell and a little more.

4. In putting on the cover-glass it is grasped with fine forceps
and the underside breathed on to moisten it slightly so that the
glycerin will adhere; then one edge of the cover is put on the cell
or slide and the cover gradually lowered upon the object. The cover
is then gently pressed down. If a cell is used, a fresh coat of cement
is added before mounting.

5. The cover-glass is sealed.

6. The slide is labeled.

7. The preparation is catalogued and safely stored.

§ 529. Order of procedure in mounting objects in glycerin jelly. —

1. Unless the object is quite thick, no cell is necessary with
glycerin jelly.

2. A slide is gently warmed and placed on the centering card
(fig. 223) and a drop of warmed glycerin jelly is put on its center.
The suitably prepared object is arranged in the center of the slide.

3. A drop of the warm glycerin jelly is then put on the object,
or if a cell is used, it is filled with the medium.

4l8 CABINETS; SLIPS AND COVERS; MOUNTING [CH. XI

4. The cover-glass is grasped with fine forceps, the lower side
breathed on and then gradually lowered upon the object and gently
pressed down.

FIG. 224. COREX GLASS SLIPS 25 x 65 MM.

The upper one shows the method of anchoring the cover-glass by means of
four drops of shellac. The lower one shows the method of irrigating a prepa-
ration. A drop of the solution is put on one side of the cover and a piece of
blotting paper on the opposite side. The arrow shows the direction of the flow
toward the blotting paper. As the irrigating liquid will be strongest or most abun-
dant in the middle, all stages of its action on the preparation may be seen on
the sides.

5. After mounting, the preparation is left flat in some cool place
till the glycerin jelly sets; then the superfluous amount is scraped
and wiped away and the cover-glass sealed with shellac (§ 530).

6. The slide is labeled.

7. The preparation is catalogued and safely stored.

§ 530. Sealing the cover-glass when no cell is used. — (A) For
glycerin-mounted specimens. The superfluous glycerin is wiped away
as carefully as possible with a moist cloth; then four minute drops
of cement are placed at the edge of the cover (fig. 224) and allowed

CH. XI] CABINETS; SLIPS AND COVERS; MOUNTING

419

to harden for half an hour or more. These will anchor the cover-
glass so that it can be sealed.

(B) For objects in glycerin jelly , Farrants' solution or a resinous
medium. The mounting medium us first allowed to harden; then
the superfluous medium is scraped away as much as possible with a
knife, and then removed with a cloth moistened with water for the
glycerin jelly and Farrants' solution; or
with alcohol, chloroform or turpentine,
etc., if a resinous medium is used. Then
the slide is put on a turn-table and a ring
of the shellac cement is added.

§ 531. Example of mounting in glycerin
jelly. — For this select some stained and
isolated muscular fibers or other suitably
prepared objects (§§ 537-541). Arrange
them on the middle of a slide, using the
centering card, and mount in glycerin jelly
as directed in § 529. Air bubbles are not
easily removed from glycerin jelly per-
parations, so care should be taken to
avoid them.

§ 532. Mounting objects in resinous
media. — While the media miscible with
water offer many advantages for mounting animal and vegetable tis-
sues, the preparations so maide are likely to deteriorate. In many
cases, also, they do not produce sufficient transparency to enable one
to use high enough powers for the demonstration of minute details.

By using sufficient care almost any tissue may be mounted in a
resinous medium and retain all its details of structure.

For the successful mounting of an object in a resinous medium it
must in some way be deprived of all water and all liquids not
miscible with the resinous mounting medium. There are two
methods of bringing this about: (A) by drying or desiccation
(§ 533), and (B) by successive displacements (§ 535).

§ 633. Order of procedure in mounting objects in resinous media
by desiccation:

Fro. 225. SMALL SPIRIT
LAMP USED AS A CON-
TAINER FOR GLYCERIN,
BALSAM, KTC.

420

CABINETS; SLIPS AND COVERS; MOUNTING [Cn. XI

1. The object suitable for the purpose (fly's wings, etc.) is thor-
oughly dried in dry air or by gentle heat.

2. The object is arranged as desired in the center of a clean slide
on the centering card (fig. 223).

3. A drop of the mounting medium is put directly upon the
object or spread on a cover-glass.

4. The cover-glass is put on the specimen with fine forceps (fig.
221), but in no case does one breathe on the cover as when media

miscible with water are used.

5. The cover-glass is pressed down gently.

6. The slide is labeled.

7. The preparation is catalogued and
safely stored (§ 548).

§ 534. Example of mounting in balsam by
desiccation. — Find a fresh fly, or, if in winter,
procure a dead one from a window sill or a
spider's web. Remove the fly's wings, being
especially careful to keep them the dorsal
side up. With a camel's hair brush remove
any dirt that may be clinging to them.
FIG. 226. CONTAINER Place a clean slide on the centering card,
FOR CANADA BALSAM, then with fine forceps put the two wings
GLYCERIN JKLLY, ETC, .., . r ,, . . T

Cover The glass cover within one of the guide rings. Leave one

to keep out dust and pre- dorsal side up, turn the other ventral side up.

vent evaporation. c , ~ . , . . f .

Rod The glass rod for Spread some C.anada balsam on the face of

transferring the contents the cover-glass and with the fine forceps place
of the container to the , . . te. \ •** *

slide. the cover upon the wings (fig. 221). Proba-

bly some air-bubbles will appear in the
preparation, but if the slide is put in a warm place these will
soon disappear. Label, catalogue, etc.

§ 635. Mounting in resinous media by a series of displacements.
— For examples of this see the procedure in the paraffin and in the
collodion methods, Ch. XII. The first step in the series in dehyrda-
tion; that is, the water is displaced by some liquid which is miscible
with both the water and the next liquid to be used. Strong alcohol
(9S% °r stronger) is usually employed for this. Plenty of it must be

JH. XI] CABINETS; SLIPS AND COVERS; MOUNTING 421

used to displace the last trace of water. The tissue may be soaked
in a dish of the alcohol, or alcohol from a pipette may be poured
upon it. Dehydration usually occurs in the thin objects to be
mounted in balsam in 5 to 15 minutes. If a dish of alcohol is used,
it must not be used too many times, as it loses in strength.

The second step is clearing. That is, some liquid which is miscible
with the alcohol and also with the resinous medium is used. This
liquid is highly refractive in most cases, and consequently this step
is called clearing and the liquid a clearer. The clearer displaces the
alcohol, and renders the object more or less translucent. In case the
water was not all removed, a cloudiness will appear in parts or over
the whole of the preparation. In this case the preparation must be
returned to alcohol to complete the dehydration.

One can tell when a specimen is properly cleared by holding it
over some dark object. If it is cleared, it can be seen only with
difficulty, as but little light is reflected from it. If it is held toward
the window, however, it will appear translucent.

The third and final step is the displacement of the clearer by the
resinous mounting medium.

The specimen is drained of clearer and allowed to stand for a
short time till there appears the first sign of dullness from evapo-
ration of the clearer from the surface. Then a drop of the resinous
medium is put on the object, and finally a cover-glass is placed over
it, or a drop of the mounting medium is spread on the cover and
it is then put on the object. For abundant examples see the next
chapter.

§ 536. Mounting in petrolatum liquidum, pure mineral oil. —
As this substance does not fluoresce, and is of nearly the refractive
index of glass it serves well for mounting unstained sections for the
ultra-microscope and also as an immersion liquid.

The unstained sections are freed from the solid paraffin as usual
(§ 638) and the oil added. No clearing is necessary. It is then
covered, and the cover-glass sealed with shellac or with ambroid or
other pyroxylin cement.

422

CABINETS; SLIPS AND COVERS; MOUNTING [Cn. XI

ISOLATION OF HISTOLOGIC ELEMENTS

§ 537. Isolation, general. — For a correct conception of the forms
of the cells and fibers of the various organs of the body, one must
see these elements isolated and thus be able to inspect them from
all sides. It frequently occurs also that the isolation is not quite
complete, and one can see in the clearest manner the relations of the
cells or fibers to one another.

The chemical agents or solutions for isolating are, in general, the
same as those used for hardening and fixing. But the solutions are
only about one-tenth as strong as for fixing, and the action is very
much shorter, that is, from one or two hours to as many days. In
the weak solution the cell cement or connective tissue is softened so
that the cells and fibers may be separated from one another, and at
the same time the cells are preserved. In fixing and hardening, on
the other hand, the cell cement, like the other parts of
the tissue, is made firmer. In preparing the isolating
solutions it is better to dilute the fixing agents with
normal salt solution than merely with water (§ 606).

FIG. 227, 228. SHELL VIAL AND COMSTOCK, BENT-NECK SPECIMEN BOTTLE.

Shell vial with turned lip. One can have almost any size and length desired.
Those of 22 X 65 mm. and 30 x 90 mm. have been found most useful. The
larger ones are excellent for staining single slides or pairs.

The Comstock, bent-neck specimen bottle is very useful for keeping small
animals straight,

§ 538. Isolation by means of formaldehyde. — Formaldehyde in
normal salt solution is one of the very best dissociating agents for

CH. XI] CABINETS; SLIPS AND COVERS; MOUNTING

423

ooo
ooo
ooo
ooo
ooo

brain tissue and all the forms of epithelium. It is prepared as
follows: 2 cc. of strong formalin (that is, a 40% solution of formal-
dehyde) are mixed with 1000 cc. of normal salt solution. This acts
quickly and preserves delicate structures like the cilia of ordinary
epithelia and also of the endymal cells of the
brain. It is satisfactory for isolating the nerve
cells of the brain. For the epithelium of the
trachea, intestines, etc., the action is suffi-
cient in half an hour; good preparations may
also be obtained any time within two days or
more. The action on nerve tissue of the brain
and myel or spinal cord is about as rapid.

§ 539. Staining the cells. — Almost any
stain may be used for the formalin dissociated
cells. For example, one may use eosin. This
may be drawn under the cover of the already
mounted preparation (fig. 224), or a new pre-
paration may be made and the scrapings
mixed with a drop of eosin before putting on
the coverglass. It is an advantage to study
unstained preparations, otherwise one might
obtain the erroneous opinion that the structure
cannot be seen unless it is stained. The stain makes the structural
features somewhat plainer; it also accentuates some features and
does not affect others so markedly. Congo red is excellent for most
isolated cells.

§ 540. Permanent preparations of isolated cells. — If one desires
to make a permanent preparation of isolated cells it may be done
by placing a drop of glycerin at the edge of the cover and allowing
it to diffuse under the cover, or the diffusion may be hurried by
using a piece of blotting paper, as shown in fig. 224. One may also
make a new preparation by mixing thoroughly some of the isolated
material with congo-glycerin. After a few minutes the cover-glass
may be put on and sealed (§ 530). If one adds congo-glycerin to a
considerable amount of the isolated material it may be kept and
used at any time.

FIG. 229. BLOCK WITH
HOLES FOR SHELL
VIALS.

The blocks are about
33 mm. thick and the
holes are bored clear
through, then a board
about 5 mm. thick is
nailed on the bottom.

424

CABINETS; SLIPS AND COVERS; MOUNTING [Cn. XI

§ 541. Isolation of muscular fibers. — For this the formalin disso-
ciator may be used (§ 538), but the nitric acid method is more suc-
cessful (§ 381). The fresh muscle is placed in this in a glass vessel.
At the ordinary temperature of a sitting room (20 degrees centi-

Fio. 230. MOIST CHAMBER AND MOIST PREPARATIONS.

A Bowl (B) inverted over a plate (P] containing water and a glass shelf
supported on glass rods. The slides (S) are supported on the glass shelf. This
makes a very efficient and cheap moist chamber.

B Cover-glasses (C) made slightly eccentric and containing between them
the^ object to be kept moist. Ky using cover-glasses the Specimen can be ex-
amined from both sides, and as part usually remains with each cover-glass, two
permanent preparations can be made.

C Slide (S) with a cover-glass (C) extending slightly over one edge so that it
can be lifted up without danger of sliding it along and thus disarranging the
specimen.

grade) the connective tissue will be so far gelatinized in from one
to three days that it is easy to separate the fascicles and fibers either
with needles or by shaking in a test-tube or shell vial with water.
It takes longer for some muscles to dissociate than others, even at
the same temperature, so one must try occasionally to see if the
action is sufficient. When it is, the acid is poured off and the
muscles washed gently with water to remove the acid. If one is
ready to make the preparations at once, they may be isolated and
mounted in water. If it is desired to keep the specimen indefinitely
or several days, the water should be poured off and 2% formal-
dehyde added. The specimens may be mounted in glycerin, glycerin
jelly or balsam. Glycerin jelly is the most satisfactory, however.

CH. XI] CABINETS; SLIPS AND COVERS; MOUNTING

42S

COLLECTION AND STUDY OF MICROSCOPIC
ANIMALS AND PLANTS

§ 542. Collection of material. — There are
many microscopic forms in nature that need no
other preparation than mounting on a glass
slide. If low powers are used, a cover-glass
may be omitted, but if high powers are to be
used, a cover-glass must be put over the ob-
ject to protect the objective as well as the
object, and to make the optical corrections of
the objective perfect (§ 254).

The easiest place to find things most in-
teresting and beautiful is in the water of pools
and along the shores of streams where the
water is quiet. Go to some pond or stream
and along the shore where it is shallow; take
some of the vegetation and the mud, put in a
pail or dish, and take to the home or labo-
ratory. Put the water and vegetation in a plate
or other shallow vessel and put it in about
the same light that it had in nature. In
a few hours, when the mud has settled the
conditions will be nearly as in nature, and
by the use of fine forceps or one of the
pipettes (figs. 221-231), gather some of the
water with scrapings from some of the vege-
tation, or some of the water and mud. Put
it on a slide, cover and examine. There
may be much to see or very little. One
must persevere and finally there will come
a kind of instinctive knowledge where to
find things. It is also a good plan to use
the tripod or other magnifier and examine
the dish. Often much can be seen in that
way, and one will get a hint where to collect

FIG. 231. PIPETTES
FOR LIQUIDS AND FOR
SPECIMENS.

A Pipette for liquids.
This is about one-third
size.

B Pipette for hand-
ling ova and other deli-
cate specimens.

/ The rubber bulb
tied to the glass part.
It is about natural size.

2 Glass rod. The
upper end is fluted so
that the rubber bulb
will not come off, and
the lower end is care-
fully smoothed by heat-
ing. To prevent small
ova and other objects
getting into the bulb,
some fine gauze may be
tied over the upper end.

3 Soft rubber tube
over the lower end.
This is not absolutely
necessary, but the soft
rubber is less likely to
injure delicate objects
than the hard glass.

4.26

CABINETS; SLIPS AND COVERS; MOUNTING [CH. XI

o o

FTC. 232. TRTPOD MAONTFTER.

the bits to put on the slide for examination. Do not use distilled

water for these organisms, but water from the source of supply.

(For food see § 543.)

§ 543. Infusoria and bacteria; In-
fusions. — One of the best ways
to get a large variety of living
forms, animal and vegetable, is to
make such a gathering as described
above and to put it into a small
fruit jar or other wide open vessel,
and to put with it some of the
stems of the grass along the
stream. If in a moderately warm

place for a day or more, this collection will be found swarming with

living things. Soon, however, the

numbers will lessen and finally there

will be very few left. These living

things need food. One of the good

foods for them is made from boiling

up some of the grass and hay found

near the natural habitat. Any good

hay may be used, however. When

the mixture is cool, add some of

it to the vessel containing the or-
ganisms, or what is better, take

another dish, and add a fair amount

of the liquid from the first gathering.

Usually this new supply will be as

rich in life as was the original gath-
ering. (See under Neutral Red

(§ 604) for experiment in staining

live forms).

§ 544. Diatoms. — These are plants

with silicious shells, and are found

in natural waters both salt and fresh. If one goes to a pond

or stream in May or June or July especially, the diatoms are verv

FIG. 233. MAGNIFIER SUP-
PORTED BY A FOCUSING, JOINTED
HOLDFR.

Base The heavy iron base to
keep the apparatus steady.

R P Rack and pinion for
focusing the magnifier.

J J Joints to make it possible
to put the lens in any desired
position.

CH. XI] CABINETS; SLIPS AND COVERS; MOUNTING 427

abundant. They may be found at any time, but in the spring most
abundantly, as with most living things. The brownish or rusty
looking substance on plants, rocks, etc., practically always contains
diatoms, and sometimes is made up mostly of them. It is most
interesting to study the diatoms alive and watch them glide around
in the water. The shells of the diatoms have been favorite objects
of study for a loig time. They are often beautifully marked.
Being silicious, they resist acids, and the living substance in and
around them can be destroyed without hurting the shells. This
may be done by placing the material containing a large number of
diatoms in a test-tube. When the diatoms have settled, pour off a
part of the liquid or draw it out with the pipette (fig. 231 A\ and
add an equal amount of nitric acid. Boil for a few minutes, let the
diatoms settle, pour or draw off most of the liquid, and add more
nitric acid and boil again. Finally, add water and gradually wash
the diatom shells by drawing off the water and adding fresh. The
shells should be clean and almost colorless and show their markings
well. One can take a sample and see if the cleaning is sufficient.
(For full and elaborate directions see Beyer's Diatomaceae of
Philadelphia and Vicinity, pp. 122-123).

§ 545. Arranging minute objects. — Minute objects like diatoms
or the scales of insects may be arranged in geometrical figures or in
some fanciful wav, either for ornament or for more satisfactory
study. To do this the cover-glass is placed over the guide. This
guide for geometrical figures may be a net-micrometer or a series of
concentric circles. In order that the objects may remain in place,
however, they must be fastened to the cover-glass. As an adhesive
substance, mucilage or liquid gelatin, this thinned with an equal
volume of 50% acetic acid answers well. A very thin coating of
this is spread on the cover with a needle, or in some other way, and
allowed to dry. The objects are then placed on the gelatinized side
of the cover and carefully put into position with a mechanical
finger, made by fastening a cat's whisker in a needle holder. For
most of these objects a simple microscope with stand (figs. 232-233)
will be found of great advantage. After the objects are arranged,
one breather very gently on the cover-glass to soften the mucilage

428 CABINETS; SLIPS AND COVERS; MOUNTING [Cn. XI

or gelatin. It is then allowed to dry, and if a suitable amount of
gelatin has been used and it has been properly moistened, the
objects will be found firmly anchored. In mounting one may use
Canada balsam or mount dry in a cell (§§ 526, 533). See New-
comer, Amer. Micr. Soc.'s Proc., 1886, p. 128; see also E. H.
Griffith and H. L. Smith, Amer. Jour, of Micros., iv, 102, v, 87;
Amer. Monthly Micr. Jour., i, 66, 107, 113; Cunningham, The
Microscope, viii, 1888, p. 237.

LABELING, CATALOGUING AND STORING MICROSCOPIC
PREPARATIONS

§ 546. Every person possessing a microscopic preparation is inter-
ested in its proper management; but it is especially to the teacher
and investigator that the labeling, cataloguing and storing of
microscopic preparations are of importance. " To the investigator,
his specimens are the most precious of his possessions, for they con-
tain the facts which he tries to interpret, and they remain the same
while his knowledge, and hence his power of interpretation, increase.
They thus form the basis of further or more correct knowledge;
but in order to be safe guides for the student, teacher, or investi-
gator, it seems to the writer that every preparation should possess
two things: viz., a label and a catalogue or history. This catalogue
should indicate all that is known of a specimen at the time of its
preparation, and all of the processes by which it is treated. It is
only by the possession of such a complete knowledge of the entire
history of a preparation that one is able to judge with certainty of
the comparative excellence of methods, and thus to discard or im-
prove those which are defective. The teacher, as well as the
investigator, should have this information in an accessible form, so
that not only he, but his students, can obtain at any time all
necessary information concerning the preparations which serve him
as illustrations and them as examples."

§ 547. Labeling ordinary microscopic preparations. — The label
should possess at least the following information:

The number of the preparation, its name and date and the thick-
ness of the sections and of the cover-glass.

CH. XI] CABINETS; SLIPS AND COVERS; MOUNTING 429

§ 548. Cataloguing preparations. — It is believed from personal
experience, and from the experience of others, that each preparation
(each slide or each series) should be accompanied by a catalogue
containing at least the information suggested in the following
formula. This formula is very flexible, so that the order may be
changed, and numbers not applicable in a given case may be
omitted. With many objects, especially embryos and small animals,
the time of fixing and hardening may be months and even years
earlier than the time of imbedding. So, too, an object may be
sectioned a long time after it was imbedded, and finally the sections
may not be mounted at the time they are cut. It would be well in
such cases to give the date of fixing under 2, and under 5, 6 and 8,
the dates at which the operations were performed, if they differ
from the original date and from one another. In brief, the more
that is known about a preparation, the greater its value.

o JO

G (y

FIG. 234. LABEL FOR A MICROSCOPIC PREPARATION.

The specimen is the myel (spinal cord) of an Amphioxus showing the dorsal
and ventral nerve roots, and some nerve cells near the middle.

G A nerve-cell with glycogen.

In the label c.ij> means that the cover-glass is 0.15 mm. in thickness; and s.
10 p, means that the section is ten microns thick. The date at the bottom shows
when the specimen was made.

§ 649. General formula for cataloguing microscopic preparations:

1. The general name and source. Thickness of cover-glass and
of section.

2. The number of the preparation and the date of obtaining and
fixing the specimen; the name of the preparator.

3. The special name of the preparation and the common and
scientific name of the object from which it is derived. Purpose of
the preparation.

430 CABINETS; SLIPS AND COVERS; MOUNTING [Cn. XI

4. The age and condition of the object from which the prepara-
tion is derived. Condition of rest or activity; fasting or full fed at
the time of death.

5. The chemical treatment, — the method of fixing, hardening,
dissociating, etc., and the time required.

6. The mechanical treatment, — imbedded, sectioned, dissected
with needles, etc. Date at which done.

7. The staining agent or agents and -the time required for stain-
ing.

8. Dehydrating and clearing agent, mounting medium, cement
used for sealing.

9. The objectives and other accessories (micro-spectroscope,
polarizer etc.), for studying the preparation.

10. Remarks, including references to original papers, or to good
figures and descriptions in books.

§ 550. A catalogue card written according to this formula:

1. Muscular Fibers of Cat; Cover 0.15 mm.; Fibers 20/4 to 40/4
thick.

2. No. 475. (Drr. IX) Oct. i, 1891. S. H. G., Preparator.

3. Tendinous and intra-muscular terminations of striated muscu-
lar fibers from the Sartorius of the cat (Felis domestica}.

* 4. Cat eight months old, healthy and well nourished. Fasting
and quiet for 12 hours.

5. Muscle pinned on cork with vaselined pins and placed in 20
per cent nitric acid immediately after death by chloroform. Left
36 hours in the acid; temperature 20° C. In alum water (J sat.
aq. sol.) i day.

6. Fibers separated on the slide with needles, Oct. 3,

7. Stained 5 minutes with DelafiekTs hematoxylin.

8. Dehydrated with 95% alcohol 5 minutes, cleared 5 minutes
with carbol- turpentine, mounted in xylene balsam; sealed with
shellac.

9. Use a 16 mm. for the general appearance of the fibers, then a
2 or 3 mm. objective for the details of structure. Try the micro-
polariscope.

10. The nuclei or muscle corpuscles are very large and numerous;

CH. XI] CABINETS; SLIPS AND COVERS; MOUNTING 431

many of the intra-muscular ends are branched. See S. P. Gage,
Proc. Amer. Micr. Soc., 1890, p. 132; Ref. Hand-book Med. Sci.,
Vol. V, p. 59-

§ 551. General remarks on catalogues and labels. — It is es-
pecially desirable that labels and catalogues shall be written with
some imperishable ink. Some form of waterproof carbon ink is
the most available and satisfactory. The waterproof ink of Higgins
or Weber answers well. For ordinary writing it should be diluted
with one-third its volume of water and a few drops of strong am-
monia added.

If one has a writing diamond, it is a good plan to write a label
with it on one end of the slide. It is best to have the paper label
also, as it can be more easily read.

The author has found stiff cards, 12! x 7! cm., like those used for
cataloguing books in public libraries, the most desirable form of
catalogue. A specimen that is for any cause discarded has its
catalogue card destroyed or stored apart from the regular catalogue.
New cards may then be added in alphabetical order as the prepara-
tions are made. In fact a catalogue on cards has all the flexibility
and advantage of the slip system of notes.

Some workers prefer a book catalogue. Very excellent book cata-
logues have been devised by Ailing and by Ward (Jour. Roy. Micr.
Soc., 1887, pp. 173, 348; Amer. Monthly Micr. Jour., 1890, p. 91;
Amer. Micr. Soc. Proc., 1887, p. 233).

The fourth division has been added, as there is coming to be a
strong belief, practically amounting to a certainty, that there is a
different structural appearance in many if not all of the tissue
elements, depending upon the age of the animal, upon its condition
of rest or fatigue; and for the cells of the digestive organs, whether
the animal is fasting or full fed. Indeed as physiological histology is
recognized as the only true histology, there will be an effort to deter-
mine exact data concerning the animal from which the tissues are
derived. (See Minot, Proc. Amer. Assoc. Adv. Science, 1890, pp.
271-289; Hodge, on nerve cells in rest and fatigue, Jour. Morph.,
vol. VII (1892), pp. 95-168; Jour. Physiol., vol. XVII, pp. 129-134;
Gage, The Processes of Life revealed by the Microscope; a Plea for

432

CABINETS; SLIPS AND COVERS; MOUNTING [Cn. XI

Physiological Histology, Proc. Amer. Micr. Soc., vol. XVII (1895),
pp. 3-29; Science, vol. II, Aug. 23, 1895, pp. 209-218. Smithsonian
Institution, Report for 1896, pp. 381-396.

CABINET FOR MICROSCOPIC
PREPARATIONS

§ 552. While it is desirable that
microscopic preparations should be
properly labeled and catalogued, it
is equally important that they
should be protected from injury.
During the last few years several
forms of cabinets or slide holders
have been devised. Some are very
cheap and convenient where one has
but a few slides. For a laboratory
or for a private collection where
, the slides are numerous, the follow-
ing characters seem to the writer
essential :

(i) The cabinet should allow the
slides to lie flat, and exclude dust

Fir.. 235. FACE AND EDGE VIEW ancl ^t.

OF A CABINET DRAWER FOR MICRO- (2) Each slide or pair of slides

SCOPIC SLIDES. shollld be ^ a separate compart-

06, 70 The number of the com- A t . P .

partment. ment. At each end of the com-

a b In the compartment a, the partment should be a groove or

slide is resting in place to show that , . . ,

the container touches the slide only bevel, so that upon depressing

in two places. either end of the slide, the other

In ft, the slide is depressed into . .. /_ .

the groove at one end of the com- may be grasped easily (fig. 235).

Partment. It is then easy to grasp ft is also desirable to have the floor

of the compartment grooved so that

the slide rests only on two edges, thus preventing soiling the slide

opposite the object.

(3) Each compartment or each space sufficient to contain one slide

of the standard size should be numbered, preferably at each end.

ji'o.% /tM
<Atn/c. Si6efi
^Cat

"'"

—

CH. XI] CABINETS; SLIPS AND COVERS; MOUNTING

433

If the compartments are made of sufficient width to receive two
slides, then the double slides so frequently used in mounting serial
sections may be put into the cabinet in any place desired.

(4) The drawers of the cabinet should be entirely independent,
so that any drawer may be partly or wholly removed without dis-
turbing any of the

others. - !

(5) On the front of
each drawer should be
the number of the
drawer in Roman
numerals, and the
number of the first
and last compartment
in the drawer in Ar-
abic numerals (fig.
236).

§ 563. Trays for
slides and ribbons of
sections. — Early in
1897 the writer de-
vised the simple tray
shown in fig. 237. It
was designed espe-
cially for the ribbons FIG. 236. CABINET FOR MICROSCOPE SLIDES.

nf QPrtinnQ in r»rpr»nr This cabinet contains 2o drawers like that shown in
oi sections m prepar- fig ^ and ag indicated at the right there are 420

ing embryologic series compartments for slides.

and for material in

class work. As will be seen by the figure, the two sides are alike

and the tray is very shallow. It was soon found that the wood

forming the bottom of the tray was too rough for ribbons of sections

and smooth white paper was put in the tray before the ribbons

were laid upon it.

These trays were soon used for the mounted preparations as well
as for the ribbons of sections. They were made of a proper size to
fit the laboratory lockers (fig. 242) and naturally came to be used for

434

CABINETS; SLIPS AND COVERS; MOUNTING [Cn. XI

storage instead of the expensive slide cabinets. For this purpose five
could be put in a single compartment of the locker or thirty-five in
an entire locker. As each tray holds fifty slides 25 x 75 mm.,
thirty-five 38 x 75 mm., and twenty-five slides 50 X 75 mm., the
saving of space was very great.

§ 554. Slide trays with tongue groove, and compartments. — In
the first trays the edges were square and sharp. These were rounded
in later trays, but there still remained a defect, for if one wished to

FIG. 237. SIMPLEST FORM OF SLIDE TRAY.

A Face view of the slide tray. The screw eye at the lower end is convenient
for pulling out a single tray.

B Sectional view of the tray showing the thin board of which it is made and
the wooden frame.

C Sectional view showing how the frame is fastened to the board.

pile up five to twenty trays on the table, they would not stay in an
even stack. To remedy this defect the long way of the frame was
tongued on one side and grooved on the other, as shown in fig. 238.
This is a great improvement, as one can make even stacks of 25 or
50 trays, and they will stay in position. Furthermore it renders the
groups of five trays stored in the locker compartments much easier
to manage, as one can remove any of the five trays without getting

CH. XI] CABINETS; SLIPS AND COVERS; MOUNTING

43S

the others disarranged, as so often occurred with the old form, lack-
ing tongue and groove.

A defect of the trays for storage is the ease with which the slides
get disarranged unless the tray is entirely full. To overcome this
defect S. P. Gage divided one face of the tray into columns (fig. 238)
by means of stout cord held in place by using melted paraffin as a
cement. Later Dr. Greenman of the Wistar Institute divided one
face of the tray into columns by wooden strips. This is the best way.
With the tray face in columns the slides in a single column may
become disarranged, but there is no mixing of the slides of different
columns. One side of the tray remains smooth and can be used for
ribbons of sections or for any other purpose. Dr. Jean Broadhurst

FIG. 238. SLIDE TRAY WITH COMPARTMENTS, TONGUE AND GROOVE.

A Face view of the new form of slide tray. It is designed for fifty slides,
25 x 75 mm. in size or twenty-five slides, 50 X 75 mm. in size used for serial sec-
tions of embryos, etc.

B Sectional view of a side piece with tongue and groove. Supports 5 mm.
wide near the end prevent the slide from contact with the tray. This is especially
important in dark-field or other work when oil-irnmersion liquid is on the lower
face of the slide. The support also enables one to depress one end of the slide thus
rendering it more easily grasped. See also figures 87, 235.

436

CABINETS; SLIPS AND COVERS; MOUNTING [Cn. XI

of Teachers College, Columbia University, has found that these trays
are admirably suited for a cabinet of lantern slides. The smooth
side will hold 13, two rows arranged lengthwise and one row cross-
wise. If a sheet of white paper is put under the slides, it is easy to
see what is on them.

§ 564a. The original maker of these trays was the H. J. Bool Co., of Ithaca,
N. Y.; there are no restrictions, however, and excellent trays of the tongue,
groove and column type (fig. 238 A) are now also available at the Clay- Adams Co.

FIG. 239. THE WISTAR INSTITUTE METAL TRAY FOR MICROSCOPIC PREPARATIONS.
The upper tray was raised up and supported by corks when the photograph
of the pile of trays was made. The picture shows the form, the rows for slides,
and the band on the edge for writing labels.

FIG. 240. THE MINOT METAL CABINET AND METAL TRAYS FOR MICROSCOPIC

SLIDES,
(Courtesy of Peter Gray & Sons, Inc. East Cambridge, Mass.)

CH. XI] CABINETS; SLIPS AND COVERS; MOUNTING

437

§ 565. Metal slide trays of the Wistar Institute (fig. 239) ; Minot
metal slide trays and cabinets (fig. 240); the Eberbach aluminum
slide trays and cabinet (fig. 241); and the Paragon C. & C. Company
Paragon cabinets for filing large numbers of slides in very small space.

FIG. 240, A. B. Two FORMS OF METAL TRAYS FOR THE MINOT SLIDE CABINET
A is for Slides 50 x 75 mm. or Two Standard Slides in Each Small Com-
partment.

Tray B is for standard size slides. The little metal tongues serve to make a
place for standard size slides (25 x 75 mm.). In both trays there is a groove in
the middle to facilitate lifting up the slides when needed. As shown also there
is a little knob for pulling out the trays, and metal clips to hold paper labels.

FIG. 241. THE EBERBACH ALUMINUM SLIDE TRAYS AND CABINET.
(Courtesy of Eberbach & Son Co.)

438

CABINETS; SLIPS AND COVERS; MOUNTING [Cn. XI

INCH HOLES.

BOARD:

REAGENT BOARDS AMD DKAWEfV* AM
INTERCHANGEA6LF TrtKOUCHOUT.

ELCVATIOh.
LOCKCR5 IN LABORATORIES.

FIG. 242. LABORATORY LOCKERS REAGENT BOARDS AND DRAWERS DESIGNED

IN 1895.

(From the Journal of Applied Microscopy, 1898, p. 127).

The lockers designed in 1899 for Stimson Hall are in banks of 12 or 9, with
three vertical tiers, not two as shown in this figure. Everything is of standard
size and hence completely interchangeable.

Measured over all, the locker banks are 329 cm. high, and 139.5 cm. wide for
the large banks and 105 wide for the smaller banks. Each individual locker,
inside measure, is 32 cm. wide, 70.5 cm. high, and 48 cm. deep. It is divided by
7 runs into 8 compartments. As indicated in the sectional view, the entire space
may be left free in the locker or partly filled or wholly filled.

Each bank of lockers is lettered, and then the individual lockers numbered
from 1-12 or 1-9, the numbering being in the order of words in a book,
i.e., from left to right. Of course vertical numbering is equally feasible. With
this form of numbering each bank is practically independent and can be changed
in position without confusion.

CH. XI] CABINETS; SLIPS AND COVERS; MOUNTING 439

REAGENTS FOR MICROSCOPIC WORK

For much of the work done with a microscope the reagents
needed are few and inexpensive, but for a large laboratory with the
diversity of investigations carried on the reagents are numerous, and
some of them expensive. Below are given some of the principal
ones with the method of their preparation.

General on preparation of reagents. — In preparing reagents both
weights and measures are used. As a rule the amounts given are
those which experience has shown to give good results. Variations
in the proportions of the mixtures are sometimes advantageous, and
in almost every case a slight change in the proportions makes no
difference. Most laboratory reagents are like food, good even under
quite diverse proportions and methods of preparation. With a few,
however, it is necessary to have definite strengths.

By a saturated solution is meant one in which the liquid has dis-
solved all that it can of the substance added. This varies with the
temperature. It is well to have an excess of the substance present;
then the liquid will be saturated at all temperatures usually found in
the laboratory.

§ 556. Solutions less than 10 per cent. — In making solutions
where dry substance is added to a liquid, if the percentage is not
over 10%, the custom is to take 100 cc. of the liquid and add to it
the number of grams indicated by the per cent. That is, for a
5% solution one would take 100 cc. of the liquid and 5 grams of the
dry substance. This does not make a strictly 5% solution. For
that one should take 95 cc. of liquid and 5 grams of the dry sub-
stance; or, if the percentage must be exact, then one should weigh
out 95 grams of the liquid and add 5 grams of the dry substance.

§ 557. Solutions of 10 per cent and more. — When the percentage
is 10% or over it is better to weigh out the number of grams repre-
senting the percentage and add to it the right amount of liquid in
cubic centimeters. For example, if one were to make a 35% aqueous
solution of caustic potash in water, then one would add 35 grams of
caustic potash to 65 cc. of water. If one wished to make a 10%
alcoholic solution of caustic potash, he would add 10 grams of caustic

440 CABINETS; SLIPS AND COVERS; MOUNTING [Cn. XI

potash to 90 cc. of alcohol. But here is a case where, the alcohol
being of less specific gravity than water, the mixture would not
weigh 100 grams; and to make the mixture weigh 100 grams, giving
therefore an exact percentage, one should take 90 grams of alcohol
and add to it 10 grams of caustic potash. In practice in making
solutions of collodion or parlodion one usually mixes ether and 95%
or absolute alcohol in equal volumes and then for a 10% solution
adds 10 grams of the dry soluble cotton or parlodion to 90 cc. of the
ether-alcohol mixture. But ether is much lighter than water and the
alcohol somewhat lighter, so that the percentage in this case would
be more than 10% because the 90 cc. of alcohol and ether would
weigh considerably less than 90 grams.

§ 668. Mixtures of liquids to obtain a desired percentage. — It
frequently happens that it is desired to obtain a lower percentage or
strength of a liquid than the one in stock. This is very readily done
according to the general formula: Divide the percentage of the
strong solution by the percentage of the desired solution and the
quotient will show how many times too strong the stock solution is.

To get the desired strength, use i volume of the strong stock
solution, and add to it enough of the diluting liquid to make a
volume corresponding to the amount indicated by the quotient
obtained by dividing the percentage of the stock solution by that of
the desired solution. For example, if it is desired to obtain a 5%
solution of formaldehyde from a stock solution of 40% strength,
the stock solution being 8 times too strong, to get the 5 % solution
i volume of the strong solution must be used and 7 volumes of the
diluting liquid (water). The solution so obtained will be | of the
original strength, or 5%.

If a 2 % solution were desired then i volume of the strong solution
would be taken and 19 volumes of water, etc.

§ 669. Mixtures of alcohol. — For alcohol if one desires a 50 %
solution it is usually near enough correct to add equal parts of 95 %
alcohol and water, but this does not actually give a 50% solution.
To find the real proportions according to the general formula: 95%
•*• 5°% = i'Q* *'e-> f°r every i cc. of 95% alcohol should be added
p.p cc. of water or for each 100 cc. of 95% alcohol, 90 cc. of water.

CH. XI] CABINETS; SLIPS AND COVERS; MOUNTING

441

Even this will not give an exact mixture of alcohol, for a mixture of
alcohol and water diminishes somewhat in volume. To get true
percentages an alcoholometer for testing the specific gravity is used.
A simple method of getting approximately correct mixtures of
alcohol is the following: Pour the strong alcohol into a graduate
glass (ng. 243, A , B) until the volume is the same as the desired per-
centage; then add water until the volume is the same as the original
percentage of the alcohol. Ex-
ample: To get 50% from 95%
alcohol put 50 cc. of 95% into
a graduate and fill the graduate
to 95 cc. with water, and the
resulting mixture will be 50%
alcohol, and so with all other
strengths. Here the shrinkage
is eliminated from consideration,
because the water and alcohol
are not measured separately and
then mixed, but one is added to
the other until a given volume
is attained.

PREPARATION or REAGENTS
§ 560. Albumen fixative

FIG. 243. GLASS GRADUATES FOR

MEASURING LIQUIDS.
A Graduate with sloping sides for
large quantities.

B Graduate with straight sides for

(Mayer's). — This consists of - - .. , - . ,
^ J ' smaller quantities and more accurate de-

equal parts of well-beaten white termination.

of egg and glycerin. To each

50 cc. of this i gram of salicylate of soda is added to prevent
putrefactive changes. Filter through absorbent cotton. It is not
to be used on slides for the ultra-violet or incineration.

§ 561. Alcohol (ethyl), QjI^OH. — Ethyl or grain alcohol is
mostly used for histologic purposes. (A) Absolute alcohol (i.e.,
alcohol of 99 %) is recommended for many purposes, but if plenty of
95% alcohol is used it answers every purpose in histology, in a dry
climate or in a warm, dry room. When it is damp, dehydration is
greatly facilitated by the use of absolute alcohol.

442

CABINETS; SLIPS AND COVERS; MOUNTING [Cn. XI

(B) 82% alcohol made by mixing 5 parts of 95% alcohol with i
part of water.

For educational and other public institutions the U. S. government
grants the privilege of using ethyl alcohol without paying the
revenue tax, but for private institutions and for individuals it would
be a great relief if the denatured alcohol could be mixed in all
proportions with water without the formation of precipitates.

§ 562. Alcohol (methyl), CH3OH. — Methyl alcohol or wood
alcohol is much cheaper than ethyl or grain alcohol on account of
the revenue tax on ethyl alcohol. It answers well for many micro-
scopic purposes. It has been refined so carefully in recent years that
the disagreeable odor is not very noticeable.

FIG. 244. GLASS-STOPPERED BOTTLES FOR THE MORE USUAL GRADES OF ALCOHOL
USED IN MICROSCOPY.

Denatured alcohol. — This is ethyl or grain alcohol rendered
undrinkable by the addition of wood alcohol and benzine (grain
alcohol 89!%; methyl alcohol 10%, and benzine £%). In some
cases the denaturing substances are somewhat different, but all render
the alcohol unusable for drinking. It is then free from internal
revenue tax.

In Great Britain " methylated spirits " consists of grain alcohol
with ib% methyl alcohol. This is used very largely in microscopic
work. In America the addition of the benzine renders denatured
alcohol also unfit for histological purposes if it is to be diluted. The

CH. XI] CABINETS; SLIPS AND COVERS; MOUNTING 443

addition of water makes it milky. If methyl alcohol alone or com-
bined with pyridin or some other substance wholly soluble in water
were used as the denaturing substance, denatured alcohol could be
used in microscopic work for all the grades. That denatured as
indicated above can be used only in full strength or very slightly
diluted.

§ 563. Alcohol, normal propyl or propanol (CH3CH2CH2OH. —
This form of alcohol has been shown by Sheridan to be especially
useful in histology to prevent the great shrinking and hardening of
tissues in imbedding by the paraffin method as it is a solvent of
paraffin. (See Jour. Tech. Methods, and Built. Internatl. Assoc.
Med. Museums, Vol XII, pp. 125-126, 1929 (Abstract in Stain Tech.,
Vol. V, 1930, p. 34. See § 641 for this method of use.

§ 564. Balsam, Canada balsam, balsam of fir. — This is one of the
oldest and most satisfactory of the resinous media used for mounting
microscopic preparations.

The natural balsam is most often used; it has the advantage of
being able to take up a small amount of water so that, if sections are
not quite dehydrated, they will clear up after a time.

§ 565. Xylene balsam. — This is Canada balsam diluted or
thinned with xylene. It is recommended by many to evaporate the
natural balsam to dryness and then to dissolve it in xylene. For
some purposes, e.g., for mounting glycogen preparations, this is
advantageous; but it is unnecessary for most purposes. Xylene
balsam requires a very complete desiccation or dehydration of
objects to be mounted in it, for the xylene is immiscible with water.

The hydrocarbon, xylene (C8Hi0) is called xylol in German. In
English, members of the hydrocarbon series have the termination
" ene," while members of the alcohol series terminate in " ol."

§ 566. Filtering balsam. — Balsam is now furnished already
filtered through filter paper. If xylene balsam is used, it may be
made thin and filtered without heat. For filtering balsam and all
resinous and gummy materials, the writer has found a paper funnel
the most satisfactory. It can be used once and then thrown away.
Such a funnel may be easily made by rolling a sheet of thick writing
paper in the form of a cone and cementing the paper where it over-

444 CABINETS; SLIPS AND COVERS; MOUNTING [CH. XI

laps, or winding a string several times around the lower part. Such
a funnel is best used in one of the rings for holding funnels, so com-
mon in chemical laboratories. The filtering is most successfully
done in a very warm place, like an incubator or an incubator room.

§ 667. Artificial resins. — There have been developed recently cer-
tain resin-like mounting media which promise to be even more satis-
factory than Canada balsam. Two of them are: Du Font's isobutyl
methacrylate polymer and the Neveliite Co.'s clarite (cycloparaffin
or naphthene polymer).

These when dissolved in xylene or toluene give a clear mounting
medium which is of approximately the refractive index of glass and
serve to give the stained sections the desired transparency.

Both plastics have been used for some months for mounting speci-
mens to show glycogen stained with iodine. The stain is preserved
perfectly.

For the clarite 60 grams in 40 cc. of xylene or toluene gives a solu-
tion of about the right consistency. The I)u Pont methacrylate
requires about equal percentage of the dry powder and the xylene or
toluene. The clarite fluoresccs somewhat in ultra-violet light and
therefore should not be used for fluorescence mounts, § 602.

§ 568. Acid balsam. — As stated above, all balsam is naturally
somewhat acid, but for various stains it is desirable to increase the
acidity. For example, specimens stained with picro-fuchsin, or
injected with carmine or Berlin blue are more satisfactory and last
longer with full brilliancy if the balsam is made more acid than it
naturally is. For this use 10 to 20 drops of glacial acetic or formic
acid to 100 cc. of balsam.

§ 569. Borax carmine for in to to staining. — Borax 4 grams;
carmine 3 grams; water 100 cc. Shake frequently for several days
and then filter and add 100 cc. of 67% alcohol. After 3 to 4 days it
may be necessary to filter again. Good for in toto staining after al-
most any fixer. Put the object to be stained from alcohol into a vial
with plenty of stain. After a day or two change the stain. Stain
4 to 5 days. Remove to 67% alcohol, adding 4 drops of HC1 to each
100 cc. of alcohol. After one day remove to 82% alcohol.

§ 570. Carmine for mucus (mucicarmin). — One can buy the dry

CH. XI] CABINETS; SLIPS AND COVERS; MOUNTING 445

powder or preferably prepare the stain. To prepare it, take i gram
of Carmine No. 40 and | gram of pure dry ammonium chlorid. If the
latter is slightly moist, dry it in an evaporating dish in a sand bath.
Mix the ammonium chlorid and the carmine and add 2 cc. of water.
Mix well and heat over a sand bath, constantly mixing with a glass
rod. Continue the heating until the carmine colored mass becomes
very dark red. It will take 3 to 10 minutes for this. The heat
should not be too great.

Dissolve the dark red mixture in 100 cc. of 50% alcohol. For use,
dilute five- or tenfold with tap water. This stains best after mercuric
fixers. One must not collodionize sections to be stained with this,
as the carmine stains the collodion very deeply. Stain the sections
first with hematoxylin as usual; then stain i to 5 hours or longer
with the dilute mucicarmin. The mucus in goblet cells, in the mu-
cous part of the salivary glands, etc., will be red. Nuclei will be
stained with hematoxylin. Mount in balsam (§ 535).

§ 571. Cedar-wood oil. — For penetrating tissues and preparing
them for infiltration with paraffin, thick oil is recommended by Lee.
For tissues fixed "with osn.ic acid for fat, the thick oil is necessary,
but for most histologic and embryologic work, that known as Cedar-
wood Oil (Florida) is excellent, also that known as Cedar-wood Oil
(true Lebanon). These forms are far less expensive than the thick
oil. The tissues should be thoroughly dehydrated before putting
them into cedar-wood oil, and they should remain until they are
translucent.

The thickened cedar-wood oil used for homogeneous immersion
should be obtained from the manufacturers of microscopes; they
naturally would supply the kind suitable for the purpose.

§ 572. Chloroform (CHCls). — This is used for clearing and im-
bedding where fats fixed with osmic acid are to be preserved in the
tissues. It is also used for hardening collodion, in collodion im-
bedding. It is an excellent solvent of cedar-wood oil and is used for
cleaning homogeneous immersion fluid (cedar-oil) from objectives,
condensers and microscopic preparations.

§ 673. Carbol-xylene clearer. — Vasale recommends as a clearer,
xylene 75 cc., carbolic acid (melted crystals) 25 cc.

446 CABINETS; SLIPS AND COVERS; MOUNTING [Cn. XI

§ 573a. Carbol-xylene and eosin. In order to counterstain with eosin during
the clearing process, the carbol-xylene is charged with eosin as follows : A satu-
rated aqueous solution of eosin is prepared, and to it is added with constant
stirring, hydrochloric acid until there is a good precipitate. Filter through filter
paper. Wash the precipitate with distilled water until the water goes through
pink. This indicates that the acid is washed out. Dry the washed precipitate.
This is soluble in the carbol-xylene and enough should be added to make that
pink. More or less can be used depending on the depth of the eosin stain de-
sired. That can be regulated also by the time the sections are left in the eosined
clearer. (Freeborn, Jour. Ap. Microscopy, Vol. Ifl, p. 1058).

§ 574. Carbol- turpentine clearer. — A satisfactory and generally
applicable clearer is carbol turpentine, made by mixing carbolic acid
crystals (Acidum carbolicum. A. phenicum crystallizatum) 40 cc.
with rectified oil of turpentine (Oleum terebinthinae rectificatium)
60 cc. If the carbolic acid does not dissolve in the turpentine,
increase the turpentine, thus: carbolic acid 30 cc., turpentine 70 cc.

This clearer is not so good as the preceding for mounting objects
which have been stained with osmic acid, as the hydrogen dioxid
(H202) present fades the blackened osmic acid.

§ 576. Clarifier, castor-xylene clarifier. — This is composed of
castor oil i part and xylene 3 parts. (Trans. Amer. Micr. Soc.,
1895, p. 361.) For the use of this clarifier, see under the collodion
method (§ 652).

§ 576. Collodion. — This is a solution of soluble cotton or other
form of pyroxylin in equal parts of sulphuric ether in 95% or abso-
lute alcohol. In using soluble cotton for infiltrating and imbedding
tissues several different strengths are used, commencing with weak
and proceeding to strong mixtures. The last in which the tissue is
imbedded is as thick a solution as can be made. All collodion solu-
tions should be kept well corked, for the ether and alcohol are very
volatile.

§ 576a. The substance used in preparing collodion goes by various names;
soluble cotton or collodion cotton is perhaps best. This is cellulose nitrate, and
consists of a mixture of cellulose tetranitrate Ci2Hi«(NO8)4O6 and cellulose pentani-
trate, CiaHuCNOa^Os. Besides the names soluble and collodion cotton, it is called
gun cotton and pyroxylin. Pyroxylin is the more general term and includes
several of the cellulose nitrates. Celloidin is a patent preparation of pyroxylin,
more expensive than soluble cotton.

An American product known as "parlodion " has recently (1915) appeared
to take the place of the celloidin not now obtainable. It is non-explosive, and is
said to be a very pure, concentrated form of collodion especially adapted to the

CH. XI] CABINETS; COVERS AND SLIPS; MOUNTING 447

needs of histology and embryology. (Advertising pages, Anatomical Record,
Dec., 1915.)

Soluble cotton should be kept in the dark to avoid decomposition. After it
is in solution, this decomposition is not so likely to occur. The decomposition of
the dry cotton gives rise to nitrous acid, and hence it is best to keep it in a box
loosely covered, so that the nitrous acid may escape.

Cellulose nitrate is explosive under concussion and under 150° centigrade heat.
In the air, the loose soluble cotton burns without explosion. It is said not to
injure the hand if held upon it during ignition and not to fire gunpowder if
burned upon it. So far as known to the writer, no accident has ever occurred
from the use of soluble cotton for microscopic purposes. I wish to express my
thanks to Professor W. R. Orndorff, organic chemist in Cornell University, for
the above information. (Proc. Amer. Micr. Soc., vol. XV11 (1895), pp. 361-370.)

§ 577. Collodion for cementing sections to the slide. — This is
a f % solution made by adding f gram of soluble cotton to 50 cc. of
95% or absolute alcohol and 50 cc. of sulphuric ether. This may
be used before deparaffining or, preferably, afterward. (See § 639.)

§ 578. Congo red. — Water 100 cc., congo red \ gram. This is
a good counter stain for hematoxylin.

§ 579. Congo-glycerin. — (Glycerin TOO cc., congo red (§578) 20
ccO- For mixing with and staining isolation preparations (§ 539)
and for a mounting medium, this is an excellent combination. It is
particularly good for nerve cells.

§ 580. Decalcifier. — One of the best is a mixture of ethyl alcohol
and nitric acid (67 % alcohol, 100 cc., strong nitric acid, 3 cc.). The
tissue or organ should first be fixed by some approved method. It
may then be put into the decalcifier. Change the decalcifier two
or three times. It usually takes from 2 to 10 days, depending on the
object and its size. One can tell when the decalcification is complete
by inserting a fine needle. If there is no gritty feeling the decalcifi-
cation is complete. Wash in two or three changes of water, and then
transfer to 67% alcohol, and in 24 hours to 82% alcohol. It is
better to imbed and section soon, as decalcified tissue is likely to
deteriorate. The original fixation and the use of alcohol in the decal-
cifier are to avoid the gelatinization of the white fibrous tissue.

One can use either the collodion or the paraffin method for sec-
tioning. For large objects perhaps the collodion method gives the
better results.

§ 581. Dissociating Liquids. — These liquids are for perserving
the tissue elements or cells and for dissolving or softening the inter-

448 CABINETS; SLIPS AND COVERS; MOUNTING [Cn. XI

cellular substance so that the cells may be readily separated from
their neighbors. The separation is accomplished by (a) teasing with
needles; (6) shaking in a liquid in a test-tube; (c) scraping with a
scalpel and crushing with the flat of the blade; (d) tapping
sharply on the cover-glass after the object is mounted. One may
find it desirable to use (d) with all the methods.

(1) Formaldehyde dissociator. — Strong formalin (40% formalde-
hyde gas in water) 2 cc.; normal salt solution 1000 cc. One can
begin work within f hour and good results may be obtained after 2
to 3 days immersion. Excellent for epithelia and for nerve cells.

(2) Muller's fluid dissociator. — Miiller's fluid i cc., normal salt
solution 9 cc. It usually requires from i to 5 days for epithelia to
dissociate in this. The action is more rapid in a warm place.

(3) Nitric acid dissociator. — Nitric acid 20 cc., water 80 cc.
This is used especially for muscular tissue. It takes from i to 3
days, depending on the temperature. The nitric acid gelatinizes the
connective tissue. Wash out the acid with water for a few minutes.
Preserve in 2% formaldehyde.

§ 682. Elastic stains. — There are four good differential stains for
elastic tissue:

(1) The orcein stain of Unna (Mon. Schr. Dermat., 1894, xix, i).
It is prepared by mixing i gram of orcein, 100 cc. of 82% alcohol, and
i cc. of hydrochloric acid. Stain for one hour or less in the solution
warmed in an oven (fig. 255). Wash well with 67% alcohol, then in
water. Dehydrate and mount in balsam or clarite. Counterstain
with haematoxylin or methylene blue. Elastic fibers stain dark
brown, nuclei purple or blue (§§ 585, 593).

(2) Weigert's basic-fuchsin-resorcin method. Basic fuchsin 2 grams;
resorcin or phenol 4 grams; water 200 cc. Boil 5 to 10 minutes.
Add to the boiling mixture 25 cc. of a 30% aqueous solution of chlorid
of iron (FeCla). Boil for 3 to 10 minutes. Then add drop-wise a
saturated solution of the iron chlorid till the color is all precipitated.
Test occasionally by letting a few drops run down the side of the
beaker used for the heating. When the color is precipitated, it will
appear as fine granules, and the liquid will be clear or slightly yellow.

When precipitated, allow the liquid to cool and the coloring matter

CH. XI] CABINETS; SLIPS AND COVERS; MOUNTING 449

to settle. Filter through filter paper. Scrape off the precipitate or
cut off the lower end of the filter. Put in a pyrex beaker; add 200 cc.
of 95% alcohol; heat over a water bath till the alcohol boils; stir
the mixture occasionally with a glass rod; boil 5 minutes or more.
Filter the hot solution into a pyrex beaker and allow it to cool. When
it is cool, add with stirring 5 cc. of hydrochloric acid.

Stain paraffin or collodion sections in this one hour or less. Wash
off the stain with 95% alcohol. This stain is good for any method of
fixation. The elastic tissue will be dark gray or black. For multiple
staining see § 66 r.

(3) Sheridan's crystal-violet (Stain Technology, Vol. 5, p. 31, 1935).
This stain is prepared the same as Weigert's except that the crystal-
violet is used instead of the basic fuchsin. Elastic fibers are stained
green. Orange G is a good counterstain.

(4) Verhoejf's iron-hcematoxylin stain (Jour. Amer. Med. Assoc.,
1908, Vol. 50, Part i, p. 876). i gram of haematoxylin crystals; 20 cc.
of 95% alcohol. Heat in a test-tube till the stain is dissolved. When
the haematoxylin is dissolved and the solution cool, add iron chlorid
(FeCl3) of a 10% aqueous solution 8 cc. Finally add 8 cc. of Lugol's
solution. (lodin 2 grams, iodide of potassium 4 grams, water 100 cc.)

Stain the sections from alcohol in this mixture till they are black
(10-15 minutes or more), then differentiate with 2% aqueous iron
chlorid solution. It takes half a minute or less to differentiate.
If the differentiation is not sufficient the nuclei will be prominent;
if it has been carried too far, the finest fibers will be too pale. They
should be black. Wash with water, dehydrate, mount in balsam or
clarite. If a counterstain is desired, picro-fuchsin (§ 610) is good.

Dr. Verhoeff says that the elastic stain with the Lugol's solution
omitted is superior to alum haematoxylin for staining nuclei, and is
very rapid. Sections are stained one minute or longer and then dif-
ferentiated with two per cent, ferric aqueous ferric chlorid, then
washed in water and counterstained with eosin or phloxine, and
mounted in balsam or one of the synthetic resins.

This stain answers well for tissues fixed in any of the standard
fixers. If the fixer contains mercury that may be removed in the
usual manner by immersing H:he slide in iodized alcohol (§ 597).

450 CABINETS; SLIPS AND COVERS; MOUNTING [Cn. XI

(5) Mallory and Wright's connective tissue stain (§ 599) gives a
bright pink color to the elastic tissue of the ligamentum nuchae, and
a blue color to its collagenous tissue.

In Dr. Mallory's new work, Pathological Technique, published in
1938, he says on page 153 that in preparing the connective tissue stain
phosphomolybdic acid was originally used, but the acid fuchsin tended
to fade. He now recommends the substitution of phosphotungstic
acid, which, he says, gives more permanent preparations. In case the
collagenous fibers are to be made as prominent as possible only the
blue collagen stain is used, the acid fuchsin being omitted.

§ 583. Eosin. — This is used mostly as a contrast stain with
hematoxylin, which is almost a purely nuclear stain. It serves to
stain the cell body, ground substance, etc., which would be
too transparent and invisible with hematoxylin alone. If eosin is
used alone, it gives a decided color to the tissue and thus aids in its
study. Eosin is used in alcoholic and in aqueous solutions. A very
satisfactory stain is made as follows: 50 cc. of water and 50 cc. of
95 % alcohol are mixed and ^V of a gram of dry eosin added. \ %
aqueous eosin is also good.

§ 584. Eosin in 95 per cent alcohol. — For staining embryos and
tissues so that the tissue in the ribbons of sections may be seen
easily a saturated solution of alcoholic eosin is made. This is also
used for staining with methylene blue.

§ 585. Eosin methylene blue. — See Mallory & Wright, Patho-
logical Technique, 8th edition, p. 102. This double stain is one of
the most useful in microscopy. It is prepared and used thus:
Eosin soluble in alcohol only, or soluble in both alcohol and water.
Saturated solution of eosin in 95 % alcohol.

Methylene blue, pure, such as is used in medicine i gram

Borax i gram

Water 100 cc.

For use dilute the methylene-blue-borax solution four or five
times with water.

Stain the slide of sections in the eosin solution 3 minutes.

Wash the eosin off with plenty of water either by flooding with a
pipette or by dipping the slide in a vessel of water.

CH. XT] CABINETS; SLIPS AND COVERS; MOUNTING 451

Stain in the diluted methylene blue 10 minutes more or less.

Rinse off the excess dye in water.

Differentiate by pouring over the slide of sections, 95% alcohol
till the sections begin to look pink. Rapidly dehydrate with abso-
lute alcohol and clear with xylene; mount in balsam. It was
pointed out by S. B. Wolbach (Jour. Amer. Med. Assoc., 1911, Vol.
56 (I), p.p. 345-346), that the addition of resin dark or light
(colophonium) to the alcohol made the differentiation more precise
and certain. He recommends the addition of i % resin for Zenker-
fixed tissue. For formalin-fixed tissue one may need to add from
5% to 10% of the resin. The use of denatured alcohol is also suc-
cessful if one uses the resin.

§ 586. Ether, ether-alcohol. — Sulphuric ether (C2H5)2O is meant
when ether is mentioned in this book. Wherever ether-alcohol is
mentioned it means a mixture of equal volumes of sulphuric ether
and 95% or absolute alcohol, unless otherwise stated.

§687. Farrant's solution. — Take 25 grams of clean, dry gum
arabic, 25 cc. of a saturated aqueous solution of arsenious acid,
25 cc. of glycerin. The gum arabic is soaked for several days in the
arsenic water, then the glycerin is added and carefully mixed with
the dissolved or softened gum arabic.

This medium retains air bubbles with great tenacity. It is much
easier to avoid them than to get rid of them in mounting.

§ 588. Flemming's Fluid. — Water 19 cc.; i% osmic acid 10 cc.;
10% chromic acid 3 cc.; glacial acetic acid 2 cc. This osmic fixer
is good for very small pieces — i to 5 millimeter pieces, thickness
not over 2 to 3 mm. Wash out with water 10 to 24 hours, then
in 67% alcohol; later in 82% and 95%.

§589. Formaldehyde (HCHO or OCH2). — -This is found in
the market under the name of " formalin," etc., and consists of a
40% solution of formaldehyde gas in water.

For fixing tissues and embryos a 5% solution is good (formalin
i cc., water 7 cc.). A common fixer is 10 cc. formalin, 90 cc. water.
This is frequently called 10% formalin; it is, however, only 4%
formaldehyde.

Tissues may stay in this indefinitely. Small pieces are fixed

452 CABINETS; SLIPS AND COVERS; MOUNTING [Cn. XI

within an hour. Before hardening in alcohol and imbedding, wash
out the formalin in running water half an hour, then harden a day or
more in 67% and 82% alcohol.

For preserving nitric-acid-dissociated muscle a 2 % formaldehyde
solution is good. Formalin i cc., water 19 cc. (§ 558). See also
§ 538 for the formaldehyde dissociator.

§590. Glycerin. Glycerol (C3H5(dH)3). — (A) One should have
pure glycerin for a mounting medium. It needs no preparation,
unless it contains dust, when it should be filtered through filter paper
or absorbent cotton.

To prepare objects for final mounting, glycerin 50 cc., water 50
cc., forms a good mixture. For many purposes the final mounting
in glycerin is made in an acid medium, viz., glycerin 99 cc., glacial
acetic or formic acid, i cc.

By extreme care in mounting and by occasionally adding a fresh
coat to the sealing of the cover-glass, glycerin preparations last a
long time. They are likely to be disappointing, however. In
mounting in glycerin care should be taken to avoid air-bubbles, as
they are difficult to get rid of. A specimen need not be discarded,
however, unless the air-bubbles are large and numerous. See also
congo glycerin (§§ 530-54°) •

§ 591. Glycerin jelly for microscopic specimens. — Soak 25 grams
of the best dry gelatin in cold water in a pyrex or agateware dish.
Allow the water to remain until the gelatin is softened. It usually
takes about half an hour. When softened, as may be readily deter-
mined by taking a little in the fingers, pour off the superfluous water
and drain well to get rid of all the water that has not been imbibed
by the gelatin. Warm the softened gelatin over a water bath and it
will melt in the water it has absorbed. Add about 5 cc. of egg albu-
men (white of egg); stir it well and then heat the gelatin in the
water bath for about half an hour. Do not heat above 75° or 80° C.,
for if the gelatin is heated too hot, it will be transformed into meta-
gelatin and will not set when cold. Heat coagulates the albumen
and it forms a kind of flocculent precipitate which seems to gather
all fine particles of dust, etc., leaving the gelatin perfectly clear.
After the gelatin is clarified, filter through a. hot flannel filter and

CH. XI] CABINETS; SLIPS AND COVERS; MOUNTING 453

mix with an equal volume of glycerin and 5 grams of chloral hydrate
and shake thoroughly. If it is allowed to remain in a warm place
(i.e., in a place where the gelatin remains melted) the air-bubbles
will rise and disappear.

In case the glycerin jelly remains fluid or semi-fluid at the ordi-
nary temperature (i8°-2o° C.), either the gelatin has been trans-
formed into meta-gelatin by too high a temperature or it contains
too much water. The amount of water may be lessened by heating
at a moderate temperature over a water bath in an open vessel.
This is an excellent mounting medium. Air-bubbles should be
avoided in mounting as they do not disappear.

§ 592. Glycerin jelly for anatomic preparations. — Specimens
prepared by the Kaiserling method or other satisfactory way may
be permanently preserved in glycerin jelly prepared as follows: Best
clear gelatin, 200 grams; Kaiserling's No. 4 solution, 3000 cc. (Po-
tassium acetate 100 grams; glycerin 200 cc.; water 1000 cc.) Put
the gelatin in the potassium-acetate-glycerin-water mixture in an
agate pail and heat over a gas or other stove. Stir. When the tem-
perature is about 55° centigrade add the whites of three eggs well
beaten, and stir them in vigorously. Make markedly acid by acetic
acid. Continue the heating until the mixture just boils, and then
filter through filter paper into fruit jars. It is best to put over the
filter paper two thicknesses of gauze. A piece of thymol in the top
of each jar will prevent the growth of fungi, or one can add 5%
chloral hydrate. Specimens are mounted in this jelly directly from
the No. 4 Kaiserling's, or alcoholic specimens can be soaked in water
an hour or more and then kept in some of the melted jelly until well
soaked; then they can be mounted permanently in the glycerin
jelly. At the time of mounting the gelatin is liquefied over a water
bath, and for every 20 cc. of the gelatin used, one drop of strong
formalin is added. This is to prevent the liquefication of the gelatin
after the specimen is mounted. Let the gelatin cool gradually after
the specimen is in place, then add some melted gelatin to make the
vessel over-full and slide a glass cover on it. This excludes all air.
The cover may then be sealed with the clear gelatin or glue used for
gluing wood, or the cement used in mending crockery. Finally ? one

454 CABINETS; SLIPS AND COVERS; MOUNTING [Cn. XI

can seal with rubber cement if desired. (See W. H. Watters, N.Y.
Med. Record, Dec. 22, 1906.)

§593. Chloral hematoxylin. — Potash alum 4 grams; distilled
water 125 cc.; hematoxylin crystals A gram. Boil 5 to 10 minutes
in an agate or pyrex dish. After cooling, add 3 grams of choral
hydrate and put into a bottle. This will stain more rapidly after a
week or two if the bottle is left uncorked. It takes from i to 5
minutes to stain sections, — sometimes a long time. Use after any
method of fixation.

It may be prepared for work at once by the addition of a small
amount of hydrogen dioxid (H2O2).

If the stain is too concentrated it may be diluted with freshly
distilled water or with a mixture of water, alum and chloral. If the
stain is not sufficiently concentrated, more hematoxylin may be
added. (Proc. Amer. Micr. Soc., 1892, pp. 125-127.)

§ 694. Iron hematoxylin. — For this stain there are three solu-
tions: (a) the mordant composed of a 2% aqueous solution of ferric
alum (iron-ammonium-persulphate); (b) a 0.5% solution of hema-
toxylin (10% alcoholic hematoxylin 5 cc., distilled water 95 cc.);
(c) the differentiating fluid composed of the ferric alum diluted
several times.

The stain can be used after any fixer, and the steps are as follows:
(i) mordant with the ferric alum i to 24 hours; (2) rinse the speci-
men 10 to 30 minutes in water; (3) stain for 3 to 24 hours in the
hematoxylin; (4) differentiate slowly, watching the effect under the
microscope. For this, dip the slide into the ferric alum in the differ-
entiator for a few seconds and then rinse with tap water. When
satisfactory, wash in running water 15 to 60 minutes. The mordant
and stain may be used several times.

§ 695. Hematein. — This is used instead of hematoxylin, as it is
believed to give more satisfactory results. Prepare as follows: Put
a 5 % solution of potash alum in distilled water and boil or leave in a
steam sterilizer an hour or two. While warm, add i per cent of
hematein dissolved in a small quantity of alcohol. After the fluid
has cooled add 2 grams of chloral for each 100 cc. of solution. (Free-
born, Jour. Ap. Micr. 1900, p. 1056.)

CH. XI] CABINETS; SLIPS AND COVERS; MOUNTING 4$$

§ 596. Iodin stain for glycogen. — Iodin i| grams; iodid of potas-
sium 3 grams; sodium chlorid ij grams; water 300 cc. For very
soluble glycogen one can use 50% alcohol 300 cc. instead of water.
The iodin stain is the most precise and differential for glycogen.
Tissues or embryos for glycogen are fixed and hardened in 95% or
absolute alcohol, and sectioned by the paraffin or by the collodion

FIGS. 245-247. BOTTLES FOR FIXING AND PRESERVING TISSUES.
Fig. 245. Wide mouth specimen bottle with glass stopper.
Fig. 246. Salt mouth bottle with glass stopper.
Fig. 247. Glass jar with screw top.

method. For permanent preparations the paraffin method is best
(§ 640). In spreading the sections use this iodin stain instead of
water. Glycogen in the sections stains a mahogany red, and the
stain remains for ten or more years in the spread paraffin sections.
Spread sections may be stained or restained by immersing the slide
in iodin stain.

456 CABINETS; SLIPS AND COVERS; MOUNTING [Cn. XI

Before mounting permanently, deparaffin with xylene, and mount
in melted yellow vaseline. Press the cover down gently. Seal with
shellac or balsam. (Trans. Amer. Micr. Soc., 1906, pp. 203-205.)

§697. lodin in alcohol. — lodin 10 grams; 95% alcohol 90 cc.
This is the strong stock solution.

For removing the pinlike or granular mercuric crystals from sec-
tions of objects fixed in any fixer containing mercury, e.g., Zenker's
fluid, etc., take 95% alcohol 500 cc. and the 10% iodin solution 5 cc.
In some cases, where the amount of mercury in the tissue is great,
one may use 10 or even 15 cc. of the strong stock solution. Rinse
the slide well in pure 95 % alcohol to remove the iodin after all the
crystals have dissolved (J an hour or more).

For embryos and tissues fixed in a mercuric fixer one can add
several drops of the stock solution to the alcohol containing the
tissue and then by changing the alcohol occasionally the mercury
will be mostly removed before sectioning. It is readily removed
from the sections as just described.

§ 598. Lamp-black for ingestion by leucocytes. — Lamp-black, 2
grams; sodium chlorid i gram; gum acacia (gum arabic) i gram;
distilled water 100 cc. Mix all thoroughly in a mortar. The gum
arabic is to aid in getting an emulsion of the lamp-black. Filter
through one thickness of gauze and one of lens paper. If for a
mammal, sterilize by boiling. If some of this mixture is injected
into an animal, the leucocytes will ingest the carbon particles. Car-
mine may be used instead of lamp-black, but it is not so good be-
cause not so enduring as lamp-black.

§ 599. Mallory and Wright's connective tissue stain. — Mallory
and Wright, Pathological Technique, 8th edition, p. 118. Mallory,
1938 edition, p. 153. Two solutions are employed:

(1) Acid fuchsin (Rubine S) Certification No. 6 0.5 gram

Water 100.00 cc.

(2) Aniline blue, water soluble 0.5 gram

Orange G 2.0 grams

i% aqueous solution of phosphotungstic acid. . . . 100.00 cc.

Keep the solutions separate. Stain first with (i), i to 5 minutes, let
the slide drain a moment and put directly in (2) without washing it.

CH. XI] CABINETS; SLIPS AND COVERS; MOUNTING 457

Leave in (2) from two to four times as long as in (i). Every tissue
seems to be a law unto itself, and one must find the best periods by
experiment.

Rinse off the water and put directly into 95% alcohol or use a
pipette and flood with 95%. Dehydrate, clear and mount in balsam.
Collagenous tissue stains blue, elastic tissue red (§ 582, 5).

This is excellent for the ligamentum nuchae, but not differential
for elastic tissue, when there are many different tissues present.

§ 600. Mercuric chlorid (HgCl2). — Mercuric chlorid y| grams;
sodium chlorid i gram; water 100 cc. The solution is facilitated
by heating in an agate dish. Fix fresh tissue in this 2 to 24 hours.
Then transfer to 67 % alcohol a day or more and then to 82 % al-
cohol. Tissues fixed in mercuric chlorid deteriorate; hence it is
better to imbed them soon after they are fixed. Crystals of mer-
cury are removed from the sections by the use of iodized alcohol

(§ 597).

§601. Methylene blue, alkaline. — Methylene blue 2 grams;
95% or absolute alcohol 50 cc.; distilled water 450 cc.; i% aqueous
caustic potash 5 cc. This stain works best after a fixer containing
mercuric chlorid, like Zenker's fluid. (See § 584 for eosin in alco-
hol.)

§ 602. Mineral oil, pure, medicinal (petrolatum). — The pure
mineral oil used in medicine does not fluoresce and is of nearly the
refractive index of glass (nD 1.4815). It is good for mounting un-
stained sections, and for an immersion liquid. (See under the ultra-
violet microscope (§§ 309, 536).

§ 603. Miiller's fluid. — Potassium dichromate 2\ grams; sodium
sulphate i gram; water 100 cc. This is one of the oldest fixers. It
must act a long time, two weeks to 10 or 12 weeks. This longer
time is for nervous tissue to be stained for the myelin. Lately this
fixer has been combined with mercury. (See Zenker's fluid, § 615.)
Before putting the tissue into 67% alcohol it is washed out in run-
ning water for 24 hours.

Miiller's fluid 10 cc.; normal salt solution 90 cc. forms an excel-
lent dissociator for epithelia, etc. (§ 537).

§ 604. Neutral red. — This is used especially for staining living

458 CABINETS; SLIPS AND COVERS; MOUNTING [Cn. XI

animals. It is used in very weak solutions: T10 gram red; 1000 cc.
of water. Put a few cubic centimeters of this solution into the
vessel containing the live animal, or animals. Infusoria stain
quickly, 10 to 20 minutes or less. Vertebrates may require a few
days. Try it on infusoria by adding a drop of the red to several
drops of the infusion containing the infusoria. Be sure that there
are many animals present. Watch them under the microscope and
the color will be seen appearing in the granules of the infusoria.
Then one may cover, and study with a high power (§ 543).
For vital stains and the technique of their use see McClung, 74-81;
Lee Microtomists Vade Mecum, 8th ed. Kingsbury, Histological
Technique, and Kingsbury and Johannsen, Conn; Biological Stains,
2nd. edition.

§ 605. Nitric acid, HNO3. — This is employed for dissociation
(nitric acid dissociator: water 80 cc., nitric acid 20 cc.); as a fixer,
especially for chick embryos in the early stages (water 90 cc.; nitric
acid, 10 cc.), and as a decalcifier (nitric acid 3 cc.; 67% alcohol 100
cc.).

§ 606. Normal liquids. — A normal liquid or fluid is one which
does not injure or change a fresh tissue put into it. The perfect
normal fluids for the tissues of any animal are the fluids of the body
(lymph and plasma) of the animal from which the tissue is taken.
The lymph or serum of one species of animal may be far from normal
for the tissues of another animal. (See also § 521.)

The commonly used artificial normal fluid is a solution of common
salt (sodium chlorid) in water, the strength varying from -fa to -r%-
per cent. As indicated above, this normal salt or saline solution is
employed in diluting dissociating liquids (§521).

§ 607. Paraffin wax. — A histologic laboratory requires two
grades of paraffin for ordinary work. These are hard paraffin,
melting at about 54° centigrade, and a softer paraffin melting at
about 43° centigrade. Usually a mixture of equal parts answers
very well. It is economical for a laboratory to buy the paraffin wax
in cases of about 100 kilograms.

All paraffin for imbedding and sectioning should be filtered
through two thicknesses of filter paper. * For this, use a metal

CH. XI] CABINETS; SLIPS AND COVERS; MOUNTING

459

funnel, heat the paraffin very hot in a water bath, and then heat
the funnel occasionally with a Bunsen flame. The warmer the
room, the easier it is to filter the paraffin.

Filter the paraffin into small porce-
lain pitchers. If the paraffin oven has
a compartment large enough, it is well
to keep one of the pitchers in the oven;
then the paraffin remains melted and is
ready for use at any time.

§608. Picric-alcohol. — This is an
excellent hardener and fixer for almost
all tissues and organs. It is composed of
500 cc. of water and 500 cc. of 95%
alcohol, to which 2 grams of picric
acid have been added. (It is a | %
solution of picric acid in 50% alcohol.)
It acts quickly, in from one to three
days. (Proc. Amer. Micr. Soc., Vol.
XII (1890), pp. 120-122.) Not recom-
mended for ultra-violet work.

§ 609. Petrolatum liquidum. See mineral oil (§ 602).

§ 610. Picro-fuchsin. — 10 cc. of a i % aqueous solution of acid
fuchsin; 75 cc. of a saturated aqueous solution of picric acid. Stain
deeply with hematoxylin first; then use the picro-fuchsin. Wash
off the picro-fuchsin with distilled water. Mount in non-neutralized
balsam, or better in acid balsam (balsam 50 cc., glacial acetic acid 5
drops). If the white connective tissue is not red enough, increase
the amount of acid fuchsin.

§ 611. Shellac cement. — Shellac cement for sealing preparations
and for making shallow cells is prepared by adding scale or bleached
shellac to 95% alcohol. The bottle should be filled about half full of
dry shellac; then enough 95% alcohol added to fill the bottle nearly
full. The bottle is shaken occasionally and then allowed to stand
until a clear stratum of liquid appears on the top. This clear, super-
natant liquid is then filtered through filter paper or absorbent cot-
ton, using a paper funnel (§ 566), into an open dish or a wide-mouth

FIG. 248. SPECIMEN JAR
WITH CLAMP.

460 CABINETS; SLIPS AND COVERS; MOUNTING [Cn. XI

bottle. To every 100 cc. of filtered shellac 2 cc. of castor oil may
be added to render it less brittle. The filtered shellac will be too
thin, and must be allowed to evaporate till it is of the consistency
of thin syrup. It is then put into a capped bottle. In case the
cement gets too thick add a small amount of 95 % alcohol or some
thin shellac. The solution of shellac almost always remains muddy,
and in most cases it takes a long time for the flocculent substance
to settle. One can quickly obtain a clear solution as follows: when
the shellac has had time to dissolve thoroughly, i.e., in a week or
two in a warm place, or in less time if the bottle is frequently
shaken, a part of the dissolved shellac is poured into a bottle and
about one-fourth as much gasoline is added and the mixture well
shaken. After twenty-four hours or so the flocculent, undissolved
substance will separate from the shellac solution and rise with the
gasoline to the top. The clear solution may then be siphoned of! or
drawn off from the bottom if one has an aspirating bottle. (R.
Hitchcock, Amer. Monthly Micr. Jour., July, 1884, p. 131.)

If one desires to color the shellac, the addition of a strong alcoholic
solution of some of the coal tar colors is good, but is likely to dis-
solve in the mounting medium when shellac is used for sealing. A
small amount of lamp-black well rubbed up in very thin shellac and
filtered is good to darken the shellac.

§ 612. Silvering. — Intercellular substance stains brown or black
with nitrate of silver. Use \ or |% aqueous solution on fresh
tissue. Stain in the silver for i or 2 minutes; then expose to light
in water till brown. Fix in 82% alcohol or 5% formaldehyde. One
may stain afterward with hematoxylin for the nuclei; mount in
glycerin, glycerin jelly, or in balsam.

§ 613. Sudan red, III for fat. — Sudan III, aminoazo-benzene-
j8-naphthol C^HieON^ was introduced by Daddi into histology in
1896 (Arch. Ital. de Biologie, t. 26, p. 142), as a specific stain for
fat. As it is soluble in all forms of fat and oils and in xylene,
alcohol, etc., it is impossible to mount specimens in balsam after
staining. As the fat of tissues is removed by the reagents used in
the paraffin and collodion methods, only teased, free-hand, or
frozen sectioned material, fresh or fixed in some non-fat dissolving

Cu. XI] CABINETS; SLIPS AND COVERS; MOUNTING 461

fixer, can be used (Miiller's fluid and 5% formaldehyde are excel-
lent). The tissues cut free-hand or with the freezing microtome or
teased can then be stained with a saturated alcoholic solution of the
Sudan. It stains all fat a brilliant red. Preparations can be pre-
served in glycerin or glycerin jelly. This stain is largely used in
pathology.

Daddi used the substance to feed animals and thus to stain the
fat which was laid down in the body while the Sudan was fed.

The fat in the body already deposited remains unstained. This
substance then serves to record the deposit of fat in a given period.
In 1907 Dr. Oscar Riddle fed Sudan to laying hens, and the fat in
the layers of yolk laid down during the feeding was stained red
(Science, XXVII, 1908, p. 495). For staining the yolks of hen's
eggs the hen may be fed doses of 20 to 25 'milligrams of the sudan.
Eggs so colored hatch as usual, and the chick, in utilizing the colored
yolk, stains its body-fat pink (Susanna P. Gage).

Sudan IV or scarlet red is also used for a fat stain. See Gage,
S. H. and Fish, P. A., Amer. Jour. Anat, Vol. 34, 1924.

§ 614. Table Black. — During the last few years an excellent
method of dyeing wood with anilin black has been devised. This
black is iusterless, and it is indestructible. It can be removed only
by scraping off the wood to a point deeper than the stain has
penetrated.

It must be applied to unwaxed or unvarnished wood. If wax
paint or varnish has been used on the tables, that must be first
removed by the use of caustic potash or soda or by scraping or
planing. Two solutions are needed:

Solution A

Copper sulphate 125 grams

Potassium chlorate or permanganate 125 grams

Water 1000 cc.

Boil these ingredients in an iron kettle until they are dissolved.
Apply two coats of the hot solution. Let the first coat dry before
applying the second.

462 CABINETS; SLIPS AND COVERS; MOUNTING [Cn. XI

Solution B

Anilin oil 120 cc.

Hydrochloric acid 180 cc.

Water 1000 cc.

Mix these in a glass vessel, putting in the water first. Apply two
coats without heating, but allow the first coat to dry before adding

the second.

When the second coat is dry, sandpaper
the wood and wash it with water. When
the wood is dry, sandpaper the surface
again and then rub thoroughly with a

FIG. 249. DRYING RACK mixture of equal parts turpentine and
WITH INCLINED PEGS FOR 1}nseed oiL The wood may appear a
BOTTLES. / \*

dirty green at first, but it will soon

become ebony black. An occasional rubbing with linseed oil and
turpentine or with turpentine alone will clean the surface. This is
sometimes called the Danish method, Denmark black or finish.
See Jour. Ap. Micr., Vol. I, p. 145; Bot. Zeit., Vol. 54, p. 326; Bot.
Gazette, Vol. 24, p. 66; Dr. P. A. Fish, Jour. Ap. Micr., Vol. VI,

pp. 2II-2I2.

§ 615. Zenker's fluid. — Muller's fluid (§ 603) 100 cc. ; mercuric
chlorid 5 grams. Just before using, add 5 cc. of glacial acetic acid
to each 100 cc. of the above. Fix fresh tissue 5 to 24 hours. Wash
out with running water 24 hours. Then place in 67% alcohol i day
or more and finally preserve in 82 % alcohol. Tissue fixed in Zen-
ker's has mercuric crystals. They may be removed from the tissue
by long treatment with iodin, or by putting the slide bearing the
sections in iodized alcohol for half an hour or more.

This is an excellent fixer, combining the good qualities of mercuric
chlorid and of the chromium compounds. Tissues fixed with this
show well the red blood corpuscles. This is called Kelly's fluid if
the acetic acid is replaced by 5 % formalin.

CH. XI] CABINETS; SLIPS AND COVERS; MOUNTING 463

COLLATERAL READING FOR CHAPTER XI

LEE, A. B. — The Microtomist's Vade Mecum, gth ed., 1928.

KINGSBURY, B. F. — Histological Technique, 1915.

MANN, G. — Physiological Histology, 1903.

EHRLTCH, P., ET AL. — Enzyklopaedie der mikroskopischen Technik, IQICX

WRIGHT, SIR A. E. — Principles of Microscopy, 1907.

CARPENTER-DALLINGER. — The Microscope and Its Revelations, 1901.

SPITTA, E. J. — Microscopy, 1907

Anatomical Record.

Journal of the Royal Microscopical Society.

Transactions of the American Microscopical Society

Journal of Experimental Zoology.

Botanical Gazette.

BOYER, C. S. — The Diatomaceae of Philadelphia and Vicinity, 1916.

DUDLEY AND THOMAS. — Manual of Plant Histology, 1894

CHAMBERLAIN, C. J. — Methods in Plant Histology, 1916.

STEVENS, W. C. — Plant Anatomy, 1915.

EWART, A. J. — Protoplasmic Streaming in Plants, 1903.

BERNARD, CLAUDE. — Lecons sur les Phenomenes de la Vie communs aux Ani-
maux et aux Ve'ge'taux. Two vols. 1878-1879.

NEEDHAM & LLOYD. — The Life of Inland Waters, 1916. This is a most impor-
tant work for all interested in water forms

WARD & WHIPPLE. — Fresh- Water Biology.

CONN, H. J. — Biological Stains, 4th ed., 1940.

MCCLUNG, C. E. — Handbook of Microscopical Technique, 2d ed., 1937.

KRAUSE, R. — Enzyklopaedie der mikroskopischen Technik. Three volumes.
3d revised edition, 1927.

MORGAN, ANNA H. — Field Book of Ponds and Streams, 1930.

MALLORY, FRANK BURR. — Pathological Technique, a practical manual for workers
in pathological histology. Philadelphia, 1938.

CHAPTER XII

FIXING AND THE PRESERVATION OF TISSUES, ORGANS, AND
ENTIRE ORGANISMS. IMBEDDING, SECTIONING, STAINING,
AND MOUNTING FOR THE MICROSCOPE
§§ 616-663; FIGURES 260-266

§ 616. Fixation and preservation of organs and tissues. — By
fixing or fixation in histology is meant the preparation of fresh
tissues, organs, embryos or small adult animals usually by means of
some chemical mixture, called a " fixer," so that the organ, etc., as a
whole and the elements or cells composing it shall retain as nearly
as possible the morphologic characters present during life. The
more perfect the fixer, the nearer will be the preservation of all
structural details.

Unfortunately no single " fixer " preserves with equal excellence
all the structural details, and therefore it is necessary to prepare the
fresh tissue in several different ways and to make a composite of the
structural appearances found, thereby approximating the actual
structure present in the living body. Changes are so rapid after

FIG. 250. WASHING BOXES FOR TISSUES FIXED IN A LIQUID CONTAINING MER-
CURIC CHLORID.
(From the Journal of Applied Microscopy).

T Small stop cock or pet cock in the usual water faucet so that a small
stream may be drawn without interfering with the large faucet.

Only the larger trays are now used, the perforated inner tray being deep or
shallow as needed.

464

CH. XII]

FIXING, SECTIONING AND MOUNTING

465

death that the fixation should begin as soon 'as possible. For the
most perfect fixation the living tissue must be put into the fixer.

With one of the larger animals, where the whole animal is to be
used for microscopic study, it is a great advantage to bring the fixer
in contact with all parts of the body quickly, and that is done by
washing out the vascular system with normal salt solution and then
filling the vascular system with the fixer. This method of fixation
by injection is of great importance in the histology of animals which
a;e large enough to inject.

KTG. 251. METAL WASHING BOXES FOR TISSUES FIXED IN A LIQUID CONTAIN-
ING MERCURIC CHLORID.
(From the Journal of Applied Microscopy).

The deeper box is now used only and depending on the size of the pieces to be
washed the shallow or the deep perforated trays and tissue baskets are used.
The deep tray serves for washing slides with Weigert and other stains which must
be in water a long time.

If the animal is too small for injection or one wishes only a small
part of a larger animal, then the pieces for fixation should be small,
say one to three cubic centimeters. Often, as for Flemming's fluid
(§ 588) and for several others, it is better to use pieces 2 to 5 cubic
millimeters in volume.

Large, solid organs must be cut into several pieces if the whole
is needed. For hollow organs the cavity may be filled with the
fixer and the organ placed in a vessel of the same.

The amount of fixer should be 10 to 50 times that of the piece of
tissue.

466 FIXING, SECTIONING AND MOUNTING [Cn. XII

Of the fixers given under " Preparation of Reagents/' picric alco-
hol, formalin and Zenker's fluid are suitable for almost every tissue
and organ. Formalin has the advantage of having strong penetra-
tion; hence it preserves whole animals fairly by immersing after
filling the abdominal and thoracic cavities. Formaldehyde is excel-
lent where a study of fat is in question, and it is much used as a fixer
where frozen sections are desired (§ 625). Remember the necessity
of removing mercury from sections of tissues fixed with a mercuric
fixer (figs. 250-251).

§ 617. Mechanical preparation of tissues, etc., for microscopic
study. — A limited number of objects in nature are small enough
and transparent enough, and a limited number of the parts of higher
animals are suitable for microscopic study without mechanical prepa-
ration except merely mounting them on a microscopic slide. Usually
the parts of animals are so large and so opaque that the histo-
logic elements or cells and their arrangement in organs can only be
satisfactorily studied with a microscope after the tissue, organ, etc.,
have been teased apart with needles, or sectioned into thin layers.

MICROTOMES AND SECTION KNIVES

§ 618. The older histologists, those who laid the foundations and
whose understanding of the finer structure of the body was in many
ways superior to the knowledge possessed by workers at the present
time, did their mechanical preparation with needles and with sharp
knives held in the hand. They dealt also with fresh tissue more
largely than we do at the present day, and learned also to distinguish
tissues by their structure rather than by their artificial coloration.

What made them so successful was not, however, the lack .of
elaborate mechanical devices for sectioning and the complicated
staining methods of the present day, but that they put intelligence
and zeal into their work.

If the reader is interested in the mechanical means for sectioning
he is referred to Dr. C. S. Minot's papers on the history of the micro-
tome in the Journal of Applied Microscopy, Vol. VI, and to Gilbert
Morgan Smith's article in the Transactions of the American Micro-

CH. XII] FIXING, SECTIONING AND MOUNTING 467

scopical Society, Vol. XXXIV, 1915, on the Development of Botani-
cal Microtechnique, pp. 71-129, 16 pages of bibliography; 18 figures,
showing early microscopes and microtomes.

§ 619. Types of microtomes. — There are two great types: (i)
The early type in which the preparation to be sectioned is held me-
chanically and moved up by a screw, the section knife being held in
the hand and moved across the object, usually with a drawing mo-
tion as in whittling.

(2) The mechanical type, in which both specimen and knife are
mechanically held and guided, and the operator simply supplies
power to the machine, or, when an electric motor is used, the
operator starts and stops the machine and uses his hands in taking
off the ribbon as it is cut. The ribbon is wound on a cylinder or
cut into the proper lengths for the slide trays (figs. 237-238).

Tn the highest types of the second class — automatic microtomes —
the operator needs only to put the knife and specimen in position
and sections of any thickness and any number may be produced in
a short time. A skilled and experienced person can get better re-
sults here as well as with free-hand sectioning or the hand micro-
tome. Even automatic machines work better for skilled workmen.

In some forms the knife of these automatic microtomes is fixed
in position and the object to be sectioned moves, while in other
forms the object to be sectioned remains fixed and the knife moves.
Furthermore, for sectioning paraffin, the knife meets the object
like a plane (straight cut), while for collodion sectioning the knife
is set obliquely and there results an oblique or drawing cut, as in
whittling. For the latest models, see catalogues of the microscope
manufacturers.

§ 620. Section knives. — A section knife should have the follow-
ing characters, (i) The steel should be good. (2) The blade should
be slightly hollow ground on both sides. (3) The edge of the knife
should be straight, not curved as in a shaving razor. (4) The back
should be parallel with the edge. (5) The blade should be long, 12
to 15 centimeters, as it takes no more time or skill to sharpen a large
than a small knife. (6) The blade should be heavy.

§ 621. Safety razor bkdes for sectioning. — Recently the Bausch

468

FIXING, SECTIONING AND MOUNTING

[Cn. XII

& Lomb Optical Co., and the Spencer Lens Co., have put on the
market holders for these blades that make them available as section

FIG. 252. SECTION RAZOR WITH HEAVY BLADE HAVING STRAIGHT BACK

AND EDGE.

knives in histology. The holders furnish the needed rigidity. Only
about two millimeters of the cutting edge projects above the holder
(fig. 2S2a). Extended personal use of these blades with the
holders on the most varied material leads the author to recommend
them strongly. They take away the time-consuming and tiresome
labor of sharpening the large section knives. They are also recom-
mended for much of the dissecting work. They may be held by the
fingers, but preferably clamped to a handle by a small bolt.

FIG. 25 2a. SAFETY RAZOR BLADE HOLDER TOR THE MICROTOME.

(About one- third natural size).

I Rigid metal the size of an ordinary microtome knife, to take the place of
the knife in the microtome.

2-3 Jaws for holding the safety razor blade. It gives such firm support that
practically only the cutting edge of the blade is free.
4 Screw head for tightening the jaws, 2-3.

As the cutting edge is not equally good in all the blades it is
worth while to examine the edge under the microscope to see that
it is smooth and free from nicks if one wishes to get the best thin
sections, $ju and less.

CH. XII] FIXING, SECTIONING AND MOUNTING 469

§622. Sharpening section knives; hones and strops. — Perhaps
it should be taken for granted that any one would appreciate the
impossibility of making good sections with a dull section knife, but
experience teaches the contrary. Students are prone to believe that
with one of the elaborate automatic microtomes, good sections may
be made with any kind of an edge on the knife. It is forgotten that
the knife is the most important part; all the other mechanism is
simply its servant.

For sharpening, select a fine yellow Belgian hone, and a very fine
Arkansas hone. As a rule hones from the factory are not sufficiently
plane. They may be flattened by rubbing them on a piece of plate
glass covered with moderately fine emery or carborundum wet with
water. Round the corners and edges of the hones on the plate glass
or on a grindstone. In using the Belgian hone for sharpening knives,
wet the surface well with a moderately thick solution of soap. With
the Arkansas stone use some thin oil — xylene or kerosene mixed
with a little olive oil or machine oil.

Honing. Before honing a section knife, make sure that the edge
is smooth; that is, that it is free from nicks. Test this by shaving
off the surface of a block of paraffin. If nicks are present the cut
surface will show scratches. It is advisable also to look at the edge
of the knife with a magnifier and with a low power (48 (2x) mm.) ob-
jective. If nicks are present remove them by drawing the edge along
a very fine Arkansas hone.

A saw edge may be all right for rough cutting and for shaving
razors, but if one wishes to get perfect sections i/z to loju in thick-
ness a saw edge will not do. In removing the nicks one should,
of course, bear on very lightly. The weight of the knife is usually
enough.

In honing use both hands; draw the knife, edge foremost, along
the hone with a broad, curved motion. In turning the knife for the
return stroke, turn the edge up, not down. Continue the honing
until the hairs on the arm, wrist, or hand can be cut easily or until
a hair from the head can be cut within 5 rnm. from the point where
it is held. The sharper the knife becomes, the lighter must one bear
on. One should also use the finest stone for finishing. If one bears

4 70 FIXING, SECTIONING AND MOUNTING [Cn. XII

on too hard toward the end of sharpening, the edge will be filled
with nicks.

In honing and stropping large section knives, there has come into
use during the last few years the so-called " honing backs." These
elevate the razor slightly, so that the wedge is blunter and one does
not have to grind away so much steel.

Strop. A good strop may be made from a piece of leather (horse-
hide) about 50 cm. long and 5 to 6 cm. wide, fastened to a board of
.about the same size.

The strop is prepared for use by rubbing into the smooth surface
some carborundum powder, i.e., 6o-minute carborundum, that which
is so fine that it remains in suspension in water for 60 minutes, or
one may use diamantine or jewelers' rouge.

Stropping. With the back foremost, draw the knife lengthwise
of the strop with a broad sweep. For the return stroke turn the
edge up as in honing. Continue the stropping until a hair can be
cut i to 2 centimeters from where it is held. (See also the hones
and strops and the methods of procedure recommended in the
catalogues of microscopical manufacturers.)

§ 623. Free-hand sectioning. — To do this one grasps the sec-
tion knife in the right hand and the object in the left. Let the end
to be cut project up between the thumb and index finger. One can
let the knife rest on the thumb or index finger nail and, with a
drawing cut, make the section across the end of the piece of tissue.
By practice one learns to make excellent sections this way. If the
whole section is not sufficiently thin, very often a part will be and
one can get the information needed. The importance of acquiring
skill in free-hand sectioning cannot be overestimated if one is to
study living and fresh tissues, and without such study no one can
gain a fundamental knowledge of structure. It is also of the highest
value in the preparation of living and fresh objects for study with
the polarizing and the ultra-violet microscopes.

§ 624. Sectioning with a hand or table microtome. — The tissue
is held by the microtome and moved up by means of a screw. The
knife rests on the top of the microtome and is moved across the
tissue by the hand. Microtomes of this kind are excellent. No one

CH. XII] FIXING, SECTIONING AND MOUNTING 471

need wait for expensive automatic microtomes to do good section-
ing. With a good table microtome, the knife being guided by the
hand or hands of the operator, he can make straight cuts as for
paraffin sectioning, or drawing cuts as for collodion work.

§ 625. Sectioning with a freezing microtome. — In this method of
sectioning the tissue is rendered firm by freezing and the sections are
cut rapidly by a planing motion as with paraffin. Now the most
usual freezing microtome is one in which the freezing is done with
escaping liquid carbon dioxid. The knife should be very rigid. A
carpenter's plane blade is often made use of. The tissue may be
either fresh or fixed. If alcohol has been used, it must be soaked out
of the tissue by placing it in water. Sometimes tissues are infiltrated
a day or two in thick gum arabic mucilage before freezing. Drop a
little thick mucilage on the top of the freezer, put the tissue in the
mucilage, and turn on a small amount of carbon dioxid. It will
soon freeze the mucilage and the tissue, as shown by the white ap-
pearance. When frozen, cut the tissue rapidly. It is well to have
an assistant turn the feed screw up while the sections are cut.
When 20 or 30 sections are cut, place them in water or normal salt
solution. This is a rapid method of getting sections much used in
pathology where quick diagnoses are demanded. In normal histol-
ogy the freezing microtome is used mostly for organs or parts of
greatly varying density. For example, if one wishes sections of the
finger and finger nail, this apparatus offers about the only means of
getting good sections. In that case the bone is decalcified before
trying to make the sections (§ 580).

Frozen sections are also very useful for demonstrating the presence
of fat by staining with Sudan III.

THE PARAFFIN METHOD OF SECTIONING

§ 626. Object of the paraffin. — In the early periods in histology
great difficulty was encountered in making good sections of organs
and parts of organs, because the different tissues were unlike in
density. At first tallow and beeswax, elder pith, liver, and various
other substances were used to enclose or surround the object to be

472

FIXING, SECTIONING AND MOUNTING

[CH. XII

cut. This gave support on all sides, but did not render the object
homogeneous. In the early sectioning, a great effort was made to
keep all imbedding material from becoming entangled in the meshes
of the tissue. This was guarded against by coating the object with
mucilage, and hardening it in alcohol. This mucilage jacket kept
the tissue free from infiltration by the imbedding mass and it
was easily gotten rid of by soaking the sections in water.

A great advance was made when it was found that the imbedding
mass could be made to fill all the spaces between the tissue elements
and surround every part, the tissue assuming a nearly homogeneous
consistency, and cutting almost like the clear imbedding mass.
Cocoa butter was one of the first substances to be used for thus
" infiltrating " the tissues. The imbedding mass must usually be
removed before the staining and mounting
processes; but in staining for glycogen by
the iodin method, the stain is applied be-
fore the paraffin is removed (§ 596).

§ 627. Infiltration of the tissue with im-
bedding mass. — The tissue to be cut in this
way is first fixed by one of the fixers used
for histology. Several good ones are given
in sections 589, 608, 615, 616.

(A) The tissue is then thoroughly dehy-
drated by means of 95% and absolute
alcohol. For most objects, especially em-
bryos and other colorless objects, it is best,

1 Upper part of the during the dehydration, first to use dilute

oven containing the covered alcoholic eosin (§ 583), as the most delicate

pitcher for the paraftin. , , J °' ,

2 Lower part contain- part shows when one cuts the sections,
ing the incandescent lamps Leave the piece of tissue to be cut over-
and supply cable (c). The .,.,,,. . , r i

oven is well insulated by night in alcoholic eosin, and a tew hours in

asbestos. Depending on uncolored 95% alcohol, using 20 times as
the temperature of the , , , , - ^ 7i_ r- i i .

room, one or both lamps much alcohol as tissue. For the final dehy-

can be used to keep the dration it should be left in absolute

paraffin melted. 1,1* /- T_ • u*.

alcohol four or five hours or overnight,

depending on the size of the object.

FIG. 253. KINGSBURY'S
PARAFFIN MELTING OVEN.
(From the Anatomical
Record).

CH. XII]

FIXING, SECTIONING AND MOUNTING

473

(B) Remove the alcohol by a solvent of the imbedding mass;
that is, by some substance which is miscible with both alcohol and
the imbedding mass. Cedar-wood oil is most generally used, but
pure xylene, chloroform, and carbol-xylene are also used, — the
chloroform and carbol-xylene when osmic acid fat is to be retained
in the tissue.

Leave the tissue in cedar oil or other clearer until the tissue sinks
and the thin parts of the specimen become translucent. If the tissue
does not sink after a time it means that the tissue was not dehy-
drated. Of course, this does not apply to lung or other spongy
tissue containing much air. It is well to change the cedar oil or
other clearer once. The used
cedar oil may be left in an open
bottle for the evaporation of
alcohol and used over and over
again.

(C) Displace the cedar oil or
other clearer by melted paraffin
wax. When the tissue is sat-
urated with the oil, transfer it
to an infiltrating dish (fig. 254)

FIG. 254. ELECTRIC INFILTRATING OVEN
WITH PROJECTING SPREADING PLATE.
(About one-eighth natural size. See

containing melted paraffin. Place
in a paraffin oven (fig. 254) and

keep the paraffin melted for from brass spreading plate projecting 8 cm.
two hours to three days, de- io***£ or tray holding the oven; and
pending on the size and character the infiltrating and paraffin dishes, (i,
of the piece to be imbedded. If *• 3, 4^, ^ ^ ^ ^ ^
the tissue is thoroughly dehy- lain receptacle for the lamp bulb.
drated and well saturated with J£^^'£±'ljgl£
cedar oil, the melted paraffin spreading plate. The dimensions are:
permeates the whole piece. See *^9*£%£ij™3t*'IiS
§ 641 for the propyl alcohol cm. high, Tray, 30 x 18 x 2 cm.
method.

§ 628. Imbedding in paraffin wax. — When the object is thor-
oughly infiltrated, imbed as follows: Make of strong writing paper
a box considerably larger than the piece to be imbedded. Nearly fill

474

FIXING, SECTIONING AND MOUNTING

[CH. XII

the box with paraffin wax, place on a copper heater (fig. 260), and
allow to remain until bubbles appear in it. Put the box on cold
water until a thin stratum of paraffin solidifies on the bottom.
Take the piece of tissue from the infiltrating dish (fig. 254) and

0 «

FIG. 255. INFILTRATING Box AND SPREADING PLATE.

1 Connection for the electric circuit.

2 Screw heads. The screws hold the electric bulb socket in place.

3 Infiltrating box. It is 30 cm. long; 17.5 wide; 12.5 deep. The ends and
sides are lined with thick asbestos. 3 mm. thick. The brass top, or spreading
plate, is 17.5 cm. wide and 38 cm. long, i.e., it projects 8 cm. beyond the box (3).

5, <5, 7 Inliitrating and paraffin dishes

8 End of the box attached to the bottom (4). This infiltrating box adopts
Dr. Kingsbur/s plan of having the top and bottom sliding apart or together to
regulate the temperature, and for ease in handling the intiltrating and paraffin
dishes. G lamp bulb of 25 to 40- watt capacity supplies sumcient heat in a room
at the ordinary temperature (20° centigrade).

arrange in the box for making sections in a definite direction. Add
hot paraffin, if necessary, and then place the box on cold water.
The more rapid the cooling, the more homogeneous will be the
block containing the tissue to be cut. For the best imbedding it is
well to drop 95% alcohol on the surface as soon as a film has
formed in cooling. In warm climates where cold water is not easy
to procure for cooling the blocks, one may float the paper box on
95% alcohol and with a pipette (fig. 264) drop strong alcohol on the
sides of the box and on the top of the paraffin as soon as a surface
film has formed.

It is very desirable to mark on the box the name of the imbedded
object and to indicate which end or face is to be cut (§ 672).

§ 629. Fastening the block to a holder. — Use one of the block
holders or object discs furnished with the microtome, or a short

CH. XII]

FIXING, SECTIONING AND MOUNTING

475

stove bolt. Heat the larger end and press the paraffin block against
the hot metal until it melts the paraffin. Hold the two together

FIG. 256. DR. KINGSBURY'S HEATING Box AND SPREADING PLATE.

1 Connection for the electric circuit.

2 Heating box, sides and ends lined with thick asbestos. Size of box: 30
cm. long; 16 cm. wide and 8 cm. deep.

3 The bottom of the box with the end (5) to close the box when the two parts
are brought together.

4 The 8 cm. projection of the spreading plate. This plate is of brass, 3 mm.
thick.

5-6 End of the heating box when the 2-3 are brought together, 6 is a knob
to grasp in separating or putting together the sliding top and bottom (2-3).

The lower box is more convenient for spreading sections than the higher box
(fig. 255), but not so large paraffin infiltrating vessels can be kept in it. The
sliding feature of top and bottom enables one to control the temperature closely.

while cold water flows over them. When cold, the block is firmly
cemented to the holder. Pains should be taken to have the axis of
the block parallel with the long axis of the holder; and one should

3V «•

L 4

E CM

B CM

4 L

/ CM

S CM

FIG. 257. DIAGRAM SHOWING How TO MAKE A PAPER Box FOR IMBEDDING.

j Lines for the first folds; these make three longitudinal strips.

2 Lines for the second folds; these make three transverse strips.

3 Lines showing where the corner folds are made.

4 The folds for the projecting end or label.

13 Bottom, S Side, E Ends and L Label of the box. The bottom occupies J
of the area.

476

FIXING, SECTIONING AND MOUNTING

[Cn. XII

not cut the block so short that the holder comes in contact with
the tissue when the paraffin and holder are cemented together.

§ 630. Fixing the imbedding block directly in the microtome.
With objects of considerable size, it is not necessary to fasten the
imbedding block to a metal holder, and then to clamp that in the
microtome, but the paraffin block itself can be put in the object
clamp of the microtome.

§ 631. Trimming the end of the block
for sectioning. — Sharpen the end to be
cut in a pyramidal form, being sure to
leave 2 millimeters or more of paraffin over
the tissue at the end as well as on the
sides. The block is trimmed in a pyra-
midal form, so that it will be rigid. Take
particular pains that the opposite faces at
the end of the block are parallel and all
the corners right angles.

In some laboratories, Dr. McClung's for
example, a cubical block of metal attached
to a rod is placed in the knife holder of
the microtome and the four sides of the
imbedding mass trimmed with great ex-
actness by the use of a straight-edged
scalpel, or better by a small chisel, the
cube of metal serving as a guide. As the

FIG. 258. SCALPEL BLADES. metal cube can be slid alonS in the knife

i, 2 with curved edges holder, and the imbedded tissue can be

for cutting ribbons; ?, with raised and iowered by turning the wheel
straight edge for trimming J 6

paraffin blocks. of the microtome, imbedding masses of

large and small sizes can be trimmed by

the same metal guide. This guide for trimming is a great help in
getting straight ribbons, and consequently good series.

§ 632. Making paraffin sections. — Put the paraffin block or the
metal holder in the clamp of the microtome. Arrange the block so
that one side of the pyramidal end is parallel with the edge of the
knife; then tighten the clamp; and if an automatic microtome is

CH. XII] FIXING, SECTIONING AND MOUNTING 477

used, make sure that the section knife is also tightly clamped by the
proper set screws. It is well to have the knife lean slightly toward
the paraffin blocks.

The knife edge meets the paraffin squarely, as in planing. The
thickness of section is provided for in the automatic microtome by
the indicator, which may be set for any desired thickness, or one
can turn up the screw by hand in the table microtome. The par-
affin and its contained tissue are cut in a thin shaving. If the
tissue was stained in toto with eosin, as suggested in § 627 A, it is
marked out with great clearness in the containing paraffin (§ 672).

As succeeding sections are cut, they push along the previous sec-
tions, and if the hardness of the paraffin is adapted to the tempera-
ture where the sectioning is done, the edges of the successive sections
will be soldered as they strike. This produces a ribbon, as it is
called, and if the paraffin block has been properly trimmed at the
end the ribbon will be straight and even. If the ribbon is curved
sideways, it indicates that one side of the block is thicker than the
other and the sections are slightly wedge shaped.

If the paraffin is too hard for the room temperature and for a
given thickness of section, the sections will curl; if it is too soft, the
sections will crumple.

The thinner the sections, the harder should be the paraffin or the
cooler the sectioning room; and the thicker the sections and the
larger the object to be cut, the softer can be the paraffin and the
higher the temperature. If, then, the sections do not ribbon, make
thinner sections or work in a warmer place. If the sections crumple,
make thicker sections or work in a cooler room. Of course, one can
reimbed in a more suitable hardness of paraffin.

In the season when steam radiators are used, one can get almost
any desired temperature by sectioning nearer or farther from the
radiator.

In the winter it is a good plan to warm the microtome and section
knife before sectioning. This can be done very easily by putting a
cloth over the radiator and the microtome something like a tent.

§ 633. Electrification of the paraffin ribbons. — Some days there
is such an accumulation of static electricity in cutting the ribbons

478 FIXING, SECTIONING AND MOUNTING [Cn. XII

that they jump toward anything brought near them. This is very
annoying and likely to be so destructive to many of the sections that
serial work cannot be done with safety.

Many devices have been tried to overcome this difficulty. One of
the simplest and most successful is to put a pan of boiling water near
the microtome or to boil some water near it. The water vapor given
off in the surrounding air prevents fairly well the accumulation of
static electricity, and the ribbons are thus free to remain where put.
See also § 634 for Land's method.

§ 634. Land's method for sectioning hardened tissues. — Some
tissues like tendon, elastic tissue of the ligamentum nuchae etc. have
a tendency to become so hard that it is practically impossible to get
continuous ribbons. Dr. W. J. G. Land found that if the paraffin
imbedded tissue in such a case had the paraffin pared away at one end
until the tissue is exposed and then the paraffin block soaked in water
for a day or more it is quite possible to cut continuous ribbons with
ease. This was demonstrated on imbedded ligamentum nuchae that
was so hard that the tissue was torn right out of the paraffin block
when an attempt to cut it was made. The hard tissue was soaked in
water for several days, and then sections, 3/z, 5/i, yju and lo/x were cut
in perfect ribbons. This method, as also pointed out by Dr. Land, has
the further advantage that the ribbons do not become electrified, and
therefore that annoyance is also obviated. (See W. J. G. Land,
Botanical Gazette, vol. 59, 1915, p. 401.)

See also remarks upon the method in Chamberlain's Plant Histology,
5th ed. p. 125 and 3d revised ed. p. 1 13. In this work, Dr. Chamberlain
discusses and recommends the use of safety razor blades for section
knives. (See § 621.)

§ 636. Storing paraffin ribbons. — The most convenient method
for caring for the ribbons as they are cut is to place them on a tray
(figs. 237-238) lined with a sheet of white paper. It is important to
write on the paper full data, giving the name of the tissue, the
thickness of the sections, the date, etc. It is well also to number
the ribbons and to indicate clearly the position of the first section or
the beginning of the ribbon.

Ribbons of sections on a tray should be covered by another tray

CH. XII]

FIXING, SECTIONING AND MOUNTING

479

FIG. 259.

ALCOHOL LAMP IN A VERTICAL AND
AN INCLINED POSITION.

if one wishes to carry them to another room. The slightest gust of
air sends them flying.

Ribbons on trays may be kept a long time, if they are stored in a
cool place. The sections do not flatten out quite so well after stand-
ing a long time.

§ 636. Spreading the
sections on water. — Par-
affin sections are almost
invariably slightly wrin-
kled or folded in cutting.
To remove the wrinkles
one takes advantage of
the expansion of paraffin
when it is warmed. The
sections may be floated
on warm water, when
they will straighten out and become smooth, or the usual method is to
stretch them on the slide upon which they are to be finally mounted.

By spreading sections on a wet slide a double operation is per-
formed, viz.: the sections are made smooth and they are also
fastened to the slide. Put a minute drop of albumen fixative on the
middle of a slide and with the ball of one finger spread it over the
slide, making a thin, even layer. It cannot be too thin. It is likely
to stain if it is too thick. Do not use albumen if for the ultra-violet
microscope (§§ 316, 318).

With a pipette (fig. 264) put several drops
of water on the slide and then place a piece
of ribbon on the water; or put the sections
on the albumenized slide and add the water

afterward. Heat the slide carefully over a
FIG. 260. LEVELING spirit lamp or gas flame bemg sure not to
METAL TABLE FOR , , ^ ^ A / ,,

SPREADING SECTIONS AND melt the paraffin. As the water warms, the
FOR IMBEDDING IN PAR- paraffin expands and stretches the sections

AFFIN. r r .

out smooth. A copper heating plate is good
(fig. 260), but an electric spreader is best (figs. 254-256). The
projecting top enables one to heat this oven with a gas or alcohol

480 FIXING, SECTIONING AND MOUNTING [Cn. XII

flame. If an electric bulb is used, one of 30 to 40 watts is suffi-
cient. All desired temperatures are possible by placing the infiltra-
ting dishes nearer or farther from the lamp; and in spreading
one can pass from a point over the lamp where the paraffin
may melt to the overhanging top which is only just warm. The
dimensions of the oven giving optimum space and the desired range
of temperature are about as follows: Length 30 cm.; width 18 cm.;
height 12.5 cm. The brass spreading plate on top is 38 cm. long,
18 cm. wide and 3 mm. thick. The tray on the bottom is about 2
cm. deep. The tray and oven are lined with asbestos.

§ 637. Drying the sections. — After the sections are spread, drain
off most of the water, arrange the sections with a needle or scalpel,
and place the slide in one of the trays (figs. 237-238). Allow it to
remain overnight or preferably longer. The longer the drying in air,
the more surely do the sections adhere to the glass slide; or use the
drying oven (fig. 274).

If one is in haste to take the succeeding steps in the preparation,
the slide may be dried by putting it into a drying oven at 38° to 40°
C. for half an hour or more.

Some tissues are very difficult to get perfectly smooth, as just de-
scribed. If fine wrinkles persist, one can sometimes overcome the
difficulty by letting the slide cool and then covering with a piece of
fine tissue paper slightly moistened ; press down firmly with the ball
of the finger on the sections. Then take hold of the edge of the
paper and roll it off the sections.

As the water dries out, the spread sections come in very close con-
tact with the glass and adhere quite firmly to it. The thinner the
sections, the more tightly do they stick.

§ 638. Deparaffining in xylene. — This is accomplished by using a
solvent of paraffin. The best and safest one to use in a laboratory
is xylene. Benzine, gasoline and even kerosene are used, but xylene
is a powerful solvent of paraffin, does not injure the tissue, and is
not very inflammable, on account of the large amount of carbon in
its molecule (see § 573) and the consequent high boiling point,
136° C. (§ 244).

It requires only a few minutes to dissolve paraffin from the sec-
tions, but a dav or more in the xvlene does no harm.

CH. XII]

FIXING, SECTIONING AND MOUNTING

481

When the paraffin is removed the staining and other operations
necessary for a completed preparation may be undertaken.

FIG. 261. SLIDE BASKET OR RACK
FOR HANDLING SERIAL SECTIONS.

FIG. 262. GLASS STOPPERED

SPECIMEN JAR WITH A SLIDE

BASKET OR RACK WITH A

SPECIMEN IN PLACE.

§ 639. Collodionizing the sections. — Except for carmine stains
and perhaps some others, collodion remains practically colorless.
While the sections remain quite firmly attached to the slide after
they have been spread and dried, thick sections are likely to come
off in the many processes of staining, and if one has many sections
on a slide, some of them may become loosened. To avoid this, the
sections are covered with a delicate layer of collodion, which holds

482

FIXING, SECTIONING AND MOUNTING

[Cn. XII

them down to the slide. The early method was to use a soft brush
and paint a thin film over the dried sections before they were
deparaffined. Now the sections are deparaffined, and then, after
draining the xylene from the slide 10-15 seconds, it is put into a
bottle containing f % collodion (§ 577). In a minute or more the
collodion displaces the xylene, penetrates the sections and forms
a delicate veil over their free surface. No harm is done by leaving
the sections in the collodion a considerable time, but a minute or
two is sufficient. The slide is removed, allowed to drain for half a
minute, and then put into a jar of 67% alcohol (fig. 262). The
alcohol fixes the collodion and removes the ether. As the 67 % alco-
hol does not hurt the tissue, it may stay in the jar a day or more,
if desired, but half an hour suffices.

The sections are now ready for the subsequent staining and other
operations to make a finished slide. One has to remember that if
mucicarmine (§ 570) is to be used in staining, the preparation must
not be collodionized, as carmine stains collodion.

§ 640. Steps in order for the paraffin method. —

Name
Animal

No.
Absl. ale

Cedar oil

Date

Infilt.

Fixer

Temp. bath. . . .
Sections cut
v Temp. room. . . .

.Imbed, in
...-M's

Time of fixation

Washed in water

67 % ale 82 % ale.

Stains

Decalc. § 580 67, 82 % ale

In toto stain

Mtd. in

Washed in

Remarks . . .

67 % ale 82 % ale. ....

95 % ale. and eosin

§ 641. Paraffin sectioning by the propyl alcohol method (§ 563).

Sheridan (Stain Technology V, 34) and Bradbury, Science, vol. 74,
p. 225, have shown that there are certain advantages in the use of
normal propyl alcohol (CHaCftCKfcOH) and isopropyl alcohol
(CHaCHOHCHs). They do not make the tissues so hard and
brittle and the imbedded mass cuts easier.

CH. XII] FIXING, SECTIONING AND MOUNTING 483

Sheridan used normal propyl alcohol and the experience of the
author has been with that quite extensively. The tissue is fixed
in any desired manner as usual and carried up to 82% alcohol in
the usual way. For the dehydration the normal propyl alcohol is
used. When dehydrated it is passed directly to melted, 43° paraffin.
It may remain in this with one or two changes for an hour or more,
depending upon its size and character. The tissue is then transferred
to melted paraffin of 50° to 56° melting point and left in it only a
few minutes. If it is moved about the displacement of the soft
paraffin by the harder paraffin will be facilitated. The tissue is
then imbedded in the usual way and sectioned when convenient.
It has proved highly satisfactory.

THE COLLODION OR PARLODION METHOD OF SECTIONING

In this method the tissue is thoroughly permeated with a solution
of collodion, which is afterward hardened. Unlike the paraffin of
the paraffin method, the collodion (§ 576a) is not subsequently re-
moved from the tissue, but always stays in the sections. It is
transparent and does no harm.

The fixing and dehydration with 95% alcohol is the same as for
the paraffin method.

The paraffin method gives thinner sections than the collodion
method and for series and large numbers of sections, is superior.

The collodion method requires, no heat for infiltration, and it does
not render the firmer forms of connective tissue so hard. It is
especially adapted for making sections of large pieces of tissue or
organs and when thick sections are desired. It is not easy to cut
sections less than lo/z with collodion, while with paraffin it is possible
to make good ribbons of small objects of delicate texture 2ju to 3/1
in thickness. With a very sharp knife and small, delicate object,
and one of the better forms of microtomes, one can cut short
paraffin series in IJJL sections and get perfect ribbons.

In plant histology paraffin is used for cytologic work, and by
many whenever possible. Collodion must be used for the hard
tissues and is used by preference in some laboratories. (See refer-
ences in the collateral reading at the end.)

484 FIXING, SECTIONING AND MOUNTING [Cn. XII

Collodion sectioning is sometimes denominated the wei method, as
the tissue and sections must always be wet with some liquid, while
the paraffin method is called the dry method, as the tissue once in-
filtrated with* paraffin keeps in the air indefinitely and in cutting
the sections no liquid is used.

§ 642. Infiltration with ether alcohol. — Transfer the piece of
tissue to be cut from 95% alcohol to a mixture of equal parts of
sulphuric ether and 95% or absolute alcohol, and leave in this for a
few hours or a day or more, as is most convenient. This is to soak
the tissue full of a solvent of the collodion.

§ 643. Infiltration with 1| % collodion. — Pour off the ether
alcohol from the tissue and add i-J% collodion. Leave in this over-
night or longer if the piece of tissue is large.

§ 644. Infiltration with 3% collodion. — - Pour off the i|% collo-
dion and put in its place 3% collodion. Leave the tissue in this
half a day or longer.

§ 645. Infiltration with 6 % collodion. — Pour off the 3 % and add
6% collodion to the piece of tissue. For complete infiltration with
this thick collodion, it requires one day at least. If the object is
large, it is advantageous to infiltrate for a week or two.

§ 646. Infiltration in strong collodion. — Many workers recom-
mend as thick a solution as can be made for the final infiltration,
and a long stay (2-3 weeks) in the infiltrating liquid.

Many also recommend a great many steps in the process, com-
mencing with i% and gradually passing up through increasing
strengths till the thickest is reached.

§ 647. Imbedding on a cork or block. — For imbedding small
pieces use a piece of wood (deck plug), vitrified fiber, glass or good
cork for a holder. Cover the end with 6 % collodion and let it get
well set in the air; then put the piece of tissue on the holder and
drop 8% collodion upon it at intervals until it is well covered all
around. If one takes considerable time for this, the collodion
thickens greatly in the air. This is an advantage, for it gives a denser
block for sectioning. After the collodion is pretty well set, place
holder and tissue in a vessel with chloroform to harden. One can
put the preparation into the chloroform, or, if the vessel is tight, it

CH. XII]

FIXING, SECTIONING AND MOUNTING

485

may be above the chloroform, the vapor then acting as the hard-
ener,

§ 648. Imbedding in a paper box. — If the object is of con-
siderable size, it is best to use a paper box for imbedding, as with
paraffin. If a very small amount of vaseline is rubbed on the inside
of the box, it prevents the collodion from sticking t& the paper
(fig. 257, § 672).

Put first some of the thick collodion in the box and let it remain
in the air until nearly solid, 2 to 3 minutes. Then arrange the speci-
men to be cut as for imbedding in paraffin, and gradually add thick
collodion until the object is well covered. Let the box stand for a
few minutes in air; then place it in a dish like a Stender dish and

FIG. 263. PERFORATED SECTION LIFTER FOR HANDLING SINGLE COLLODION OR
FROZEN SECTIONS.

pour some chloroform on the bottom of the dish. Cover and the
collodion will harden, partly by the chloroform vapor and partly by
that which soaks through the paper. It is well to change the
chloroform at least once. The used chloroform will contain some
ether alcohol, but is good for killing animals.

After 24 or 48 hours the collodion should be firm all through.
Then it is placed in 67% alcohol where it may be left a day or
more. If it is to be left an indefinite time, the 67% alcohol should
be changed for 82%.

§ 649. Sectioning by the collodion method. — For this one can
use a table microtome or one of the sliding microtomes. The sec-
tions are made with a knife set obliquely and hence with a drawing cut.

The holder with the small piece of tissue is clamped in the micro-
tome and arranged as desired; then the sections are made with an
oblique knife which is kept w^ with 82 % alcohol. The best way

486 FIXING, SECTIONING AND MOUNTING [Cn. XII

to keep the knife wet is to have a dropping bottle over the object,
the drops falling about every two seconds. As the sections are cut,
they are drawn up towards the back of the section knife with a soft
brush. They can be kept in order in this way and do not interfere
with succeeding sections.

Some operators in drawing the knife across the tissue use a slight
sawing motion. However one proceeds, the knife is drawn rather
slowly, not rapidly as with paraffin work.

If the imbedding was done in a paper box, remove the box and
trim the collodion block suitably. Dry the end opposite the tissue,
then wet it with 3 % collodion. Use a piece of wood, a cork or other
holder of suitable size. Put some 6 % collodion on the holder and let
it dry for a minute or so; then press the collodion block down on the
holder. Leave in the air for a minute or two and then put into 67 %
alcohol to harden the cementing collodion. After 15 minutes, or
longer if convenient, put the mounted specimen into the clamp of
the microtome and cut as above.

Sometimes when the imbedded object is of sufficient size and the
collodion block is firm, the block itself is put into the microtome
clamp, no wood or cork holder being used.

§ 650. Transferring sections from the knife to the slide. — When
one has cut the number of sections for one slide, they should be
transferred to the slide as follows: Take a piece of white tissue
paper about 3X6 centimeters in size and lay it on the knife over
the sections. Press down slightly so the paper is in contact with all
the sections. Take hold of the paper beyond the edge of the knife
and gradually pull it down off the knife.

If there is the right amount of alcohol on the knife, the sections
adhere to the paper and move with it. This transfers the sections
from the knife to a piece of tissue paper. Place the tissue paper
with the sections down on the middle of an albumenized slide.
Cover with another piece of paper and press down gently. This
presses the sections against the slide and absorbs a part of the
alcohol. Take hold of one edge of the paper and lift it with a
rolling motion from the slide. The sections should stay on the
slide (§ 65oa).

CH. XII] FIXING, SECTIONING AND MOUNTING 487

§ 650a. — Various forms of paper have been used to handle the collodion
sections. It should be moderately strong, fine-meshed, not likely to shed lint,
and fairly absorbent. One of the first and most successful papers recommended
is closet or toilet paper. Cigarette paper is also excellent. In my own work the
heavy white tissue paper has been found almost perfect for the purpose. Or-
dinary lens paper or thin blotting paper for absorbing the alcohol or oil may be
used with it.

§ 651. Fastening the sections to the slide. — With a pipette,
drop 95 % alcohol on the slide of sections, then use a pipette full of
absolute alcohol if it is at hand. Drain most of the alcohol away
and add a few drops of ether alcohol. The collodion should melt
and settle down closely on the slide. If the collodion does not melt
the dehydration was not sufficient and more alcohol must be used.
After the collodion has melted down upon the slide, let the slide
remain a minute or two in the air, and then transfer it to a jar
of 67% alcohol (fig. 262).

After half an hour or longer the preparation is ready to stain.

§ 652. The castor-xylene method of sectioning. — The prepara-
tion of the tissue is the same as described in §§ 642-646, except that
when the collodion is hardened in chloroform, the block is trans-
ferred to castor-xylene (§575). In a few days the collodion gets as
transparent as glass and one can see the tissue within with great
clearness. It can remain in the castor-xylene indefinitely.

In cutting one proceeds exactly as in § 649, except that the block
is kept wet with castor-xylene, and not with alcohol. The sections
are arranged on the knife and transferred to the slide in the same
way as for alcohol sectioning (§§ 650-651).

For fastening the sections to the slide, as no water is present, one
can add the ether alcohol at once. It is advantageous here to have
a mixture of ether two parts and absolute alcohol one part for melting
the collodion in these oil sections.

Allow the slide to remain in the air till the collodion begins to
look dull; then the slide may be transferred to a jar of xylene to
remove the oil. From the xylene it is transferred to 95% alcohol
and then the slide is ready to be stained, etc., as described below
(§ 654).

88 FIXING, SECTIONING AND MOUNTING [Cn. XII

Steps in order for the collodion method. —

Name
Animal

No.
95 % ale

Date

Ether-ale

Fixer

\\% col. .

3% col.

Time of fixation

6 % col

8 % col

Washed in water

Imbedded . .

67% ale.

82 % ale

Chloroform . . .

67 % ale

Decalc. § 580

Or castor-xylene

67 % ale

82 % ale

Sections cut . . .

. . . ,/i's

In toto stain

Stains

Washed in

Mounted in. . . .

67 % ale

82% ale

Remarks

DOUBLE IMBEDDING IN COLLODION AND PARAFFIN

§ 653. Need of double imbedding. — Some objects like ova with
considerable yolk and other objects in which the different parts are
of unequal density or are very loosely bound together are advanta-
geously imbedded first in collodion so that there will be a tough
matrix to hold the parts in place, and then for ease and rapidity of
sectioning, paraffin imbedding is added.

Steps in double imbedding:

1. Fix in any desired way.

2. Dehydrate with absolute alcohol half a day or more.

3. Put into ether alcohol half a day or more.

4. Put into f % collodion half a day or more.

5. Put into 2\% collodion i to 2 days.

6. Put into 5% collodion for one day or longer.

7. Imbed in the 5% collodion, using a paper box (fig. 257).
Take the precaution to vaseline lightly the inside of the paper box

(§§648,672).

8. Float the imbedded tissue on chloroform in a glass dish.

9. When the collodion is hardened by the chloroform, remove the
paper box and transfer to the castor-xylene (§ 575) clarifier to finish
hardening and clarifying the collodion mass.

10. Put into melted paraffin for infiltration. Leave in the infil-

CH. XII]

FIXING, SECTIONING AND MOUNTING

489

trating oven (fig. 254) a day or two. There is advantage, according
to some, in transferring to pure xylene or to cedar-wood oil for half
a day before putting into the imbedding paraffin. Section in rib-
bons as with paraffin (§ 632).

The sections are spread and stained exactly as for the paraffin
method, except that carmine cannot be used without staining the
collodion.

STAINING AND PERMANENT MOUNTING

§ 654. Generalities on stains. — From the standpoint of the
object to be stained, dyes may be divided into two great groups:

(1) (a) Those which differentiate certain parts of the tissue and
make them prominent. Such dyes are called then differential or
selective. If the nucleus is the part selected, the

dye is frequently called a nuclear dye.

(b) General or counter stains. These stain all
parts of the tissue, and are usually contrasting
in color; blue or purple and bright red are fre-
quent combinations, e.g., hematoxylin and eosin.
There is an appearance of differentiation even
with a general stain, as the denser portions of the
tissue seem more deeply stained ; that is, there is
more substance and more stain is taken up,
hence the color is deeper.

(2) From the standpoint of the solvent used
in preparing the stains they are called (a)
aqueous, and (b) alcoholic.

If one uses an aqueous stain, the object must
be well wet with water before the stain is ap-
plied, and afterward well washed with water
before it is put again into alcohol. If an alco-
holic stain is used, the object to be stained should
be from alcohol of the same strength as that
used in making the dye. The dye is also washed away from the
tissue with the same strength of alcohol; it may then be put
into the stronger alcohols for dehydration.

FIG. 264. RE-
AGENT BOTTLE WITH
PIPETTE.

4QO FIXING, SECTIONING AND MOUNTING [Cn. XII

With reference to the now much used anilin dyes, Wright, Prin-
ciples of Microscopy, p. 34, gives this excellent general statement:
" Anilin dyes may be regarded as salts containing a" coloring element
or chromophore, united to a base or acid, according as the chro-
mophore in question possesses, in the particular case, acid or basic
properties. In the case where the chromophore functions as an acid,
the dye is denoted an acid dye (e.g., eosin). In the case where the
chromophore functions as a base, the dye is designated a basic dye."
Eosin is used as an example where the chromophore functions as an
acid, and methylene blue where the chromophore functions as a base.

The tissue elements and their parts are named from their affinity
for acid or basic dyes. For example, in the blood, the red corpuscles
and the granules of some, of the leucocytes have an affinity for acid
chromophores and hence stain strongly with eosin. They are accord-
ingly said to be acidophil or oxyphil, sometimes also eosinophil. The
nuclei of all the leucocytes, and of the red corpuscles when nucleated,
and the granules of some of the leucocvte?, have an affinity for basic
dyes and hence stain with methylene blue, and are designated
basophil.

§ 655. Staining with hematoxylin. — Take a slide of sections pre-
pared by the paraffin or the collodion method from the jar of alcohol
and plunge it into a vessel of water to remove the alcohol. For
staining put the slide of sections into a jar or shell vial of the
hematoxylin solution, or lay the slide flat on the staining rack or
some other support and add the stain to the sections (figs. 265-266).
It usually takes from 2 to 10 minutes to stain sufficiently with
hematoxylin.

A good plan when one is learning the process is to wash off the
stain after one minute, either with a pipette or by putting the slide
in a dish of water. Wipe off the bottom of the slide and put it un-
der the microscope. Light well, use a low power, and one can see
the nuclei stained a bluish or purple color, as hematoxylin is a
nuclear dye. If the color is faint, continue the staining until the
nuclei stand out boldly. Sometimes it takes a long time to stain
well with hematoxylin. In such a case the jar of stain may be put
into the paraffin oven and the heat will accelerate the staining.

CH. XII]

FIXING, SECTIONING AND MOUNTING

491

One may also heat the individual slides as for spreading sections,
but one must be careful not to let the stain dry on the sections.
As the stain evap- .N^t ^ ..,,v^vv

orates, add fresh

. . , .

stain with a pipette.
When the sections
are well stained
\\ ith hematoxylin,
wash off the hema-
toxylin with water.
If the slide is
allowed to stand
some time in or-
dinary water the
color is likely to be
brighter. This is
due to the action of
the alkali (ammonia,
etc.) usually pres-

FIG. 265.

BOWL WITH DRAINING RACK AND FUNNEL
FOR STAINING SKCTIONS.

ent in natural wa-
ters. One could use

distilled water, adding a few drops of a saturated solution of

lithium carbonate.

Dehydrate in 95 % alco-
hol and absolute if neces-
sary; clear and mount in
balsam as described in

§535-

Hematoxylin is so

FIG. 266. SMALL AQUARIUM JARS FOR STAIN- nearly a pure nuclear

ING SERIAL SECTIONS. • r

R Rack for the top of the jar and contain- stam for most tlssues

ing a small draining funnel. and organs that the cell

bodies are not very evi-
dent with this done;
hence some counterstain is generally used also.

§ 656. Counterstaining with eosin. — One of the solutions of

boule.the

492 FIXING, SECTIONING AND MOUNTING [Cn. XII

eosin (§ 583) is dropped upon the sections after the hematoxylin has
been washed away with water. This stains almost instantly. One
rarely needs to stain with eosin over 10 or 30 seconds. The excess stain
is then washed away with a pipette or by dipping the slide into water.

§ 657. Dehydrating and clearing. — Put the slide directly into
95% alcohol after it is rinsed with water. Leave it in the alcohol
a short time and transfer to fresh 95 % alcohol or to absolute alcohol
a few seconds, 10-20. One must not leave the sections too long in
the alcohol or the eosin will dissolve out.

Remove the slide from the alcohol and put it into a jar of clearer
(§ 573) or P^ it on the rack (figs. 265-266) and add enough clearer to
cover the sections. Soon the clearer will displace the alcohol and
make the sections translucent. It usually requires only half a
minute or so. The clearer is drained off and balsam put on the
sections, and then a clean cover-glass is added. One soon learns to
use the right amount of balsam. It is better to use too much than
too little (§ 535).

§ 657a. — In the past the plan for changing sections from 95 % alcohol to
water, for example, was to run them down gradually, using 75, 50 and 35% al-
cohol, successively. Each percentage might vary, but the principle of a gradual
passing from strong alcohol to water was advocated. I have found that the safest
method is to plunge the slide directly into water from the 95% alcohol. The
diffusion currents are almost or quite avoided in this way. There is no time for
the alcohol and water to mix; the alcohol is washed away almost instantly by the
flood of water. So in dehydrating after the use of watery stains, the slide is
plunged quickly into a jar of 95 % alcohol. The diffusion currents are avoided in
the same way, for the water is removed by the flood of the alcohol. This plan
has been submitted to the severe test of laboratory work, and has proved itself
perfectly satisfactory (1895-1931).

§ 658. Counterstaining with the eosin in the clearer. — With this
method the eosin is dissolved in the carbol-xylene clearer (§ 573a),
and the hematoxylin stained sections are dehydrated with 95% alco-
hol and absolute alcohol if necessary and then placed in the clearer.
The sections are cleared and stained in eosin at the same time. It
usually takes half a minute or more for the double process. When
the sections are clear and sufficiently red, the slide is removed and
the Clearer drained off by holding in the forceps or in the draining
funnel (figs. 265-266). Then the balsam is added, and covered ac
described above.

CIL XII] FIXING, SECTIONING AND MOUNTING 493

It is a good plan to rinse off the stained clearer by pure xylene
before adding the balsam. This is not absolutely necessary, how-
ever.

§ 659. Hematoxylin and picro-fuchsin. — Picro-fuchsin is so selec-
tive in its general staining that it is frequently used after hematoxy-
lin. The hematoxylin staining should be intense and after the
hematoxylin is washed away add the picro-fuchsin (§ 610). It takes
only 10 to 30 seconds for it to act. Wash with distilled water, or
natural water very faintly acidulated. The acid fuchsin is very
sensitive to alkalies and fades easily.

Dehydrate in 95% and absolute alcohol, clear and mount in acid
balsam. Acid balsam injures hematoxylin, but is necessary for the
red in the picro-fuchsin.

Look out for mercuric chlorid crystals in the sections (§ 669).

§ 660. Hematoxylin and mucicannine. — Tissues and organs are
best fixed in Zenker's, or mercuric chlorid. Small intestine is one of
the most striking and instructive organs for this double stain.
Make the sections by the paraffin method, but do not fasten them
to the slide with collodion, for collodion stains with mucicarmine

(§ 570).

Stain i to 24 hours in mucicarmine. Wash off the stain with
water and then stain with hematoxylin. Do not stain too deeply.
Wash with water, dehydrate, clear and mount in natural balsam.
Nuclei will be bluish or purple and the cells containing mucus will
be rose red. The goblet cells of the villi stand out like small red
goblets, and if any mucus is streaming out of them, it will be red.

§ 661. Combined elastic mucicarmine and picro-fuchsin stain. —
For this, one should take some object that is known to contain
elastic tissue, mucus, white fibrous tissue and muscle. (The non-
cartilaginous part of the trachea is excellent.) The organ should
have been fixed in mercuric chlorid or Zenker's fluid (§§ 600, 615)
for this preparation. The sections should be made by the paraffin
method and no collodion should be used for fastening the sections
to the slide (§ 639), for collodion is stained by mucicarmine.

(i) Stain first in the elastic stain. Wash well with 95% alcohol
and then with water.

494 FIXING, SECTIONING AND MOUNTING [Cn. XII

(2) Stain in a shell vial or jar of mucicarmine (§ 570) from i to 24
hours. Wash well with water, but one must be careful in treating
these sections, as they have no collodion mantle to protect them.

(3) Stain 15 to 30 seconds with picro-fuchsin of one-fourth
strength (§ 610). Dehydrate with 95% and if necessary absolute
alcohol. Clear in carbol-xylene and mount in acid balsam (§ 568).
The elastic tissue will be black or blue black. Mucus will be carmine
or rose red; white fibrous tissue will be magenta red; muscle, epithe-
lium and blood will be yellow.

§ 662. Eosin methylene blue. — One of the best objects for this
stain is a hemolymph gland. Such a gland is easily and surely found
by a beginner if he takes the heart and lungs of a veal. In the fat
around the heart and behind the pleura will be found red bodies
looking almost like blood clots. Remove carefully; fix in Zenker's
fluid or mercuric chlorid (§§ 600, 615). Section by the paraffin
method, make the sections 5ju and loju thick. Use collodion for
insuring the fixation to the slide (§ 639). Stain with eosin methyl-
ene blue (§ 585).

Eosin-methylene blue staining is also excellent for demonstrating
mucus.

Do not forget that mercury is likely to be present in sections of
tissue fixed with any mercuric fixer. Remove it with iodized alco-
hol (§ 597). This should be done before the staining. One can tell
whether the tissues contain mercury by looking at the unstained
sections. The mercury looks black by transmitted light, white by
reflected light. Seen by transmitted light, the substance is often in
the form of delicate black pins.

§663. lodin stain for glycogen. — Use tissue fixed in 95% or
absolute alcohol. Cut by the paraffin method. Mount the sections
in serial order. Do not use water for spreading the sections, but one
of the iodin stains for glycogen (§ 596). The glycogen will be
stained at the same time that the sections are spread.

Let the sections dry thoroughly after spreading. Deparaffm with
xylene and mount in yellow vaseline or use thin xylene balsam, but
do not put a cover-glass over the balsam preparations.

The iodin stain remains in the spread sections for ten years or

CH. XII] FIXING, SECTIONING AND MOUNTING 495

longer. One can restain any time by putting the slide with the
spread, but not with the deparaffined sections, in a shell vial of the
iodin stain. It is possible also to stain the nuclei with hematoxylin
in the same way. If this is done, the hematoxylin should be used
first and washed off with water and the iodin stain be used last, but
not washed off with water.

For collateral reading see the references given in the preceding
chapter (Ch. XI).

CHAPTER XHI

SERIAL SECTIONS OF ORGANS, SMALL ANIMALS AND EMBRYOS;

PREPARATION OF MODELS; EXAMPLES AND EXPERIMENTS

§§664-702; FIGURES 267-277

ADVANTAGES OF HISTOLOGICAL SERIAL SECTIONS

§ 664. General on series. — It is coming to be appreciated more
and more that in histology as well as in embryology one can only get
a complete knowledge of structure by having the entire organ cut in
microscopic sections and each section mounted in order. Further-
more, it is necessary to have the organ cut in three different planes.
In this way one can see every aspect of the structural elements and
their arrangement in the organs.

In single sections one gets only a partial view. For example, how
many students have any other idea of a ciliated cell than that it is
a cell with triangular outline with a brush of cilia at the broad end.
Probably many would be puzzled if they had a top view of the
ciliated end; and the attached end would be even more puzzling.

It may not be possible for every worker to make serial sections
of all the organs in ail the three planes, but every one who is work-
ing seriously in histology can make all his preparations serial; that
is, the sections which are mounted can be in serial order; then a
puzzling appearance in one section may be perfectly intelligible in
one a little farther along.

To get the greatest benefit from serial, as indeed also from single
sections, the sections should be made in a definite manner; that is,
they should be exactly across the long axis of an organ or parallel
with the long axis (Transections and Longisections).

Or with such an organ as the liver, the skin, etc., the sections may
be parallel with the surface (Surface Sections) or at right angles to
the surface (Vertical Sections).

§ 665. Order of serial sections. — Some plan must be adopted in

406

CH. XIII]

SERIAL SECTIONS AND MODELS

497

arranging the series or only confusion will result. An excellent plan
is to arrange the short pieces of ribbons for a given slide as the words
on a page are arranged. That is, section No. i is at the upper left-
hand corner. The next row of sections begins where the first row
left off, etc. (fig. 267).

As the paraffin stretches considerably one must cut the ribbons
into pieces considerably shorter than the cover-glass to be used.

P.Q

Ss.

Pig 6

SI 23

Seo 253

10// "260

1900

FIG. 267. A SLIDE OF SERTAL SECTIONS SHOWING THE ARRANGEMENT AND ORDER
OF THE SECTIONS; ALSO THE LABELING OF THE SLIDE.

Both the paraffin and collodion methods are adapted to the prepa-
ration of series. The paraffin ribbons are easier to manage and
easier to make than the serial sections in collodion.

By arranging the collodion sections as they are cut on the knife
in collodion sectioning (§ 649), one can put them on the slide in per-
fect series by the tissue paper method (§ 650).

If the sections are large, as in cutting serial sections of the central
nervous system, the series can be kept in order in a small dish by
putting a piece of tissue paper over each section and piling them up.
If the vessel is small enough, the papers and sections will not shift
and get out of order. Or one might put a single section in a Syra-
cuse watch glass or a Petri dish. Then in mounting, the sections can
be taken in order.

§ 666. Numbering the serial slides. — For temporary numbering
a fine pen with Higgins' or Weber's waterproof carbon ink serves
well. If the end of the slide is varnished, one can write on it as
well as on paper. When the ink is dry it should be coated with thin
xylene balsam or with any good varnish like valspar i part, xylene
9 parts. It is also important to write the number of the slide with

498 SERIAL SECTIONS AND MODELS [Cn. XIII

a writing diamond. The double marking is desirable because with
wet slides the diamond number is hard to see, while the ink marks
are clearly visible. One is not so likely to wipe off the sections if the
ink mark is present.

FIXING AND STAINING FOR SERIES

§ 667. Fixing. — The two most used fixers for embryos are
Zenker's fluid and formaldehyde (§§ 589, 615). For those unskilled
in microscopic technic, or for one who is exceedingly busy, the best
results are obtained by putting the embryos in formaldehyde (10
parts of formalin, the formalin of the pharmacy, and 90 parts water
answers well). If there is plenty of this the embryos are likely to be
well preserved even though they are left in the membranes, and that
is far the best way for small embryos.

§ 668. Fastening the sections to the slide. — For all serial work
it is especially desirable to fasten the sections to the slide with collo-
dion (§ 639). This should always be done unless some stain like car-
mine is to be used on the slide after the sections are fastened. With
thin sections, if one is careful enough, an entire series can be carried
through without losing a section, but with thick sections (15/4 and
thicker) some are almost sure to separate from the slide if not fas-
tened by collodion.

§ 669. Removal of mercuric chlorid from sections. — It should be
remembered that if a fixer containing mercuric chlorid is used, the
sections are almost sure to contain mercury. By transmitted light
the mercury appears dark. Often the appearance is as if a multitude
of delicate black pins were in the section. Sometimes the mercury
is in rounded masses. This should be removed by putting the slides
of sections into alcoholic iodin (§ 597). < After half an hour or an
hour, wash off the iodized alcohol with pure 95% alcohol and the
sections are ready for staining.

If the embryo was stained in loto and contains mercury, the sec-
tions should be passed from the deparaffimng xylene to the iodized
alcohol (§ 597). After half an hour or more the slides are passed
through pure 95% alcohol, and back to the xylene or to carbol-
xylene. Then they can be mounted in balsam.

CH. XIII] SERIAL SECTIONS AND MODELS 499

§ 670. Staining for series. — There is a great advantage in point
of time and safety in staining the entire embryo in some good stain
like borax carmine (§ 569). Carmine is a very permanent stain.
For bringing out special structural details the sections are stained
on the slide as described in §§ 655-656. The slide baskets are al-
most a necessity for serial work (figs. 261-262), as the slides are
handled individually only twice, (i) when they are spread and dried
and put into the baskets, and (2) after all the processes are com-
plete and the sections are to be mounted in balsam.

The sections are mounted in balsam directly from the deparaffin-
ing xylene. No alcohol is used unless it is necessary to remove
crystals of mercuric chlorid (§§ 597, 669).

COMPLETE SERIES OF EMBRYOS AND SMALL ANIMALS IN THE THREE
CARDINAL PLANES, — TRANSECTIONS; SAGITTAL SECTIONS;
FRONTAL SECTIONS

§ 671. Serial sections of entire animals. — With improvement in
means for making thin sections of objects, the long-desired ability to
see the entire organism in complete series is now easily realized.
What was formerly determined with so much difficulty in dissecting
embryos can now be attained with ease in a complete series. It is
almost too easy, and with a lively imagination structural arrange-
ments are described and depicted which never actually existed in
the animals or embryos themselves. It is so difficult for most people
to add the third dimension accurately when working with flat
specimens that it is now appreciated that the older workers had a
great advantage in dissecting the entire animal or embryo because
they were there dealing with an obviously three-dimensional object
and true relations in space were seen. There is now a wholesome
tendency toward the retention of the advantages of dissection of
entire forms with the advantages of serial sections. Hence embryos
are now dissected entire almost as much as in the old days, and
enlarged models of the series are made so that the object can be
seen in three dimensions, the models also serving to make it easy to
follow out the relations of parts with the naked eye. But one should

500 SERIAL SECTIONS AND MODELS [Cn. XIII

not forget that a model, like a drawing, is after all only the interpre-
tation of the artist and the thing itself must be referred to whenever
there is to be real advancement in knowledge. Furthermore, as it is
not possible to both dissect and serial section the same objects, and
sometimes very few are available, anatomists have decided on the
three planes which give the greatest information, — transections or
cross sections, sagittal sections and frontal sections. With sections
in these three spatial planes it is possible to gain some just con-
ception of the actual relation of parts and structures in the ob-
ject.

§ 672. Orientation of imbedded objects. — In order that sections
may be made in any desired plane the object must be so arranged
or oriented in the imbedding mass that one can attach the imbedding
block to the microtome holder, and then arrange for sectioning in a
definite manner. With translucent or transparent collodion where
the position of the object can be seen after it is imbedded, this is not
particularly difficult, but with paraffin, which is nearly opaque, one
cannot see distinctly enough the position of the object to give the
exact arrangement necessary to make precise sectioning possible.
The embryo or animal or other object must, therefore, be arranged
in the imbedding box in a very definite manner.

To overcome the difficulties Dr. Kingsbury, ten to fifteen years
ago, devised the method of making a diagram of the object to show
its exact shape and position. (Anat. Record, Vol. XI, 1916, p. 294).
The method is as follows: A natural-size diagram of the object is
made on the inside of the bottom of the imbedding box before any
paraffin is put into it. This is most easily done before the box is
folded, or the folded box can be unfolded and made flat again. For
making the diagram a soft lead pencil can be used or one of the or-
dinary colored crayons or a colored glass pencil. In any case enough
of the lead pencil or the crayon mark adheres to the paraffin to
make a clear diagram on it of the object.

In imbedding, the object should be arranged exactly over the dia-
gram. The solidified layer of paraffin formed before the object is
placed in the box (§ 628) is no hindrance, as the diagram shows
through it clearly.

CH. XIII] SERIAL SECTIONS AND MODELS 501

For embryos and small animals, of which serial sections are to be
made, there should always be a photograph natural size.

The diagram for orientation is easily made from such a photo-
graph by the use of the drawing shelf (fig. 277, A.D.S., §§ 415, 417).
As the embryo or animal is always imbedded with the right side
down, left side up, one must be sure to have the diagram in the
same position. This is easily accomplished, as one can draw equally
well with the photographic print whichever side is up. That is, if
the embryo was photographed left side down, the print should be
face down on the drawing shelf to bring the diagram in the imbed-
ding box with the left side up. On the other hand, if the photo-
graph was made with the embryo right side down, then the print
should be face up when making the diagram on the bottom of the
imbedding box.

With the definite outline of the embryo or animal on the bottom
of the imbedding mass one has a good guide for arranging the object
for sectioning any desired plane.

§ 673. Thickness of serial sections. — The thickness of the sec-
tions of a series should be known in all cases; and for modeling it is
absolutely necessary (§§ 680, 684). The thickness usually depends
somewhat upon the size of the object to be made into series. If the
object is small, the sections can be thin without having an un-
manageable number of slides. With larger objects the sections are
naturally made thicker to keep the length of the series within
bounds.

One of the following thicknesses will be found to meet nearly all
requirements and make modeling easier than as if some odd number
of microns were used: 5/1, ioju, ISM, 20;*, 25/4, 30^, 40/1, 5°M> 75M,
loo/x. Of course every investigator decides for himself the thickness
of section which will serve his purposes best.

§ 674. Arrangement of sections on the slide. — (i) A satisfactory
and widely adopted method is to arrange the sections like the
printed words in a book. This brings the first section at the upper
left-hand corner of the series, and the last section at the lower right-
hand corner (fig. 269).

(2) It is a great advantage to have the sections so arranged on

502 SERIAL SECTIONS AND MODELS [Cn. XIIT

the slide that under the compound microscope the aspects will be
as in the observer's body; then it will be easy to locate objects at
the right or left, dorsal or ventral.

(3) Remember that in the ribbons the surfaces are somewhat
unlike in appearance. The lower surface, that is, the surface facing
the section knife, is shiny, while the opposite surface is dull. This
knowledge is important, for sometimes sections get turned over acci-
dentally. It is unfortunate to have part of the sections of a series
wrong side up.

(4) The aspect cut first will face upward on the slide; that is, if
the head is cut first the cephalic aspect will face up; if the left side
is cut first the sinistral aspect will face up, and if the dorsal side, the
dorsal face will be up.

(5) The aspect of the embryo which first meets the edge of the
knife will be at the beginning of the series. If arranged and cut as
here directed, transactions would have the right side of each section
toward the left on the slide (fig. 269). Under the compound micro-
scope it would appear on the right.

For sagittal sections where the caudal end meets the knife, the cau-
dal end of the section would be toward the left on the slide (fig. 272).

For frontal sections (fig. 270) where the right side meets the knife
edge first, the right side of each section will be toward the left end
of the slide.

§ 675. Mounting. — Cut the ribbons into segments of equal
length, using preferably a curved knife (fig. 258). Transfer to
albumenized slides with fine forceps (fig. 221). Make parallel with
the long axis of the slide, and put the first section at the upper left-
hand corner (fig. 267).

In a word, decide on some good plan for mounting series and fol-
low the plan consistently.

§ 676. Size of slides and cover-glasses for series. — (i) If the
object is small, the standard slide 25 x 75 mm. (fig, 217) is good and
the cover-glass can be either 22 or 23 mm. wide and 50 or 60 mm.
long. The smaller sizes are to be preferred when convenient, for
more space is left to the label, and the cover-glass is not too near the
edge as with wide covers.

CH. XIII]

SERIAL SECTIONS AND MODELS

503

(2) If the embryo or animal is of moderate size, that is, not over
30 to 35 mm. long, one can use advantageously the intermediate size
of slides (fig. 216), that is, those 38 X 75 mm. A suitable cover-
glass is 35 X 5° or 35 X 60 mm.

(3) For objects of considerable size, i.e., over 35 mm. in length,
if sagittal or frontal sections are to be made, and if they are to be
mounted crosswise, the slide must be of sufficient width. Ordinarily
the large standard, 50 x 75 mm., will answer (fig. 215). For the
large slides the covers can be 48 X 60 or 48 X 65 mm. For special
large sizes of object, special slides can be made of lantern slide
covers or old negative glass, etc., and for cover-glasses one can go
back to the earlier workers and use mica.

Do not use too thick cover-glasses, or high powers cannot be em-
ployed in studying the sections (§§ 101-106).

TRANSECTIONS OR CROSS SECTIONS

§ 677. Transections are those made by dividing the body into sec-
tions made across the long axis of the body. This divides the em-

FIG. 268. SERIAL TRANSACTIONS.

At the right is the embryo in the imbedding mass and attached to the micro-
tome holder.

At the left is a glass slide showing how the sections are to be mounted.

Imbedded embryo It is in the proper position for transactions.

In section i, the word cephalic shows that the section is cephalic face up; the
caudal face rests on the slide. In the middle section the words indicate the edges
of the section. Under the microscope the words will be erect. Invert the book
and the appearance will be the same as under the microscope.

bryo or animal into equal or unequal cephalic and caudal segments.

With microscopic sections, of course, the segments of the entire body

are very unequal, although each section may be of equal thickness.

(i) Imbed the embryo or animal with the right side down, taking

504

SERIAL SECTIONS AND MODELS

[Ca. XIH

the precaution to have a layer of partly solidified paraffin at the
bottom of the box (§ 628); and arrange the object exactly over the
orientation diagram in the bottom of the imbedding box (§ 672).

(2) Mount the block of paraffin containing the embryo so that
the caudal end is next the microtome holder. The head is then cut
first, and the caudal surface of the sections will rest upon the slide,
bringing the cephalic face up (fig. 268).

(3) Place in the microtome so that the right side of the embryo
or animal meets the edge of the knife.

(4) Mount the sections like the words in a printed line. This
will bring the first or most cephalic section at the upper left-hand
corner. The cephalic face will be up, and the dorsal aspect next the
upper edge of the slide.

_3
40

Homo 3
SI 40

See 493

20/4 504

1900

FIG. 269. A SLIDE OF SERIAL TRANSECTIONS SHOWING THE ARRANGEMENT AND
THE LABELING OF THE SLIDE.

Under the compound microscope the rights and lefts will appear
as in the observer's own body, as will also the dorsal and ventral
parts.

FRONTAL SECTIONS

§ 678. Frontal sections. — These are sections made by dividing
the body into equal or unequal dorsal and ventral parts.

(1) Imbed the animal or embryo with the right side down in the
imbedding mass (§ 628) ; and arrange the object exactly over the
orientation diagram in thef bottom of the imbedding box (§ 672).

(2) Mount the block of paraffin containing the embryo so that
the ventral aspect of the embryo or animal is next the disc of the
microtome holder (fig. 270). The dorsal part is then cut first, and

CH. XIII]

SERIAL SECTIONS AND MODELS

505

the ventral surface of the sections will rest upon the slide, bringing
the dorsal face up.

(3) Place in the microtome so that the right side of the object
meets the edge of the knife first.

(4) Mount the sections like the words in a printed book. This
will bring the first or dorsal section in the upper left-hand corner

FIG. 270. FRONTAL SERIAL SECTIONS SHOWING THE ARRANGEMENT OF THE
EMBRYO IN THE IMBEDDING MASS, THE CONNECTION WITH THE MICROTOME
HOLDER AND THE POSITION OF THE SECTIONS ON THE GLASS SLIDE.

Microtome Holder The metal disc and stem for holding the imbedded embryo
in the microtome while sectioning.

Imbedded Embryo The embryo in the proper position for frontal sections.

Frontal Sections A slide showing the proper arrangement of frontal sections.

z, 2, 3, 4 Serial order in which the sections are arranged like the words in a
printed book.

In section i the word dorsal indicates that the section has its dorsal face up-
ward away from the slide while the ventral face is down in contact with the slide.

In section j, the words cephalic, caudal, dextral, sinistral are wrong side up so
that they will appear erect under the compound microscope.

of the series. The dorsal face will be up, the right side to the left,
and the cephalic end toward the lower edge of the slide (figs. 270-
271). Under the compound microscope the cephalic end will be
away from the observer or in front, and the rights and lefts will be
as in his own body.

P'Q

460 491 4S2 4M

PtQ
SI

Sec^ 440
10 /U 453

1900

FIG. 271. FRONTAL SERIAL SECTIONS SHOWING THE ARRANGEMENT AND THE
NUMBERS OF THE SECTIONS ON THIS SLIDE. THE ^ SLIDE is PROPERLY LABELED.

$o6

SERIAL SECTIONS AND MODELS

[CH. XIII

If the sections are too long to mount crosswise, they can be cut
apart and mounted lengthwise of the slide, the order being like that
of the words in a line of print as with all serial sections.

SAGITTAL SECTIONS

§ 679. Sagittal sections are those made parallel with the long axis
of the body and from the dorsal to the ventral surface, thus dividing
the object into equal or unequal right and left (dextral and sinistral)
parts.

(i) Imbed the animal or embryo with the right side down in the
imbedding mass (§ 628); and arrange the object exactly over the
orientation diagram in the bottom of the imbedding box (§ 672).

FIG. 272. SERIAL SAGITTAL SECTIONS SHOWING THE POSITION OF THE EMBRYO
IN THE IMBEDDING MASS, THE CONNECTION WITH THE MICROTOME HOLDER AND
THE ARRANGEMENT OF THE SECTIONS ON THE GLASS SLIDE.

Microtome Holder The metal disc and stem for holding the embryo in position
while it is being cut.

Imbedded Embryo The imbedded embryo in the proper position for sagittal
sections.

Sagittal Sections A slide of sagittal sections in the proper position on the slide.

i, 2 Serial order in which serial sections are arranged on the slide.

In section 2, the word sinistral indicates that the left surface of the section faces
directly upward. The right side rests upon the glass.

The words cephalic, caudal, dextral and sinistral are inverted under the com-
pound microscope, the sections are reinverted, and will appear like this picture, if
the book is turned upside down.

(2) Mount the block of paraffin containing the embryo so that the
right side will be next the disc of the microtome holder. The left
side will then be cut first, and look up when mounted (fig. 272).

(3) Place in the microtome so that the caudal end will first meet
the edge of the knife.

(4) Mount the sections in the order of the print on a page. This

CH. XIII]

SERIAL SECTIONS AND MODELS

507

will bring the caudal end to the left, the cephalic at the right, ven-
tral aspect up and dorsal down toward the lower edge of the slide.
The dextral face of the section will rest on the slide, and the sinistrai
face will look up.

Under the micrdscope the head will be at the left and the dorsal
side will appear toward the upper edge of the slide — away from the
observer. It will appear like the figure when the book is turned
upside down.

If the embryo is large it may be better to turn it around so that
the ventral side meets the edge of the section knife. If this is done
the sections will have to be cut apart and mounted one by one on the
slide, otherwise they would be crosswise of the slide like the frontal
sections (fig. 270).

FIG. 273. SLIDE OF SERIAL SAGITTAL SECTIONS SHOWING THE ARRANGEMENT

\\TD LABELING.

§ 680. Labeling serial sections. — The label of a slide on which
serial sections are mounted should contain at least the following:

The name of the embryo and the number of the series; the num-
ber of the slide of that series; the thickness of the sections, and the
number of the first and last section on the slide; the date. It is
also a convenience to have the information repeated in part on the
left end (figs. 267-273).

MODELS FROM SERIAL SECTIONS

§ 681. General considerations on modeling. — Anatomists have
for a long time produced models of gross anatomic specimens, and
enlarged models for minute details.

508

SERIAL SECTIONS AND MODELS

[CH. XIII

Naturally, after serial sections of embryos and organs came to be
made with considerable accuracy and of known thickness, there was
a desire to make enlarged models which should be exact representa-
tions of the original rather than the generalized approximations
built up as an artist produces a statue.

Further, the difficulty of getting a true conception of the object
by studying only two dimensions in the sections is very great; hence
a model giving all three dimensions becomes almost a necessity for
the beginner in embryology, and is of enormous advantage to an in-
vestigator in working out the true form and relation of complex

FIG. 274. DRYING OVEN FOR SLIDE TRAYS.
(From the Anatomical Record).

1 The oven showing all the parts, the oven proper (i) is lifted up to show
the electric lamps in the base (2).

2 Sectional view of the oven (i) and base (2) showing the construction and
the air currents. One tray (5) is in position.

A The asbestos lining of the outer shell. B One of the numerous ventilating
holes. C Flue for the escape of air. H Runs for the slide trays.

D Door of the support or base (2). W-L Wiring for the lamps. One can
vary the heat by turning out one or more of the incandescent bulbs.

structures. For modeling a series it is of great advantage to have
photographs of the object to be modeled. If possible, the object
should be photographed in the fresh state and after fixation. The
more aspects photographed, the better.

CH. XIII] SERIAL SECTIONS AND MODELS 509

The principles involved in the construction of a model are exceed-
ingly simple: —

1. It is necessary that the embryo or other object to be modeled
should be cut into a series of sections of definite thickness.

2. The sheets of modeling material must be as much thicker than
the sections as the model is to be larger than the original.

3. The sections must be drawn as much larger than the actual
specimen as the model is to be larger than the object.

4. The drawings with the desired outlines must be made directly
upon or transferred to the sheets of modeling material which are
then cut out, following the lines of the drawing.

5. The different plates of modeling material representing all the
sections are then piled up, in order, thus giving an enlarged model
of the object with all its parts in proper positon and in true pro-
portions.

MODELS OF WAX

§ 682. Wax models. — For making wax models, beeswax 820
grams, paraffin 270 grams and resin 25 grams are melted together
and thoroughly mixed.

To get the sheets of wax of the proper thickness two methods are
available: —

(1) The hot wax is poured into a vessel containing hot water.
The wax spreads out into an even layer over the hot water and is
allowed to cool. While it is solidifying, it should be cut free from
the edges of the vessel. Of course, by calculation and experiment
one can put in the right amount of wax to get a plate of a given
thickness.

(2) One must have a wax-plate machine consisting of a flat sur-
face — planed cast iron is good — with some means of obtaining
raised edges. If these are adjustable by a micrometer screw, it is
simple to set them properly for the desired thickness of plate. Then
there must be a hot roller. The hot wax is poured on the plate, and
with the hot roller resting on the raised edges, the wax is rolled out
into a plate. It cools quickly and may be removed for another
plate. This is the most rapid and satisfactory method of preparing

SERIAL SECTIONS AND MODELS

[CH. XIII

the plates. By using a brush with turpentine, the paper with the
drawing can be wet and then with the hot roller cemented to the
plate before that has been removed from the machine.

The wax plate is cut with a sharp instrument, following the out-
lines of the object which has been traced upon it by the aid of a
camera lucida or the projection microscope. The sections are piled
together, some line or lines obtained from a drawing or photograph
of the specimen before it was imbedded and sectioned being used as a
guide. Finally the whole is welded into one by the use of hot wax
or a hot instrument. Models which illustrate complex internal
structures are difficult to prepare, but numerous devices will occur

to the worker, as the representation of
blood vessels and nerves by strings or
wires. A large model will need much
support which can be given by wire
gauze, wires, pins or paper, according
to the special needs.

A practical method for wax modeling
was first published by G. Born, Arch,
f. Mikr. Anat., Bd. xxii, 1883, p. 584.
The most detailed statements of im-
provements of the method have been
published by Born (Bohn u. Oppel),
1904, and by Dr. F. P. Mall and his
assistants. See contributions to the
Science of Medicine, pp. 926-1045.
Proceedings of the Amer. Assoc. Anat-
omists, 1901, i4th session (1900), p.
193. A. G. Pohlman, Zeit. wiss. Mikro-
skopie, Bd. xxiii, 1906, p. 41.

To overcome the difficulty of cutting
out the wax plates, Dr. E. L. Mark of
Harvard University uses an electrically
heated wire moved rapidly by a modified sewing machine (Amer.
Acad. Arts and Sciences, March, 1907; Science, vol. xxv, 1907;
Anat. Record, April, 1907).

FIG. 275. KINGSBURY'S
MOVABLE STAND FOR SLIDE
TRAYS AND REAGENTS.

(From the Anatomical
Record).

r, r, r Reagent boards with
bottles and jars.

st, st Slide trays.

The stand has furniture
slides on the legs and is easily
moved on the floor.

CH. XIII] SERIAL SECTIONS AND MODELS 511

SUSANNA PHELPS GAGE BLOTTING-PAPER MODELS

§ 683. Comparison of wax and paper models. — Wax has certain
inherent defects for models: It is expensive, heavy and fragile. It
is easily deformed by the temperature of summer, and the amount of
time necessary for the preparation of the plates is great. A wax-
plate machine is expensive and bulky.

It therefore seemed worth while to see if there was not some other
material obtainable in the open market which would be more suit-
able and more generally available.

Blotting paper seemed promising, and an actual trial showed it
to be admirably adapted for the purpose. Since making the first
model in 1905 it has been constantly used in the laboratory of em-
bryology in Cornell University. Models made from it were demon-
strated before the Association of American Anatomists in 1905 and
before the International Congress of Zoology in 1907.

" The advantages of blotting-paper models are the ease and
cleanliness of their production and the lightness and durability of
the product. The models are broken with difficulty, are easily
packed or transported, and when they cleave apart are easily re-
paired, thus contrasting with the weight and fragility of wax models
and their deformation by heat.

"By this process are secured for the original model reconstructed
from microscopic sections the same qualities which have made the
Auzoux models molded from papier-mache such useful and lasting
additions to laboratory equipment; and, in the hands of Dr. D wight
and Mr. Emerton, of Harvard University, have aided so much in the
demonstration of structure and form of special anatomic preparations."

§ 684. Thickness of blotting paper. — Blotting paper of a uniform
thickness of i mm. T\ mm., and | mm. was found in the market.
The i mm. is known as 140 Ib. A. and costs about two cents for a
sheet 61 x 48 centimeters (24 x 19 in.).

The thickness is easily tested by cutting out 50 small pieces, piling
them, dipping one end in melted paraffin, and pressing them to-
gether. The whole pile should of course measure 50 mm. if the
paper is millimeter paper (§ 684a).

Si2 SERIAL SECTIONS AND MODELS [Cn. XIII

§ 684a. — Book-stores, paper dealers and job printers are supplied by the
paper manufacturers with samples of blotting paper. One can look these samples
over, select and order the kinds desired. The millimeter blotting paper men-
tioned in the text is one of the cheaper grades, costing by the package of 500
sheets about two cents a sheet (sheets 61 X 48 centimeters, 24 x 19 inches).

§ 686. Size of the model. — In deciding upon the size of the
model to be made from a given series of sections one should select
the largest section and with the projection microscope throw the
image on the table (fig. 276), By using different objectives and
different distances from a microscope one can find a size which
seems suitable. The magnification may be found by § 409. Then
by multiplying the whole number of sections by the thickness of
the sections and this by the magnification, one can get the length
or height of the model. One must take these preliminary steps and
decide upon the magnification to be used or the model is likely to
be too large to be manageable or too small to show well the neces-
sary detail.

(1) Suppose the model is to be 100 times the size of the original
object, and the object has been cut into a series of sections IOJJL
thick. Then each section must be represented by a plate or sheet
100 times as long, broad and thick as the object. As the sheets of
blotting paper are so large (61 x 48 cm.), one need be solicitous
only about the thickness.

As each section is actually lo/x thick and the model is to be 100
times enlarged, the thickness representing each section must be
loju X ioo - loooju or i millimeter, i millimeter blotting paper is
used and every section of the series is drawn.

(2) If the blotting paper were only T%- mm. thick, it would be
simpler to make the model 90 times the size of the original. If,
however, one wished the magnification to be ioo, it could be ac-
complished thus: Each section in the series should be represented
by i mm. or looo/x in thickness. But if one uses blotting paper of
-f-$ mm. thickness or 900/4, there is a loss of looju for each section
and for 9 sections there would be a loss of 900;* or the thickness of a
sheet of the blotting paper. To remedy this one uses 10 sheets of
blotting paper for 9 sections. This keeps the model in true pro-
portion. In practice each of the sections is drawn upon one sheet

CH. XIII] SERIAL SECTIONS AND MODELS 513

except one of them, and for that two sheets of the blotting paper are
united and the sections drawn upon the double sheet.

§ 686. General rule for the use of blotting paper. — Divide the
thickness by which each section is to be represented in the model by
the thickness of one sheet of the blotting paper available. The quo-
tient shows the number of sheets or the fraction of a sheet required
for each section.

If a quotient is a mixed number reduce it to a fraction. The
numerator represents the number of sheets required and the denomi-
nator the number of sections to go with the sheets.

Examples: (a) With a series of IO/JL sections to be modeled at 100
enlargement each section of the series must be represented in the
model by a thickness of lo^u x 100 = IQOOJJ, or i millimeter. If one
uses millimeter or IOOQ/J paper, then 1000/1 -5- roooju == } , and one
must use i sheet for i section.

(6) With a series of IOJJL sections to be made into a model 100
times enlarged, and with blotting paper of -}\ mm. or 900/4 thickness,
each section must be represented by loju X 100 = looo/z. If the
blotting paper is 900/4 thick, then it requires for each section: 1000
•*• 900 = 1 1 sheets of paper or V sheets for one section or 10 sheets
for 9 sections, that is, a double sheet for one of the nine sections.

(c) With a series cut 15/4, for a 5o-fold model, each section is
represented by a thickness of 15/4 X 50 = 750/4. If one uses i mm.
or 1000/4 blotting paper, then each section requires 750 -*- loooju =
f of a sheet for one or 3 sheets for four sections. In this case one
omits every fourth section in drawing, thus: ist, 2d and 3d sections
would be drawn; then the 5th, 6th and 7th; 9th, loth, nth, etc.,
eVery fourth being omitted.

(d) If for the model just considered one had \\ mm. or 900/4
paper, then 750 4- 900 = f . That is, there must be 5 sheets of the
paper for each 6 sections. In that case every sixth section would
be omitted in the drawing, as every fourth section was omitted in

fc).

It is, of course, best to use sheets of exactly the right thickness to
represent the necessary thickness in the model (a), but one can pro-
duce models with accuracy by duplicating one or more sheets for

514 SERIAL SECTIONS AND MODELS '[Cn. XIII

a group of sections (6) or by omitting certain sections of the series
in drawing (c, d).

DRAWINGS FOR MODELS

§ 687. — The methods given for drawing microscopic preparations
in Ch. VIII are all applicable except the free-hand method. This is
not applicable, because it is not possible to draw a uniform and ac-
curate enlargement in that way. But the camera lucida method
(§ 408) or the projection apparatus method (§ 419) is good. With
the perfecting of projection apparatus that method is far the best
because one can sit in a comfortable position and use both eyes.
It is, indeed, as simple as tracing the outline of actual pictures.

By making negative prints directly on one of the developing
papers (§ 488), drawing for models may be wholly avoided.

§ 688. Avoidance of distortion and of inversion. — In the draw-
ings for models one must, of course, avoid all distortion (§ 402) and
the inversion of the image (§ 430). Both these defects are easily
avoided if one keeps in mind the optical principles involved, and
follows the directions given in Ch. IX.

§ 689. Use of the 6-volt, concentrated filament lamp as a source
of light. — From the experience of the author nothing equals the
direct-current arc light for all exacting work in drawing and projec-
tion, and for the dark-ground illuminator, but the care required to
keep the arc lamp going and to keep the crater centered is so great
that the less brilliant light from the 6-volt lamp which requires abso-
lutely no adjustment after being once properly arranged is, very
acceptable (§ 487). The 6-volt lamp with a transformer is used only
on an alternating circuit. As most lighting circuits are now alternat-
ing, it is a great advantage; and as this lamp with its transformer
can be used anywhere wherever there is an ordinary electric light
socket, it is exceedingly convenient. If it is to be used on a direct
current circuit, no transformer is used, but the current must be
drawn from a storage battery, not from a no or a 2 20- volt circuit
from a dynamo.

§ 690. Connections of the transformer. — If alternating current

CH. XTII]

SERIAL SECTIONS AND MODELS

515

and a transformer, are used, the transformer must be connected to
the supply by means of the small connecting wires. The connection
with the lamp is by the large terminal wires. Ordinarily the ter-
minals of the transformer are marked so that no mistake need be
made. Theoretically the transformer does not modify the energy;
it either raises or lowers the voltage or pressure. For the purposes
here used the transformer lowers, the voltage, and is called a step-
down transformer. As the activity or wattage of which the current
is capable is not changed by the transformer, and as the wattage is
the voltage multiplied by the amperage used, if the voltage is
lowered, the amperage is raised proportionally; hence the need of
the large wire on the side toward the lamp beyond the transformer
where the amperage is
increased.

§ 691. Lamp for 6-
volt current. — There
are in common use two
lamps, one of 72 watts
and one of 108 watts.
Now as the wattage
is the voltage times the
amperage, for the 72-
watt lamp the amper-
age with a 6- volt cur-
rent must be 72 divided
by 6 or 12 amperes.
For the 1 08- watt lamp
in like manner the am-
perage is the wattage divided by the voltage, — 108 divided by
6 = 1 8 amperes. This shows at once why the large wires must be
used between the lamp and the transformer. If the usual small
wires are used the resistance is too great and part of the energy is
used up in heating the wires instead of in heating the filament to
supply the light.

§ 692. Arrangement of the lamp for the large projection outfit. —
Tf the lamp is to be used in the lamp-house instead of an arc lamp

FIG. 276. DRAWING AND PROJECTION OUTFIT
WITH LARGE MIRROR ON SEPARATE DRAWING
TABLE.

For full explanation see Fig. 181. Instead of
the arc lamp here shown the 6-volt incandescent
lamp can be used for most purposes (§ 691).

5i6 SERIAL SECTIONS AND MODELS [Cn. XIII

for the large projection outfit, it must be centered carefully and put
the right distance from the large condenser. The filament takes the
place of the crater of the arc lamp and hence should be in the focus
of the first element of the condenser, so that the beam between the first
and second elements of the condenser will be approximately parallel.

If a two-lens condenser is used, the lamp-filament is slightly
within the focus, making the light slightly diverging between the
two lenses of the condenser.

A concave mirror or reflector behind the lamp is of considerable
advantage, for the light which extends backward is reflected forward
to the condenser and is thus available for illuminating the object.

§ 693. Large condenser for drawing. — If the three-lens con-
denser is used (fig. 179), and it is much to be preferred, the second
element which converges the parallel beam should be of long focus.
One of 38 cm. (15 in.) focus has been found very satisfactory. The
reason for using the long focus lens is discussed in Ch. IX, § 423,
fig. 184.

If a two-lens condenser is used, the second element should also be
of longer focus than for ordinary magic lantern work, for the same
reason as for the three-lens condenser.

§ 694. Drawing with the small projection outfit. — If one has no
large projection outfit, drawings for models and for publication can
be made very satisfactorily with the 6- volt lamp as follows: It is
a great advantage to have the lamp in one of the metal lanterns
like those used for daylight glass (figs. 46, 53), then scattered light
willjt>e avoided. There should be a condenser like that used for the
small arc lamp (fig. 78). As the microscope must be horizontal and
is ordinarily raised to make the drawing distance 250 mm., the
lantern containing the 6-volt lamp must be supported on a box or
block to bring the filament of the lamp in the optic axis of the
microscope.

When horizontal, the microscope is unstable; hence a weight or
better a clamp is put over the feet to hold the microscope firmly
so that when once centered it will not move easily. A table with
the drawing shelf on the legs is very convenient for getting the de-
sired magnification (fig. 277).

CH. XIII]

SERIAL SECTIONS AND MODELS

517

§ 695. Relative position of the lamp and microscope. — This can
be as with the small drawing outfit and arc lamp (fig. 182), or it can
be put in line, as with the large outfit. If in line (fig. 179) the
mirror is not used, and care must be taken to get all parts lined up
to one axis. With the mirror slight deviations from centering can
be overcome by inclining the mirror accordingly.

§ 696. Condensers to use with the small outfit. — For low powers,
50 to 1 6 mm. (3.2x-iox), the substage condenser of the microscope
can be turned aside and the small condenser with the lamp alone
employed. In many cases no ocular is used for the sake of the large
field. For powers of 8 to 2 mm. (2ox~9ox) when the ocular is used,
it is necessary to use the substage condenser to light with the proper

Microscope

FIG. 277. DRAWING AND PROJECTION OUTFIT.

For full explanation see Fig. 180. For drawing the 6-volt lamp can well take
the place of the arc lamp here shown (§ 691).

aperture. And if the oil immersion is used, it is a great advantage
to make the substage condenser homogeneous immersion also; that
is, to have some of the homogeneous immersion fluid between the
lower side of the slide and the condenser as well as between the
objective and the cover-glass (§ 265).

§ 697. Making the drawings. — One can draw directly upon blot-

5i8 SERIAL SECTIONS AND MODELS [Cn. XIII

ting paper, but it is so important to have a drawing to refer to that
one or more duplicates should be made. This is easily accomplished
by putting a sheet of carbon manifolding paper on the blotting
paper and a sheet of thin paper over the carbon paper, using thumb-
tacks to hold the blotting paper and the duplicating sheets in position.

One should take the precaution to number each drawing as it is
made; then confusion in the later processes will be avoided.

§ 698. Cutting out the sheets for the model. — " With the blot-
ting paper, if the drawings are small, the cutting is easily done with
scissors or a knife. When the drawings are large and especially
when the model is to be made by representing each section by two
or more thicknesses of blotting paper, it has been found that an
ordinary sewing machine can be used to do the cutting. By setting
the regulator for the shortest stitch, an almost continuous cut is
made and the parts are easily separated. If a large sewing-machine
needle is sharpened in the form of a chisel, the cut becomes con-
siderably smoother. It has been found advantageous when long
continued or heavy work is to be done to attach to the machine an
electric sewing-machine motor. Skill in guiding the work is soon
acquired. There are some details of a complicated drawing which
are more easily cut by the scissors or a knife after the main lines
have been cut by the machine."

§ 699. Contrasting colors for marking groups of sections. — " It
is a great advantage in any working model to have sections at
regular intervals in marked contrast with the body of the material.
Blotting paper of a large variety of colors (black, red, blue, pink) is
easily obtained in the market. In the models made every tenth
plate was a bright or light color and every one-hundredth was black,
rendering rapid numeration easy."

§ 700. Putting the sheets together to make the model. — " When
the paper sections are thus prepared, they are piled and repiled as is
usual until the shape conforms to an outline predetermined from
photographs, drawings, or measurements made before the specimen
was cut.

" It has been found that an easily prepared support and guide for
the model in process of setting up is made by cutting the outline

CH. XIII] SERIAL SECTIONS AND MODELS $IQ

to be followed from a block of four or five sheets of blotting paper,
marking upon it the lines of direction of every tenth or twentieth
section. The colored numerating plates must, of course, conform to
the spacing and direction of these lines.

" The preliminary shaping having been accomplished, more exact
modeling is undertaken. The paper sections slide very easily upon
one another. The most satisfactory means of fastening them to-
gether is by the use of ribbon pins, ordinary pins, or wire nails of
various sizes, depending on the size of the model. No kind of paste
or glue was found suitable for this purpose."

§ 701. Finishing the model. — " When the model is well formed,
inequalities are best removed by rubbing with the edge of a dull
knife and smoothing with sandpaper. Any dissections of the model
for showing internal structures should be planned for at this stage,
for it is now more easily separated than later. It is also at this
time that superfluous 'bridges,' which have been left in place to
support detached parts, would better be removed.

" To finish the model it is held together firmly and coated with
hot paraffin either by a camel's hair brush or by dipping in paraffin
and removing the superfluous coating by a hot instrument. One
might use a thermo-cautery for this purpose.

" The paraffin renders the model almost of the toughness of wood
without destroying the lightness of the paper."

§ 702. Coloring the surface; dissecting the model. — " For color-
ing the surface of the model, it was found most desirable to use
Japanese bibulous paper, lens paper (§54) which had been dipped
in water color and dried. Any of the laboratory dyes or inks can
be used, such as eosin, picric acid, methylene green, black ink,
etc. The colored lens paper molds over the surface with ease and
is held in place by painting with hot paraffin. All color and enumer-
ation lines and fine modeling show through the transparent paper.

" When the model ceases to be a working model it can be covered
with oil paints mixed with hot paraffin and rubbed to any degree of
finish desired.

" One can dissect a model by a hot knife run along the planes
of cleavage or cut across them by a saw."

520 SERIAL SECTIONS AND MODELS [Cn. XIII

For the literature of blotting-paper models see: Susanna Phelps
Gage, Amer. Jour. Anat., vol. v, 1906, p. xxiii; Proceedings of the
International Zoological Congress for 1907; Anatomical Record, Nov.,
1907. (From this paper the above quotations were made.) Zeit.
wiss. Mikroskopie, Bd. xxv, 1908, pp. 73-75.

Blotting-paper models have also been made and demonstrated
by Dr. J. H. Hathaway and by Dr. J. B. Johnston at the Association
of American Anatomists, 1906 (Proc. Assoc. Amer. Anatomists,
Anat. Record, April i, 1907); in 1909 by Dr. J. Parsons Schaeffer
(Anat. Record, 1910); and in 1916 by Dr. Charles Brookover and
Dr. H. Saxon Burr (Anat. Record, 1917).

CHAPTER XIV

MICRO-INCINERATIONS AKD THE OPTICAL APPLIANCES
FOR THEIR EXAMINATION
§§703-730; FIGURES 278-299

INTRODUCTION

Ever since chemistry has given a clue to a method of penetrating
into animal and plant composition, there have been ever-increasing
efforts made to determine what chemical elements and compounds
are present in the different tissues and organs. Because bones and
teeth are evidently so largely made up of mineral substances, they
were the first structures to be studied to find out exactly what are
their mineral constituents. However, the method soon came to be
applied to the soft tissues where mineral matter is not so obvious, and
these too were found to have a distinct mineral content. Naturally
in the beginning the object of study was to determine the total mineral
matter without any attempt to give the exact location of the different
chemicals found in the tissues. Such general knowledge, important
as it is, was not wholly satisfactory, and more and mere the aim has
been to go beyond the animal or organ as a whole to the individual
tissues and cells. And now the investigations are being extended tc
the constituents of the cells, cell membranes when present, cytoplasm
and nucleus. Finally researchers have tried to go still further and
determine the chemical constituents of the chromosomes and the
mitochondria. (See especially the work of Bensley in the Anat
Record, vol. 60, pp. 251-256, 449-455.)

Micro-incineration is for the purpose of locating the different fixec
minerals in the structural units of the body. As in ordinary histologl
cal procedure, sections or isolations must be made to see the individua
tissue elements in multicellular animals and plants, therefore suet
preparations must be so treated that the organic matter is eliminatec

521

$22 MICRO-INCINERATIONS, OPTICAL APPLIANCES [Cn. XIV

and the fixed or non-volatile inorganic matter left in place. As one
studies these incinerated preparations, there comes the feeling that if
the entire amount of organic matter could be removed from an animal
or plant without disturbing unduly the mineral matter, the entire
tissue, organ, or animal as a whole would be as recognizable as are the
bones under similar conditions.

Among the first attempts to locate the mineral matter in the tissues,
the structures were heated red hot to burn off the organic matter, and
to the astonishment of the experimenter, the French chemist, Raspail,
1833, the shape of the tissue did not seem changed. (Raspail, in the
collateral reading.) While this crude method gave much information,
the present refined and successful method of micro-incineration came
only when Policard invented the small, regulated electric furnace
(Policard, collateral reading). In America the chief exponent of
micro-incineration and the results to be attained with it, is Dr. Gordon
H. Scott of Washington University, St. Louis. He had the great ad-
vantage and privilege of working with Dr. Policard, and learning at
first hand the refinements of the method. Dr. Scott has also devised
a much improved micro-incinerator (fig. 278) by which any laboratory
worker can get excellent results.

§ 703. Chemical constituents of the organism, animal or plant. —
One might fairly expect that owing to the marvelous activities of
animals and plants during life they would require some of the most
rare and subtle chemical components; but the truth is that the chemi-
cal elements found in organic bodies are few in number, only about 20
of the 90 or more already known, and these few are among the com-
monest, the rarer ones being wholly absent. These elements are:
calcium, carbon, chlorine, copper; fluorine; hydrogen; iodine, iron;
lithium; magnesium, manganese; nitrogen; oxygen; phosphorus,
potassium; silicon, sodium and sulfur. Some others are occasionally
found, but they are thought to be accidental or due to the special
environment (Starling's Physiology, yth ed., p. 33).

In the living organism these elements exist mostly in compounds.
In incinerated preparations only the fixed or non-volatile compounds
remain. (See the references in the collateral reading.)

While a chemist can determine with ease and certainty the compo-

CH. XIV] MICRO-INCINERATIONS, OPTICAL APPLIANCES 523

sition of the bulk ash in organic matter. Determining the exact
character of the chemical constituents in the individual cells has
proved a difficult task. The refined methods of chemical microscopy
and the use of spectrum analysis and finally of the electron microscope
have all been called into requisition with only partial success up to
the present time. (See the papers from 1930 to 1940.)

PREPARATIONS FOR MICRO-INCINERATION

§ 704. Fixation. — It is self-evident that for the determination of the
amount and character of the mineral matter in tissues and cells, the
preliminary treatment should not add anything nor remove anything
cf mineral nature. That is, the ideal fixative would render permanent
the structural constituents exactly as in life. It would realize in
modern histology what the ancients in their mythology ascribed to
Medusa of the snaky locks. This ideal fixative has not as yet been
discovered. Of the hundreds of combinations which have been tried,
not one is universal; all are more or less selective. For example, if
one wishes to determine the presence of glycogen, strong alcohol is an
excellent fixative, but if lipoid substances are to be sought for, it is
very poor.

For the determination of the mineral constituents, the standard
fixative is 9 parts of absolute alcohol and i part of strong, neutral
formalin. Small pieces of tissue or parts of organs are placed in this.
Small pieces are used so that the fixer will penetrate quickly and pre-
serve all the cells. Twenty-four or thirty-six hours is usually sufficient.
Either a relatively large amount of the fixer is used, or if a smaller
amount as compared with the tissue, then it should be changed two
or three times for fresh fixer. If one cannot proceed at once with the
sectioning, the tissue may remain in absolute alcohol, but it is better
to imbed the tissue at once after it is fixed.

§ 706, Imbedding for sectioning. — The paraffin method is practi-
cally always used, as the paraffin is wholly removed in the subsequent
steps, and therefore adds nothing to the sections. It is essential for
the imbedding that the tissue shall be wholly freed from water. This
can be attained by two or more changes of the absolute alcohol.

524 MICRO-INCINERATIONS, OPTICAL APPLIANCES [Cn. XIV

§ 706. Clearing before paraffin. — The purpose of this is to remove
the alcohol by a liquid which is a solvent of paraffin. It is usually
accomplished in two steps: From the absolute alcohol the tissue is
passed to a mixture of equal parts of absolute alcohol and xylene for
an hour or more, and then it is placed in pure xylene till it appears
translucent, or it may be passed from the absolute alcohol to cedar
oil and left in the cedar oil till it is translucent. When it is translucent
by either of the above methods, the tissue may be transferred to
melted paraffin. This is also done in two steps by many workers.
The first step is to transfer the cleared tissue to low melting point
paraffin (40° to 45° melting point), and after an hour or more in this,
it is transferred to melted paraffin of 56° to 58° melting point, and kept
in this melted paraffin in an infiltrating oven or box for several hours.
It is then fully infiltrated with the hard paraffin and is ready to be
put in a block for sectioning. Whatever method is used for finally
blocking the tissue for sectioning, it should be remembered that
paraffin quickly cooled is more nearly homogeneous, i.e. has finer
crystals, than paraffin cooled slowly. The finer the texture of the
paraffin the more successful the sectioning.

§ 7070. Sections for incineration. — These must be thin. Rarely
will one get good results with sections over lo/z thick, and the usual
experience is that sections 3ju, 5ju or 7jic give even better incinerations
than thicker ones.

Of course, for such thin, perfect sections the section knife must be
sharp, and the microtome a good one. The room temperature should
not be over 20° centigrade, and for the thinnest sections a temperature
of 12° to 15° c. is more favorable. In his Plant Histology y Dr. Chamber-
lain advises the cool room, and safety razor blades in a suitable holder
for sectioning. Many others, including the author, have also found
the safety razor blade satisfactory (see §§ 621, 634, and Chamberlain^
5th ed., p. 122).

§ 708. Glass slips for incineration preparations. — Not all brands
of glass slips have been found of sufficiently high melting point to
remain undistorted during the incineration which goes up to 600°
centigrade or hotter, that is, to red heat. Hence it is wise before wast-
ing time and losing valuable specimens to make sure the glass in the

CH. XIV] MICRO-INCINERATIONS, OPTICAL APPLIANCES 525

mounting slips will not become distorted by the heating. The author
has found the Corex D glass recommended for preparations to be
studied under the ultra-violet microscope (§ 308) and Pyrex micro-
scope slips to remain undistorted in every case. If the glass slips are
of high enough melting point, it is unnecessary to use sheet platinum
to prevent their sticking to the supporting quartz plate.

§ 709. Spreading sections for incineration. — As nearly all sections,
especially thin ones, are more or less wrinkled in sectioning, it is
advantageous to flatten or spread ' them. Some recommend that
petrolatum or absolute alcchcl be used, but nothing is so satisfactory
as the usual water method. A suitable length of ribbcn is put on a
perfectly clean slide (the Stitt method cf cleaning with bon ami
§§ 512, 515, has been found satisfactory).

The slide is then warmed on a spreading box or plate (fig. 255-256)
and with needles the sections are drawn out flat and arranged. After
this the excess water is drained off and the sections are left to dry
completely. If they are left over night in a dry, warm place, they
will be completely adherent to the slide. It is important to remem-
ber that no albumen or other material is to be put on the slide.
There is no danger of the sections getting loose during the incineration.

In seme cases it has proved advantageous, after the excess water
has drained away, to use tissue paper and press the sections down
firmly upon the slide with the ball cf a finger. The paper is then
rolled off the sections by lifting one edge and turning it in a circular
ir.anncr. If cne is skillful, the sections v/ill remain firmly attached to
the slide (§ 637).

It is recommended that every ether slide cf sections be prepared for
staining and mounting in the best way experience has shown for the
particular tissue. This is important for the stained preparations have
the mere familiar appearance, and special features are found easily.
Cf course, for these sections the usual albumen coating on the mount-
ing slide is permissible, but not for the slides to be incinerated.

§ 710. Incinerator and incineration. — The electric furnace found
most satisfactory for micro-incineration is Dr. Scott's modification of
Policard's (fig. 278). The heating is regulated by an adjustable rheo-
stat. On a 110-115. volt circuit it reaches 637° C. in 41 minutes if one

526

MICRO-INCINERATIONS, OPTICAL APPLIANCES [Cn. XIV

follows the settings in the table. For the cost of appliances needed for
the micro-incineration work see at the end of this chapter.

FIG. 278. SCOTT MICRO-INCINERATOR; FRONT VIEW.

A End view of the quartz tube in which the sections are incinerated. It is
surrounded by a heating coil.

Q Quartz plate for supporting the slide of sections to be incinerated. The
platinum plate (PL) shown on its upper face is to prevent slides from sticking.
For the corex and pyrex slides this expensive part may be omitted.

B Baseboard of the incinerator; it fits the laboratory lockers.

C Cap or lid to cover one end of the quartz tube.

CE Cap for connecting with the electric circuit.

F Rubber support between the insulating plate and the base.

H Handle of the insulating cover of the quartz tube.

M-I Insulating block containing the quartz tube.

Q Quartz tube; it is surrounded by a heating coil and is imbedded in insulating
material.

R Variable rheostat with its rider (SL).

SL Rider on the adjustable rheostat to vary the resistance.

T Top of the insulating material covering the quartz tube. It is shown lifted
off to hasten the cooling of the quartz tube after an incineration is completed.

i-/o Scale of 10 centimeters to show the amount of reduction.

The furnace is designed to be connected to the usual house lighting
circuit. As stated in the legend of fig. 278, there is associated with it
an adjustable rheostat by which the heating is gradually increased.
This is important, for if heated too rapidly, the ashes do not remain
in position.

When ready to incinerate a slide of sections, place the slide on the
quartz plate and push the plate bearing the sections into the heating

CH. XIV] MICRO-INCINERATIONS, OPTICAL APPLIANCES 527

tube to its middle. This tube is long enough to admit two slides at
once, but the incineration is usually more successful if but a single
slide is placed in the middle.

For sections where no mercury was used in the fixation, the paraffin
need not be removed from the sections. The heat in incineration will
burn it off. (See also § 712 for mercury fixatives.)

While incinerating, it io found in practice that an interval timer is
convenient to mark the different steps, then one can attend to other
duties during the intervals.

§ 711. Time of incineration. — There is considerable difference in
the ease of burning off the organic matter with different specimens.
As there has not yet been sufficient experience to standardize the
method for all objects, the individual worker must do considerable
experimenting with his particular material.

In general, it takes about 41 minutes for a successful incineration
when the schedule in the table is followed. In a dark or dimly lighted
room the quartz tube will show a red heat over its entire length at
the 7th or 8th interval. The current is then turned off and the cap C
pulled back to allow free access of air. The insulating top T is lifted
off to hasten the cooling.

The following table shows the different settings of the rider (SL)
on the resistance coil (R) for each of the eight intervals and the time
of each interval. It also shows the amperes, volts of the line, the
v/atts used, the millivolts when the incinerator is in the circuit, and
finally, the temperature centigrade at the end of each setting.

With some tissues a slower increase in heating and a longer time
than given in the table give better results.

It is seen from the table that it takes 25 minutes for the incinerator
to increase from room temperature of about 20° to 300° C. and an ad-
ditional 15 minutes to increase the temperature to 600° C. With the
current turned off, the insulating top removed and the cap turned
back from the quartz tube, it takes 30 to 40 minutes for the furnace
to cool down to 300°, when it is safe to remove the incinerated slide
with forceps. The slide will soon cool in the free air. If to the naked
eye the ashes are gray or white and have the form of the incinerated
sections, the incineration is usually successful.

MICRO-INCINERATIONS, OPTICAL APPLIANCES [Cn. XIV

Sometimes the ashes look confused or brown. This is likely to
happen if the sections are too thick or not completely dry before the
incineration begins. Like other operations in microscopy, micro-
incinerations require much exactness in detail, and even then the
results are sometimes disappointing. When success is obtained, how-
ever, one feels well paid for all the trouble.

For the calibration of the electric furnace shown in figure 278 a
Chromel-Alumel thermocouple and a potentiometer with the neces-
sary ammeter and voltmeter were employed and the observations
made in the laboratory with the alternating electric current available
where the incinerations were to take place.

Table Showing the Calibration of the Micro-Incinerator.

Setting

Minutes

Amperes

Volts

Watts

(AxV>

Millivolts

Temper-
ature, ° C.

i. 60

113

180.80

4-32

104

1.90

113

214.70

6.29

150

2.18

113

246.34

9.68

235

2.50

113

282.54

12.80

2.82

113

318.66

16.20

394

3-25

113

367-25

20.40

492

3-70

113

418.10

25.00

60 1

3-82

113

431.66

26.50

637

§ 712. Further methods of fixation for incineration. — As stated,
it is axiomatic that ideally the fixer should not add anything or remove
anything from the tissue to be incinerated. But as every worker with
the incineration method soon realizes, alcohol-formalin preparations
are far from ideal histologically, and some tissues become so hard that
they can scarcely be cut (e.g., ligamentum nuchae).

One of the simplest and best fixers that have been used for a great
variety of tissues is a mixture of a 3 % aqueous dichromate of potash
solution to which has been added neutral formalin in the proportion
of 90 cc. dichromate solution, 10 cc. strong, neutral formalin. Small
pieces are fixed in this two or three days, changing the freshly pre-
pared mixture each day. Then it is washed several hours in running
water, transferred to 67% alcohol for one or two days, then 82%
alcohol until one is ready to proceed with the imbedding. The dehy-

CH. XIV] MICRO-INCINERATIONS, OPTICAL APPLIANCES 529

dration should be thorough in 95 % and absolute alcohol. The clear-
ing is then by means of absolute alcohol and xylene, then pure xylene
or cedar oil. Infiltration is accomplished by low-melting and then
high-melting paraffin as in § 706. With these sections the paraffin
need not be removed. If a fixer with mercury, such as Zenker's fluid
cr Kelly's fluid, is used, then the paraffin must be removed and the
n:ercury got rid cf by soaking in iodin. (See § 597.) After the alcohol
used to remove the iodin, the sections are allowed to dry in the air,
and may then be incinerated. In any of these methods only small
traces cf chromium and potassium salts are added, but the amount
is only a trace, and the general result is vastly superior to the alcohol
formalin method both for the histological and for the incineration
appearance as cne can see by comparing preparations made by the
different methods.

§ 713. Preservation of incinerated sections. — It cannot be too
highly emphasized that the ashes of the incinerated specimens are
very delicate and can be easily disarranged if brushed or the fingers
put upon them. If one is careful the uncovered preparations can be
examined, but to avoid injury it is far safer to put a cover-glass over
them at cnce. To do this a clean cover is placed over the specimen
and held in place at one end by the thumb and finger. Then with a
hot wire in the other hand a seal of beeswax is run along the four edges.
The best way is to get the wire quite hct in a bunsen flame and then
press the wire against a mass cf beeswax to get it well coated. Then
it is run along the edges. The beeswax cools almost instantly and
makes a good seal which can later be covered with shellac cement foi
added strength and permanence. Many advocate the use of paraffir
for the seal, but the high melting point of the beeswax makes it more
suitable for the purpose, and less likely to melt and run under the
cover and spoil the preparation.

One can also make a shallow cell of shellac or balsam or other cemenl
about the ashes; and when nearly dry the slide can be warmed anc
the cover pressed down all around against the cement till it adheres
The beeswax method is preferable, however.

§ 714. Mounting medium for incinerations. — As described above
incinerated preparations are mounted in air. This is the most satis

530 MICRO-INCINERATIONS, OPTICAL APPLIANCES [Cn. XIV

factory way, for any mounting medium so far suggested is likely to
disarrange the ashes or to obscure the finer details. It is instructive,
however, to be able to compare the ashes in air and in some mounting
medium. This is easily accomplished if the slide has upon it several
sections of a ribbon. Before adding the cover-glass a small drop of
petrolatum (§§ 536, 602) is put upon one of the end sections. If the
cover is then added as directed, the mineral oil will spread over one
or two of the sections, the remainder being in air. Then it is easy to
compare the ashes in the air with those in the petrolatum.

§ 715. The mineral matter of plant tissues. — Judging by the lit-
erature, much less work has been done in incinerating plant tissue than
animal, but as stated above, it was plant tissue that Raspail found so
interesting when the organic matter was burned away. In the limited
experiments carried on in the Cornell laboratory, the plants lent
themselves as readily as the animals to this form of investigation.
The presence of much silica in many cases adds to the striking appear-
ance of the ashes. For example, the teeth or serrations along the
edges of grass leaves are almost completely unchanged and have the
same clear outlines as in the stained preparation, and as shown later
the mineral remains of plant tissues polarize almost as strongly after
incineration as before, thus being in strong contrast to the mineral
matter of animal tissues.

In preparing the tissues for incineration one must take the same
precautions as for animal tissues (§§ 704-712).

§ 716. Minerals in pathological material. — The incineration
method has been utilized with informing results for pathological
tissues. As one might expect from arteriosclerosis in the blood vessels,
the mineral contents in pathological material is often considerably in
excess of that in normal tissue.

OPTICAL APPLIANCES FOR THE STUDY OF MICRO-INCINERATIONS

§ 717. Dark-field microscopy. — From the nature of the material,
a dark-field is almost a necessity for the study of the ash after micro-
incineration. Fortunately this study can be made most successfully
with the rather simple apparatus found in every laboratory. For the

CH. XIV] MICRO-INCINERATIONS, OPTICAL APPLIANCES

531

general understanding of dark-field microscopy the reader is urged to
go carefully over the discussion of the dark-field microscope in Ch. III.

FIG. 279. PARABOLOID DARK-FIELD CONDENSER.

SB Solid beam of light from the mirror (M) to the ccntr.il stop, which allows
only a hollow beam to pass on to the condenser.

Cst Central stop to intercept all but the border rays.

EC Hollow cone passing on to illuminate the object.

AS Silvered outside surface of the paraboloid.

//, //, // Homogeneous media below the slide, co\ cring the object and above the
cover-glass.

eg, gs Cover-glass and glass slide.

Obj The first element of the objective showing the light deflected from the ob-
ject by dotted lines.

For the special work with incinerations, the following observations
upon their illumination are added after much experience.

Micro-incinerations are most satisfactorily studied with moderate
powers, therefore rather small apertures and large fields are utilized.

The proper illumination may be most easily obtained by the use of
the ordinary condenser and a central stop (§ 719).

532

MICRO-INCINERATIONS, OPTICAL APPLIANCES [Cn. XIV

Furthermore, while much of the ash is in optical contact with the glass
slide, the overlying mineral substance is in air. This makes the ordi-

FIG. 280. REFRACTING CONDENSER WITH A FIXED DARK-STOP
BELOW THE UPPER ELEMENT.

1 Rays of light from the object in the focus of the dark-field condenser.

2 Front lens of the objective.

3-3 Sectional view of the hollow cone from the condenser. It lights the object

at its focus, and is of greater aperture than the objective. (See figs. 294-296.)
4-4 Glass slide supporting the object. It should be of a thickness to bring the

object at the focus of the hollow cone, and should be in immersion contact

with the top of the condenser.
5 Numeral placed just above the dark-stop, which eliminates the central part of

the light cone.
6-6 First or lower element of the condenser. Most often the dark-stop is below

this element. (See fig. 279 )
7-7 The plane and concave faces of the mirror.
8-8 Parallel rays from the light source.

nary refracting condensers now found on nearly all laboratory mi-
croscopes entirely adequate. It is well to recall that before Wenham

CH. XIV] MICRO-INCINERATIONS, OPTICAL APPLIANCES 533

FIG. 281. REFRACTING CONDENSER WITH CENTRAL STOP UELOW TO *ORM A
HOLLOW CONE OF LIGHT FOR DARK-FIELD ILLUMINATION.

SB Solid beam of light from the mirror (M) to the central stop (Cs/), which
cuts out all but the border rays.

HC Hollow cone focusing on the object on the slide (SI). Compare with
figure 70 where only the border rays seem to be extending from the mirror to the
condenser. This is the usual method of representing all dark-field condensers.

FIG. 282. CENTRAL DARK-STOPS OP 10, 15 AND 20 MILLIMETERS TO USE WITH
REFRACTING CONDENSERS AND OBJECTIVES OP DIPPERENT POWERS.

534 MICRO-INCINERATIONS, OPTICAL APPLIANCES [Cn. XIV

283

284

285 286

FIGS. 283-286. TRANSKCTIONS or THE LIGAMENTUM NUCIIAE OF THI<: Ox.
(All at a magnification of 250.)

283 Specimen stained with VerhoefTs hematoxylin. Bright-field photomicro-
graph.

284 Unstained specimen incinerated to show the mineral matter. Dark-field
photograph.

285 The same specimen photographed with a light-field.

286 The same specimen photographed with a dark-field.

(in 1850-1856) introduced the paraboloid condenser (fig. 281) for high
power dark-field illumination, the English microscopists were making

CH. XIV] MICRO-INCINERATIONS, OPTICAL APPLIANCES

535

much use of the refracting condensers for dark-field lighting by in-
serting an opaque central stop below the condenser to eliminate the
central part of the light ccne, and thus light the object by rays at such
great obliquity that none of them could enter the objective, hence the
objects seemed to shine by their own light in a dark field. The re-
fracting condensers also light a relatively large field, and the specimens
need not be on a slide of such definite thickness as is required by the
special dark-field condensers.

§ 718. Relative numerical aperture of condenser and objective
for dark-field illumination. — By glancing at figures 291-292 and
294-296, it will be seen that the aperture of the objective must be
considerably less than that of the condenser or the rays of light from
the condenser will enter the objective and render the field light. This
involves two requirements: There must be some means (i) of varying
the size of the dark-stop under the condenser and (2) of varying the
aperture of the objective by means of a reducing diaphragm, most
conveniently of the iris type in the objective.

Generally speaking, an aperture of less than 0.65 N.A. is most
successful in objectives to be used with refracting condensers.

Table of the diameter of the central dark-slop below the condenser of 1.20 or 1.40 N.A.
and the aperture of objectives to give the best ejffects with incineration specimens.

Objective

Full N.A.

Best N.A. for
Incinerations

Size of Sub-
stage Stop

1 6 mm.

0.25

10 mm.

8 mm.

0.50

0.40-0.50

10-15 mm.

4 mm.

0.66

0.45-0.60

15-20 mm.

3 mm.

0.85

0.50-0.60

20 mm.

1.8 mm. im.

1-25

0.50-0.60

20 mm.

In figures 294-296 is shown the central part of the light cone
eliminated by the different central stops of 10 mm., 15 mm. and
20 mm. diameter, and by the dotted lines is shown the angle of the
dark center which is utilized by the different objectives for dark-field
observation. These diagrams show convincingly that the dark area
in the cone cannot all be utilized for dark-field illumination by the

536 MICRO-INCINERATIONS, OPTICAL APPLIANCES [Cn. XIV

objective. Apparently the diffracted light along the edges of the
hollow cone is sufficient to give a light halo around the margin of
the dark field if the aperture of the objective is too great. The dia-
grams show also the thickness of the hollow cone of light remaining

287

288

289

290

FIGS. 287-292. CONES OF LIGHT IN URANIUM GLASS IN IMMERSION
CONTACT WITH THE TOP OF A CONDENSER RATED AT 1.40 N.A.

U Uranium glass with refractive index no 1.5069.

287 Cone of light with full aperture. Plane mirror.

288 Cone of light with substage iris open 10 mm. Plane mirror.

289 Cone of light with iris open 10 mm. Concave mirror.

290 Cone of light with iris of 10 mm. Plane mirror tipped.

CH. XIV] MICRO-INCINERATIONS, OPTICAL APPLIANCES 537

291

292

291

2Q2

Hollow cone with a 10 mm. substage dark-stop. Plane mirror. Iris of

condenser wide open.

Hollow cone with a 20 mm. dark-stop. Plane mirror. Iris of condenser wide

open.

By comparing fig. 287 with 203, and 201-292 with 205-296 it will be seen that the
greater the aperture of the condenser the thicker will be the hollow cone of light
with a given dark-stop and consequently the greater the amount of light to illumi-
nate the object at its focus.

with the different substage stops. Of course the thicker the hollow
cone of light, the greater is the amount of light available for illuminat-
ing the object at its focus. It is also seen, except for the 16 mm.
objective, that the available angle is less than the numerical aperture
of the objective used.

§ 719. Best dark-field effects. — For obtaining the best dark-field
effects two great principles must be kept constantly in mind: (i) That
the illumination must be of sufficient intensity to render the finest
details of the object visible, and (2) That the aperture of the objective
must be great enough to resolve the visible particles, i.e., to show the
details (§ 264). One can see an unforgettable demonstration of these
principles as follows: Get by trial the most favorable illumination
and the best aperture of the objective to give the clearest view of the
details, then keeping some fine dot or other detail in sight, gradually
dim the light by putting neutral glasses or ground glasses in the path
of the light source. As the light is dimmed the fine details are lost.
Then restore the light to give the clearest image. Now gradually close

538 MICRO-INCINERATIONS, OPTICAL APPLIANCES [Cn. XIV

295

296

FIGS. 293-296. DIAGRAMS TO SHOW THE CONES OF LIGHT IN URANIUM

GLASS (U) PROM A CONDENSER RATED AT 1.20 N.A. THE

DOTTED PORTION SHOWS THE APERTURE

AVAILABLE FOR DARK-FIEID.

It is

Uranium glass in homogeneous contact with the top of the condenser,
fluorescent and has a refractive index of nD 1.5069.
293 Cone of light with the substage iris wide open. As indicated u} or half the
angular aperture, = 47°, It is the same in all.

CH. XIV] MICRO-INCINERATIONS, OPTICAL APPLIANCES 539

294 Hollow cone with a 10 mm. dark-stop. The dark hollow has u, 19°, but only
9° 30' of this is available for the best dark-field effects.

295 Hollow cone with a 15 mm. dark-stop. This gives a dark hollow of u 26°,
but only u 17° 30' gives the best effects.

296 Hollow cone with a 20 mm. dark-stop. The dark-center is u 34° 30' of which
only about u 19° is available for the best dark-field effects.

The smaller the dark-stop the more the light for illumination, but the smaller
must be the aperture of the objective.

the iris in the objective and soon the aperture will become too small
for resolving the fine details, and they will disappear. On increasing
the aperture, they will reappear.

It must not be forgotten too, that for the best dark-field effects with
all forms of condensers the slide must be in immersion contact with
the top of the condenser, otherwise only an aperture of i.oo N.A. can
pass into the slide to illuminate the object on its upper face. As
many of the particles are in optical contact with the slide, the rays
of an aperture greater than i.oo are very important for illuminating
the object. (See fig. 73 and § 190.)

§ 720. Determination of the aperture of the objective. — For the
information given in the above table (§ 718), the best light and the
most favorable aperture of the objective was found by trial in each
case, then the aperture of the objective actually used was determined
by removing the objective with care not to change the objective iris,
and employing the apertometer. (See for using the apertometer
§ 266.)

§ 721. Change from dark-field to bright-field illumination; combin-
ing bright- and dark-field illumination. — The refracting condensers
have a great advantage over the special dark-field condensers in that
with them it is easy, without disturbing the preparation in the least,
to change the illumination or to combine the dark- and the bright-
field lighting. If the central stop is in place, the field will be dark, but
if that is removed and the substage iris is used, the field will be light.

Furthermore, if one wishes to see the effect of a combination of
light-field and dark-field, that is also readily accomplished as follows:
A small central dark-stop is used, say one of 5, 7, or 10 mm. which
will eliminate only a small central cone. For example, one might use
a 10 mm. dark-stop and the 3 mm. or the 4 mm. objective. If the iris
of the objective is closed sufficiently, there will be a dark-field; but

540 MICRO-INCINERATIONS, OPTICAL APPLIANCES [Cn. XIV

if it is opened it will include in the aperture of the objective some of
the edge rays of the hollow cone. The central stop gives the dark-field
illumination and the edge rays of the hollow cone the bright-field
illumination.

If, when the iris is closed sufficiently to give a dark-field one looks
into the microscope and gradually opens the objective iris, there will
first appear a bright halo all around the field. This will gradually
spread over the whole field as the iris is opened. It is instructive also
to reverse the process and attain a dark-field again.

§ 722. Comparison cf stained and incinerated tissues. — As
recommended above, every other slide of a ribbon is mounted for
staining. With the thin sections the tissue on the stained slide and
that on the incinerated one are nearly identical so that one can see
the histological elements in the stained sections, and the same ele-
ments represented by the mineral matter in the incinerated ones. It
is cf advantage to have two rdcrcsccpcs near together, the one for
the stained preparation lighted with the bright-field and the inciner-
ated one with dark-field illumination. One can look from one to the
ether and make sure that the same elements are being studied. If
one has a comparison ocular (fig. 142) one can see the two fields at
the same time and thus make the comparison more exact. Two
wholly different microscopes answer very well, however. By repeated
comparison, one soon learns to detect special structures by the ash in
the incinerated specimens with the sarr.c ccrtcJnty as with stained
specimens.

§ 723. Dark- and bright-field appearances. — In the accompany-
ing photomicrographs (figs. 283-286), the appearances are strikingly
different for the same tissue, depending upon the method of prepara-
tion and also upon the method of illumination. Figures 283 and 285
were photographed with the bright-field microscope, while figures

284 and 286 were made with a dark-field microscope. Figures 284,

285 and 286 are of identical parts of the same specimen. The stained
specimen (fig. 283) is of the same elastic tissue, but could not be quite
identical with the other figures. The cut ends of the fibers were
stained black by VerhoefTs method, and are markedly larger than the
mineral matter in each fiber although all were magnified exactly the

CH. XIV] MICRO-INCINERATIONS, OPTICAL APPLIANCES 541

same (250 diameters). In the stained specimen there seems to be
empty space between the black fibers, but this is not the case. The
white spaces are filled with collagenous connective tissue as can be
seen in a specimen stained with Mallory's connective tissue stain,
which colors the elastic tissue red and the ordinary connective tissue
blue. In unstained preparations under the dark-field, also in a prepara-
tion like this, the dark- field illumination reveals the collagenous tissue;
and with the ultra-violet microscope the elastic tissue in unstained
sections mounted in petrolatum fluoresces blue-white, but the inter-
vening collagenous tissue does not fluoresce and therefore the area
between the elastic fibers appears black. On the other hand, with
the dark-field microscope the elastic fibers are dark and the collage-
nous tissue a brilliant white (fig. 130, A B). These observations will
also emphasize the necessity of using many methods if one is to gain
a true insight into the real complexity of organic structure.

FIG. 297. CHALET MICBOSCOPE LAMP.
(See figs. 46-47.)

LAMPS FOR ILLUMINATION

§724. Intensity and visibility. — As stated above (§719) there
must be sufficient intensity of illumination to render visible the
objects one wishes to see. For this with all powers, both for light-
and for dark-field study, one of the research lamps (figs. 298-299)
answers well. For bright-field observation with all powers, and for
the lower powers in dark-field observation the Chalet Lamp (fig. 297)
is adequate.

542 MICRO-INCINERATIONS, OPTICAL APPLIANCES [Cn. XIV

For both lamps the plane mirror is usually the one to employ, and,
if dark-field illumination is desired, the substage iris diaphragm is

FIG. 298. RESEARCH MICROSCOPE LAMP WITH io8-WATT
6- VOLT BULB AND ACCESSORIES.

(For full description see fig. 80.)

opened fully (figs. 287, 293). As seen by fig. 288, if the substage iris
is partly closed, the aperture of the condenser is lessened and a dark
central stop would eliminate all the light.

In figure 289 the concave mirror is used, and in 290, the light is
oblique. Figures 291-292, 294-296 show that with the substage iris
wide open, a dark, central stop leaves a rather thick shell or hollow
cone to light the object at its focus.

The research lamps are too brilliant for some specimens. The light
can be softened as desired by introducing neutral tint glasses or
ground glasses in the path of the beam.

While the lighting recommended above gives the best results, it is
quite marvelous how much can be seen with lights of lower intensity.
This was demonstrated to the author on one occasion when snow and
floods eliminated the electric lights. Then a kerosene lamp, a naked
candle flame, and an electric flash light were tried out of curiosity.
The results were astonishingly good for both bright- and dark-field
observation with objectives as high as a 4 mm. Such experiences give

CH. XIV] MICRO-INCINERATIONS, OPTICAL APPLIANCES 543

one an inkling that the old histologists were not so badly handicapped
as is sometimes thought, and the insight they gained into histological
structure with their simple appliances is not quite so astonishing.

FIG. 299. RESB&RCH MICROSCOPE LAMP OF THE SPENCER LENS COMPANY.

1 Knob with which to incline the lamp-house when it is hot.

2 Lamp-house.

3 Screw head for focusing the condenser.

4 Handle of the iris diaphragm in front of the condenser; the 4 is on the water-
cell container.

5 Pyrex water-cell partly raised in its holder.

6 Neutral tint glasses in the color-screen holder.

7 Holder for neutral tint and color screens.

8 Lamp-socket.

9 Lamp-house standard on which it can be raised and lowered.

10 Base of the lamp-house support and electric plug cap.

§ 725. Micro-incinerations under the polarizing microscope. —

The micro-incinerations of animal tissues with the abundant mineral
matter after incineration must be in an amorphous condition as usually
there is no double^ refraction shown under the crossed nicols of the

544 MICRO-INCINERATIONS, OPTICAL APPLIANCES [Cn. XIV

polarizing microscope. With plant tissues, however, the polarization
after the incineration was almost as good as before. The serrations
along the edge of grass blades were apparently quite unchanged by
the incinerating heat both in form and in reaction in polarized light.
This striking difference in the minerals left by incineration of animal
and plant tissues was quite unexpected, but was present in all the
cases examined except in artericsclcrcsis.

§ 726. Micro-incinerations in ultra-violet radiation. — In both
animal and plant tissues the ashes show no fluorescence in the numer-
ous examples tested. Comparison specimens unstained and unin-
cinerated and mounted in petrolatum after the removal of the paraffin
by xylene, gave brilliant fluorescence in both animal and plant tissues.

In the ultra-violet, then, the animal and plant tissues agree, but the
ashes are ordinarily in striking disagreement in polarized light.

SPECIAL APPARATUS NEEDED FOR MICRO-
INCINERATION INVESTIGATION

§ 727. Policard Incinerator Modified by Scott. — This is shown in
figure 278 with its regulating rheostat. The cost is approximately
$4o-$5o.

§ 728. Optical appliances. — It is assumed that the laboratory or
private worker already has a good laboratory microscope with a
refracting, substage condenser, and low-power objectives up to 16
mm., also a good lamp for use with bright-field.

Objectives needed and desirable: It will be noted by comparing
the price here given with that in the manufacturer's catalogues that
it costs $5.00 additional in each case to have the iris diaphragm
present.

Objectives: 8 mm. achromatic, N.A. 0.50, iris $20.00

4 mm. achromatic, N.A. 0.66, iris $22.00

3 mm. achromatic, N.A. 0.85, iris $25.00

1.8 mm. oil immersion, N.A. 1.25, iris. . .$40.00

For most work with incinerations the 16 mm., and the 8 and 4 mm.
objectives with iris are sufficient. If one wishes to carry the investiga-
tion as far as possible, the 3 mm. dry, and the 1.8 mm. oil immersion

CH. XIV] MICRO-INCINERATIONS, OPTICAL APPLIANCES 545

with iris will be needed. It is also desirable to possess one of the
special, dark-field condensers of the paraboloid or cardioid type.

For the refracting condensers, substage dark-stops of 10 mm.,
15 mm. and 20 mm. are needed. One can make them if necessary.
(See § 180.)

§729. Research microscope lamp (figs. 298-299). — The cost is
about $60.00. For photomicrographs and for dark-field work the
type with 6-volt io8-watt lamp bulbs requiring a step-down trans-
former is much to be preferred to any other type. (See pp. 146-150.)

The lamps and the objectives here recommended may be obtained
from the Bausch & Lomb Optical Company, Rochester, N. Y., or
from the Spencer Lens Company, Buffalo, N. Y.

§ 730. Uranium glass for showing the form and path of light
beams. — For teaching purposes and for the individual worker it is
of great advantage to see exactly how the light from the condenser
actually appears with different arrangements. The pictures shown in
figures 287-292 represent some of the appearances. As described in
the legend of those pictures, they were obtained by placing a plate of
uranium glass of refractive index nD 1.5069 in homogeneous contact
with the upper face of the condenser. Because of its fluorescent
character one can see in this glass just how the cone of light from the
condenser appears with the plane and the concave mirror, with the
light central or oblique, with the full aperture and with the aperture
reduced by the substage iris, or by a dark-stop to cut out the central
part of the light cone. After experimenting with this help one will
always have a clear conception of just what happens to the light under
different conditions.

A plate of uranium glass 50 mm. square and 12 to 1 8

mm. thick with all the faces polished costs $5.00

A cube with 25 mm. sides all polished also costs $5.00

This glass is called fluorescent canary, and may be had of the
Corning Glass Works, Corning, N. Y.

The microscope slips of high melting point required in § 708 which
will not scf ten and become distorted in the incineration process may
be had of the Corning Glass Works also.

That these high melting-point slips may not be confused with the

546 MICRO-INCINERATIONS, OPTICAL APPLIANCES [CH. XIV

ordinary glass slips used in microscopy, the author has found the
size of 25 x 65 mm. used with the ultra-violet microscope satisfactory
and convenient (see § 308, and figs. 218, 224).

The cost per 100 with cut edges (i.e., not ground) is approx-
imately $6.00

COLLATERAL READING FOR CHAPTER XxV

RASPAIL, FRANCOIS- VINCENT. 1833, Nouveau systeme de chimie organique fonde
sur des methodes nouvelles d'observation. p. 528, et seq. Raspail has been
claimed as the founder of chemical microscopy or microchemistry. He applied
the microscope to the study of plant and animal structures with great success,
and among his methods was that of incineration to show the presence of
mineral matter in soft tissues after the organic matter had been burned away.

. 1838. Same, 2d edition in three volumes.

LIBSEGANG, R. E. IQIO. Die Veraschung von Mikrotomschnitten. Biochem.
Zeitschr., Vol. 2S, p. 413. He used sections 20/1 thick and heated over a
Bunsen flame. Such sections do not give good results at present.

POLICARD, A. 1023. Sur une methode de micro-incineration applicable aux rc-
cherches histochirniques. Bull. Soc. Chim. de France, 4th ser., 33, 1551.

. 1923. La mineralization des coupes histologiques par calcination et son

mteret com me me"thode histochirniques ge'ne'rale. Compt. Rend. Acad. Sci ,
176, 1012. In these first papers Policard describes his incinerator and gives
explicit directions for the procedure in micro-incineration.

- — . 1932. Some new methods in histochemistry. The Harvey Lectures de-
livered under the auspices of the Harvey Society of New York, 1931-32, under
the patronage of the New York Academy of Medicine, 204-26. In this lecture
Policard describes the micro-incineration process, but points out that it is
only the first step in the analysis of the minerals present.

POLICARD A., AND OKKELS, H. 1930. Localizing inorganic substances in micro-
scopic sections. The Micro-Incineration Method. Anat. Record, 44, 349-
61. This paper contains a picture of Dr. Policard's micro-incinerator and some
excellent photomicrographs of incinerated specimens with a good discussion
of the process and the findings. There is also an extended bibliography.

POLICARD, A. 1938. La Me*thode de la Microincineration, expose* pratique.
Actuality's Scientifiques et Industrielles 765. Histophysiologie.

SCOTT, GORDON H. 1933. A critical study and review of the method of micro-
incineration. Protoplasma, 20, 133-51. This paper is full of useful informa-
tion concerning the history and method of incineration, and contains 74 refer-
ences in all fields where micro-incineration has been applied.

. 1933. The localization of mineral salts in cells of some mammalian tissues

by micro-incineration. Amer. J. Anat., 53, 243-79. This paper contains
46 figures of micro-incinerations in three plates and gives references to 46 other
papers, including those of special historical interest.

GAGE, S. H. 1938. Apparatus and methods for micro-incineration. Stain Tech-
nology, Vol. 13, PP- 25-36.

HINTZSCHE, ERICH. 1938. Das Aschenbild tierscher Gewebe und Organe. Er-
gebnisse der Anatomic und Entwicklungsgeschichte. 32 Band, pp. 63-136.
This monograph gives a general review, and refers to 208 other papers.

UBER, FRED. 1940. Microincineration and Ash Analysis. This paper deals
especially with plant tissues. Botanical Review, Vol. 6, pp. 204-226.

CHAPTER XV

BRIEF HISTORY OF LENSES AND MICROSCOPES
FIGURES 300-313

Lenses. It is difficult to think of a world without lenses. All
apparatus like the moving picture machine, magic lantern, photo-
graphic camera, the microscope and telescope and spectacles, would
be no more. But it is not to be forgotten that the most splendid
creations in the world of art, as those of the Greeks; and in the
world of literature, as those of the Hebrews, the Greeks and the
Romans; the architecture of the Orient, of Egypt, Greece and
Rome; and the feats of engineering of the ancient world were all
independent of lenses and the optical instruments which they make
possible. But what immeasurably greater insight into the real
world has come with these "optic glasses"! What revelations as
to the cause of disease, the structure of the universe in its smallest
details by the microscope, and in its larger ranges by the telescope;
and greatest of all for the common man, has come the power, by
means of spectacles, to make good use of the years that hygiene has
added to the average human life.

That nature made lenses during every rain-storm and every
heavy dew and in the tears of every gum and balsam tree, we know
now; and for the almost infinite years which man has been upon
the earth, the learned and the ignorant were equally unmindful of
the marvel before their very eyes; as unmindful as are the vast
majority of men and women at the present day.

All who have made a study of the question are unanimous in the
opinion that optical instruments, other than mirrors, were unknown
to the ancient world; and that lenses were wholly unknown.

In the first and second centuries of the Christian era there was an
abundance of knowledge of mathematics and of optics to make
possible the invention of the simple microscope and of appreciating

547

548 BRn-F HISTORY OF LKNSKS AM) MICROSCOPES [Cn. XV

it as such. In works of literature there are hints that men were on
the track. For example, Seneca, in his Questiones Naturales
(L. I, q. 6), says that " Letters however small and dim are com-
paratively large and distinct when seen through a glass globe filled
with water/' and that apples in a vase of water are far more beauti-
ful. He is trying to account for the size of the rainbow and sums it
all up by saying that " anything, in fact, that is seen through
moisture appears far larger than in reality it is." To Seneca the
magnification was the effect of the water and not the effect of the
refraction at curved surfaces.

Ancient theories about Vie eyes. — The microscope and all other
optic instruments are intimately bound up with the eyes of the ob-
server, and the brain behind the eyes which gives the final judgment
concerning the appearances. This takes us a long journey back into
the past for the first understanding of the means by which knowl-
edge of the external world comes to our consciousness.

It was 2500 years ago in the age when ^Eschylus (525-456),
Sophocles (495-406) and Euripides (480-406) wrote their immortal
poetry; Phidias wrought forms of beauty out of marble; and Soc-
rates, Plato and Aristotle spoke words of wisdom, that Hippocrates
(460-360), the greatest of all ancient physicians, asserted that the
so-called " sacred disease," epilepsy, was no more sacred than any
other disease; and then he added the brain is the organ by which we
think, taste and smell, hear and see; through which are joy and
sorrow, laughter and tears and, when it is diseased, it brings terror
and despair and all insanities.

Nothing in physiology to-day is on firmer ground than that the
brain is the final seat of consciousness; and without its healthy
action no good vision is possible. Of course, it has always been
known that the eyes and light are necessary for vision, but at this
time there was much discussion as to the precise means by which
objects in the external world could gain their contact with the brain
through the eyes. Empedocles thought there must be rays of visual
spirits extending from the eyes out to the object and feeling of it,
so to speak; Aristotle asserted that the rays of light from the object
to the eyes were sufficient; but Plato, to be absolutely safe, as-

Or. XV] BRIEF HISTORY OF LENSES AND MTCRCSCOrilS 549

sumed that there were needed both the visual rays and the rays of
light to make the vision complete.

Six hundred years later, Galen, next in importance to Hippocrates
among the ancient physicians, agreed with Empedocles that vision
\\ as by means of %the visual rays or spirits from the brain and eyes
to the object, and gave the cogent argument that all men could
appreciate then as now, namely, that objects far off, small or in
dim light required much effort to see well as though it were hard
work to squeeze out enough visual spirits to make them fully
visible.

It was Galen also who argued that the chiasma in the optic
nerves from the eyes to the brain was for two great purposes: First,
so that if one eye were lost by accident, all the visual spirits could
be sent to the remaining eye; and second, it was to answer the
puzzling question why with two good eyes two images of everything
were not seen. The chiasma, said Galen, is so that the visual rays
from the object through the eyes to the brain can be mingled and
united so that the brain will have but one image and not two.
Furthermore, it is so that the axes of the visual cones will cross and
be in one plane, for if these axes are not in one plane there will be
seen two images and not one. He gave the simple device of proving
this by displacing one axis by pressing on one of the eyeballs with
a finger. He asserted also that while normally only one image of an
object was seen with the two eyes, this image included more than
the image of either eye alone, and gave the experiment of looking
at a column first with one eye and then the other, and then both
eyes.

Galen gave an excellent anatomical description of the eye and its
parts. We still use most of the names he applied, and appreciate
what he said about the retina's similarity to brain tissue. He
described the vitreus and its hollow cup above to receive the crystal-
line body. Even before Galen, this crystalline lens was called a
lentil-like body, and Galen knew that the curvature was not the
same on the two sides. Galen called the eye a most divine instru-
ment, and expressed unbounded admiration for the perfection of the
eye for its purpose. Apparently, some people had been finding fault

BRIEF HISTORY OF LENSES AND MICROSCOPES [Or. XV

with the eye and saying that they could make a better one them-
selves. Galen remarks that if they are so much more skillful than
the Creator, he would like to see some of the eyes they could make.

Ancient theories about the physical properties of light. — Turning
to the physical side, Galen refers to Euclid as the mathemat-
ical authority for the straight course of the rays of light, and
says that the visual rays are straight like the light rays. He also
appeals to the experience of every one who has seen the rays of the
sun streaming out through a rift in the clouds.

During the first part of Galen's life there was working in the field
of science another giant intellect, Ptolemaeus, whose system of
astronomy dominated the world for more than 1500 years.

Ptolemaeus wrote a book on optics which, it seems to me, is one of
the chief landmarks in the history of the subject. Like Euclid he
showed that the angles of incidence and reflection were equal, and
that the incident and reflected rays were in the same plane; but what
for our purposes is of far greater importance, he showed that when
light passed from one transparent medium to another, the incident
and refracted rays, while they are in the same plane, do not have
equal angles with the normal, but that the angle is always less in
the denser medium; and he measured the angles for air to water,
air to glass and glass to water, and the reverse, and found that no
matter in which direction the light passed, the an^le was always less
in the denser medium. He explained by this bending of the light
why it was that a coin in an empty basin, which could not be seen
over the edge, became visible when water was added.

For measuring the angles of incidence and refraction, he used a
divided circle, and the tables he prepared fill us with admiration for
the closeness with which they agree with the results attainable
to-day with the most refined apparatus. He not only discussed re-
fraction in bodies with plane surfaces, but also with concave and
convex surfaces, and found the rule to hold whatever the shape.
It is true that he did not discover the mathematical expression for
refraction — that took 1500 years longer — but he showed the facts
and stated them with a clearness never since excelled.

Not only did he apply the knowledge to the explanation of the

CH. XV] BRIEF HISTORY OF LENSES AND MICROSCOPES 551

visibility of the coin in the basin, but he showed that from the
refraction of the earth's atmosphere, the heavenly bodies were not
where they appeared to be, unless they were directly overhead.
He showed, too, that if the eye is in the air and the body in a denser
medium it will appear enlarged, but if the eye were in the denser
medium and the body in air, it would appear smaller. Why, with
all his optical knowledge, Ptolemaeus did not find the way to make
magnifying glasses with curved surfaces, is hard to understand. It
took over a thousand years more of effort for that to be accom-
plished.

In passing, while every one must have the deepest appreciation
for the service to the world that the Arabians gave in preserving the
science of the Greeks, their additions to scientific knowledge seem
very small. For example, in our subject of optics and vision, their
statements are almost wholly based on the geometry and optics of
Euclid, the optics of Ptolemaeus and the structure and functions of
the eye as stated by Galen. Their greatest exponent in optics, Al-
hazen, went back to Aristotle in declaring that vision is by light rays
from the object to the eye. He also applied the optics of Ptolemaeus
to the eye and saw that there must be refraction at the curved sur-
face of the cornea as the light entered the eye.

While the principles of optics and the devising of optical instru-
ments besides mirrors did not command much attention during
noo years following Ptolemaeus and Galen, still some progress had
been made by some one, but by whom no one knows.

Theories and experiments of Roger Bacon. — Roger Bacon advo-
cated with the deepest earnestness that the only sure guide to truth
was experiment. Every theory must stand that acid test before it is
wise finally to accept it.

That great i3th century, as it has been called, was one of intense
intellectual activity, and was as full of wild guesses and vague
dreams as any period in the history of the world, including our own.
Roger Bacon tried to put the scientific guesses to the test of experi-
ment as far as he could.

I know that many of us will hear with the sympathy of personal
experience what he says in speaking of these experiments. For that

5$2 BRIEF HISTORY OF LENSES AND MICROSCOPES [Cii. XV

time, he was comparatively well off, and he asserts that most of his
fortune had been spent for copyists to get the needed books; for
calculators to prepare the desired astronomical tables; and, last but
not least, to buy the apparatus with which to try the experiments.

The most extravagant claims have been made for Bacon. One
would think to read the claims that he was the originator of all
scientific knowledge, and the inventor of every piece of scientific
apparatus devised before or during his time. He made no such
claims. What he believed with all his strength was that the prog-
ress of civilization is bound up with a knowledge of science, already
at hand or to be gained, and he was filled with zeal to make the
knowledge available so that progress might begin at once and pro-
ceed with ever increasing speed.

In his enthusiasm he mentioned some things which he thought
might be found out, such as flying machines, ships without sails,
combinations of lenses to see what was too small or too far off to
see with the naked eye, engines of power by the use of explosives.
He has, as you know, been credited with the invention of gun-
powder, cannon, etc. Here is what he himself says in that connec-
tion: "Then wonders can be done by explosive substances. There
is one used for amusement in various parts of the world made of
powdered saltpeter, sulphur and the charcoal of hazel-wood. For
when a roll of parchment about the size of a finger is filled with this
powder, it produces a startling noise and flash. If a large instru-
ment were used, the noise and flash would be unbearable, and if
the instrument were made of solid material, the violence would be
much greater.0

To deal specifically with optics, Roger Bacon expounded with
great clearness the laws of refraction given by Ptoiemaeus, and the
structure of the eye as given by Galen, and more, he applied the
knowledge of refraction to the curved surfaces and structures of
the eye in explaining vision. He stated that all the rays reaching the
curved surfaces of the cornea and the crystalline lens, except the
axial ray of the visual cone, must be bent toward the axis on enter-
ing the eye. But this seemed to bring on a trouble which he tried
to avoid. The trouble was that if the rays crossed, there would be

Cn. XV] BRIEF HISTORY CF L KNITS AND MICROSCOPES 553

an inversion, so that what was right would be left, and what left
right, and what up would be down, and what down would be up.
Here then was a second puzzle to add to that of the single vision with
two eyes.

Roger Bacon showed as much skill in getting out of a seemingly
tight place as the scientific men of the present day. He assumed
that the vitreus with its outer concave surface to receive the crystal-
line lens was designed on purpose to keep the rays from crossing, and
thus to prevent the inversion of the image. It is not so, but it satis-
fied not only Roger Bacon, but such brilliant minds as Leonardo da
Vinci and Maurolycus, and a host of others during the next 400
years.

We may ask what was the fundamental step in optics that Bacon
showed. In his own words it was this: " If a man looks at letters
and other minute things through crystal glass or other transparent
substance in the form of the small part of a sphere ... he will see
the letters far better, and they will appear larger to him, for the
angle under which they are seen is greater, and the image is con-
sequently greater. Such an instrument is, therefore, useful for old
men and those with weak eyes, for they can see the letters, however
small, with sufficient magnitude." . . . Here then is the simple
microscope and convex spectacles. For the unnumbered centuries in
which the human race had been upon the earth, there never had
been any help for giving the sight of youth to the aged and experi-
enced, and the wisest years of life had to be spent in looking at
distant things; the near and the minute were only a blur.

So far as I have been able to find, this statement of Roger Bacon
concerning the action of artificial lenses for an aid to vision is the
first in scientific literature. He does not call these segments of
spheres lenses, although he uses the adjective lenticular in de-
scribing their form as had been done for the crystalline lens of the
eye for over a thousand years.

In leaving the contributions of Roger Bacon to optics, there are
two remarkable statements by him of the profoundest significance.

(i) He says light is not composed of material particles, but is a
kind of motion, and is not instantaneous in its propagation, but

554 BRIEF HISTORY OF LENSES AND MICROSCOPES |_Cn. XV

requires time, although the time is very short. It is transmitted
more rapidly in a rarer than in a denser medium on account of the
resistance of the density.

(2) He described and gives a diagram showing the passage of the
rays of the sun through a flask filled with water, such as had long
been used by the physicians for cauterizing; and he says that if any
inflammable substance is put at the point where all the rays come
together beyond the flask, they will be set on fire. Later he makes
this significant statement: " In the fifth place we have to speak of
light's action in all its degrees. Its propagation is unequivocal when
as light it produces light; but there is equivocal action when it
makes something of a different essence, as when light produces
heat."

Development of Optical Instruments in Two Groups. — At the time
of Roger Bacon's Opus Majus, not only were the principles of
reflection and refraction well understood, for plane and curved
surfaces, but lenses were actually in hand and it seems as if the
way was fairly open for the production of optical instruments.
Progress has been from that time on in two closely parallel roads.
Sometimes progress has been rapid on one road, and sometimes on
the other, depending upon human need.

The two roads serve for two groups of instruments:

The first group contains instruments in which the eye of the
observer forms an integral part of the optical train, as with specta-
cles, the simple and the compound microscope, and the telescope.
The second group includes the optical instruments which form real
images entirely independent of the eye, like the magic lantern, the
projection microscope, the moving picture machine and the photo-
graphic camera.

As the most pressing human need was for aid to defective vision,
the first development was with spectacles. It is astonishing how
soon spectacles came into use after the publication of Roger Bacon's
Perspectiva or Optics. This was widely copied and found in many
libraries, so that the knowledge soon became available. Even as
early as 1299, only about 32 years after Bacon put out his work,
there appeared in a manuscript this remarkable passage:

CH. XV] BRIEF HISTORY OF LENSES AND MICROSCOPES 555

" I am so affected by years that I cannot read or write without
those glasses they call spectacles, lately found out for the benefit of
poor old men when their eyesight gets weak."

It is also quite modern that cheaper means of producing spectacles
were sought. In 1300 the superintendent of arts in Venice found it
necessary to forbid the use of glass for making " reading stones "
and eye-glasses, for it was believed at that time that only those
made of beryl or rock crystal were really effective and not harmful.
But in 1301 permission was given to use glass provided the specta-
cles and reading glasses were sold as glass, not as crystal or beryl.

Naturally, in the beginning the needs of mature persons were
especially considered. Their chief difficulty was their growing lack
of accommodation that comes with advancing years, and to over-
come this, convex spectacles were constructed. Concave spectacles
came in later. Two early references to them have been found, the
first in the works of Cardinal de Cusa in a chapter called " Beryllus
oculare specillum, " which reads: "The beryl is a resplendent,
colorless and transparent stone to which is given a convex or a
concave form, and those that look through it succeed in discovering
things at first invisible." De Cusa died in 1464, therefore this
reference is of a date prior to that.

The second reference to concave spectacles is in the work of
Barbaro (1568), p. 192, in which the statement is very specific, for
he says, in connection with the construction of a camera for drawing
by projection: " Take an old man's glass, convex on both sides,
not concave like the glasses of youths of short sight."

The next radical step in the development of spectacles was taken
by two English astronomers. The first was Thomas Young, a man
of many accomplishments, honored equally by the archaeologists,
physicists, astronomers and physiologists.

In 1800, Young, in experimenting with distances at which lines
were sharp to him, found that, when held vertically, the lines were
sharp at a distance of twenty-five centimeters, but when- horizontal,
they had to be held at a distance of only eighteen centimeters. He
knew that this meant that some of the refracting surfaces in his
eyes had unequal curvatures for the vertical and the horizontal

55(> BRIEF HISTORY OF LENSES AND MICROSCOPES [Cn. XV

ZACHARfAS JANSEN 300

Inventor of the Dutch Compound Microscope with convex objective and con-
cave ocular (1500). It gave erect images (fig. 309). Portrait from Petrus Bo-
rellus, De Vero Telescopii Inventore, 1655. See also Harting, Mayall, Petri and
Carpenter-Dallinger.

JOHANNES KEPLER 301

Astronomer and Optician. Inventor of the compound microscope with convex ob-
jective and convex ocular. It gave inverted imaecs (fie:. 310), 1611. Portrait from
Kepler's Opera Cmnia. See Juannis Kepleri, Dioptrice, 1611. "Problema
LXXXVI, Duobus cotwexis majora et distincta przestare visibilia, sed eversa."
Opera Omnia, p. 540.

GALILEO GALILEI 302

Astronomer and Physicist. Adaptation of the Dutch telescope construction to
a compound microscope with convex objective and concave ocular giving erect
images (1610"). $ee C arpenter-Dallinger, Jour. Roy. Micr. Soc., 1889, p. 574;
Sedgwick and Tyler, Hist. Science. Portrait, Operc, Vol. I. Milano, 1808

CHRTS'ITAAN IIUYGENS 303

Mathematician. Astronomer and Physicist. Inventor of the Huygenian ocular
(1681-1687). Portrait from O.uvrcs comp. t. vii. See Sedgwick and Tyler, [list.
Science; Encyc. Brit.

CHARLES A. SPENCER 304

Pioneer American Optician. Teacher of Tolles and II. R. Spencer. Producer
of microscope objectives of high aperture for resolving power. Manufacturer of
glass with special optical qualities, and user of flitorite in lens combinations for its
optical effects (1851). Proc. Amer. Micr. Soc., i8qi, p. 248-249. Memoir by H. L.
Smith, Proc. Amer. Micr. Soc., 1882, by Wm. C. Krauss, 1901. Portrait from
the original negative in the author's possession.

ROBERT B. TOLLES 305

Student of C. A. Spencer. Producer and advocate of homogeneous immersion
objectives for an aperture above 180° in air for their superior resolving power,
(1874). Portrait from the memoir of Dr. Blackham. Amer. Micr. Soc., 1884,
pp. 41-46. See also General Cox, same volume, pp. s~39> and Dr. Krauss, 1901,
pp. 19-30 with portraits. Mayall, p. 95.

FRANCIS II. WENHAM 306

Inventor of the Dark-Field Microscope, 1850-1856, by the use of a paraboloid
condenser and central stop to give a hollow cone of light. Advocated necessity
of immersion contact of condenser and glass slip for high apertures. Thickness
of slip must be equal to the working distance of the condenser. Trans. Micr.
Soc. London, III, 1850, pp. 83-90; Quart. Jour. Micr. Soc. 1854, pp. 145-158;
1856, pp. 55-60. Obituary. Jour. Roy. Micr. Soc., 1908, pp. 693-697. Portrait
by the courtesy Roy. Micr. Soc. & Ross Ltd.

HERBERT R. SPENCER 307

Son and student of Charles A. Spencer. Founder of the Spencer Lens Com-
pany. Continued the optical work and traditions of his distinguished father.
Portrait from the memoir by Dr. Wm. C. Krauss, Trans. Amer. Micr. Soc.,
IQOI, pp. 19-30. Used fluorite, 1864-1865. Proc. Amer. Micr. Soc. 1891, p. 248.

ERNST ABBE 308

Inventor of the Apochromatic Objectives, and Compensation Oculars for the
microscope (1885). Clarifier of discussion and understanding by the use of the ex-
pression ''Numerical Aperture." Creative genius at the foundation of the
Jena Glass Works (1881-1884). Constructive humanitarian in the Zeiss Optical
Works. Portrait from Vol. I of the Abhandlungen. See also Jour. Roy. Micr.
Soc., 1905, pp. 156-163.

CH. XVJ liRTEF HISTORY OF LENSES AND MICROSCOPES 557

JANSEN 300
1590

KEPLER 301
1571-1630

GALILEO 302
1564-1642

HUYGENS 303
1629-1695

C. A. SPENCER 304
1813-1881

TOLLES 305

1822-1883

WENHAM 306
1823-1008

H. R. SPENCER 307
1849-1900

ABBE 308
1840-1905

558 BRIEF HISTORY OF LENSES AND MICROSCOPES [Cn. XV

axes. He found that this could be compensated by holding a
spectacle obliquely before the eye, when the lines would be sharp at
the same distance whether they were held vertically or horizontally.

Twenty-five years later, apparently without knowing of Young's
experience, George B. Airy found the same difficulty with one of his
eyes. The other was short sighted, but otherwise normal. Airy
understood the condition as had Young and found obliquity of a
spectacle a corrective. He reasoned also that if his eye had a cylin-
drical instead of a perfect spherical curve, and if a cylindrical
spectacle which just balanced the cylindrical curve in the eye were
used, there ought to result good vision, and so it proved and
has proved for every one corrected for astigmatism since that
time.

Up to the end of the i6th century the manufacture of lenses of all
shapes was in the hands of the spectacle makers, and from the
nature of the work the artisans were men of good intelligence.
Naturally, many experiments were tried, and at last in 1590, Jansen,
one of the opticians of Middleburg, Holland, got a combination with
convex objective and a concave ocular which realized the dream of
Roger Bacon, inasmuch as it made small things appear large, and
distant things near. As the same instrument served both as a
microscope and as a telescope, a little later it was called a microscope-
telescope. The possibility of seeing distant objects clearly seemed
of immense military importance, so naturally the telescope side was
first intensively developed.

Simple microscope. — Every convex lens is or may be used as a
microscope, as it aids the eye in seeing an object under an increased
visual angle, and hence makes it appear larger than it would if
viewed by the naked eye. Hence, when considering the history of
the simple microscope, it is evident that that history is the same
as the history of convex lenses. The date of the invention is some
time before the date of the Opus Majus of Roger Bacon. He speaks
of them, not as a wholly new invention of his own time, but as one
of the means by which wonderful things can be done. His whole
purpose in the discussion was to induce the church to make the full-
est use of all the products of science to give the superiority which he

Cu. XV] BRIEF HISTORY OF LENSES AND MICROSCOPES 559

felt was the right and the privilege of the Christian world to possess
in its efforts for advancing civilization.

The simple lens or the combination of lenses making up a simple
microscope may be held in the hand, but ordinarily there is some
metal binding and support for the protection of the lens or lenses,
and their easier handling or focusing. The common reading glass
with its convenient handb (fig. 4) and the tripod (fig. 232) and focus-
ing lens holder (fig. 233) are good examples.

In reading the older literature one often meets with the expression
" single microscope." This means a simple microscope, composed
of one lens (fig. 145), and is in contrast with the " double micro-
scope," or compound microscope of two lenses or two combinations
(objective and ocular, fig. 146).

Dutch and Keplerian compound microscopes. — Each has a convex
lens for objective. For ocular the Dutch form has a concave and
the Keplerian form a convex lens. The ocular for the Keplerian
form is properly a magnifier of the real image, while the concave-
lens ocular of the Dutch microscope acts as an amplifier for the
objective.

The virtual image is erect with the Dutch, but inverted with the
Keplerian microscope.

The Dutch compound microscope. — So far as known at present
the first compound microscope invented was composed of two
lenses, a convex lens for the objective and a concave lens for the
ocular (fig. 309). The convex lens is placed in a position to give a
real image of the object, that is, the object is outside the principal
focus of the objective, but before the real image is formed, a concave
lens (the ocular) is placed in the path of the beam. This makes the
rays less convergent and therefore acts as an amplifier, and serves
to increase the size of the real image which would be formed by the
objective alone. The eye is placed close to the ocular and focuses
the real image on the retina. This retinal image is inverted and,
therefore, when projected out into space, it seems erect as with the
simple microscope.

Very early the two lenses were put into tubes and made capable
of being brought together or separated, depending upon the distance

560 BRIEF HISTORY OF LENSES AND MICROSCOPES [CH. XV

of the object to be examined. The nearer the object, the farther
apart must be the ocular and objective. There still remains in the
ordinary opera glass the original Dutch telescope. If one has an
opera glass it is easily demonstrated that it can be used as a micro-
scope by unscrewing the ocular so that it may be separated a
considerable distance from the objective. If now the objective is held
within 10 to 20 centimeters of an object and the ocular moved back

Ocular

OtyMt

FIG. 309-310. DUTCH AND KEPLERIAN COMPOUND MICROSCOPES FOR
COMPARISON.

Each has a convex lens for objective. For ocular the Dutch form has a con-
cave and the Keplerian form a convex lens. The ocular for the Keplerian form
is properly a magnifier of the real image, while the concave-lens ocular of the
Dutch microscope acts as an amplifier for the objective.

The virtual image is erect with the Dutch, but inverted with the Keplerian
microscope.

and forth along the axis, the place will be soon found where the
image is distinct and it will be seen much enlarged.

The name telescope was given sometime before 1618, and the
designation microscope in 1625. As every one who used the instru-

CH XV] BRIEF HIS TORY OF LENSES AND MICROSCOPES 561

ment found that it could be used as a microscope or as a telescope
it soon came to be called a telescope-microscope, or a microscope-
telescope.

77 e Kephrian compound microscope. — When the Dutch telescope
came to the attention of the astronomer and optician, Kepler, he
very quickly saw that the same effect could be brought about by
using a convex ocular as well as a convex objective, but that
the image would be inverted, the objective serving to produce
an enlarged real image and the ocular to magnify that image
U-g. 310).

The demonstration of the principles on which such a microscope
or telescope could be constructed is to be found in the Dioptrica of
Kepler, Proposition LXXXVI. The proposition is: With two
convex lenses to show objects larger and inverted.

In Prop. LXXXIX, it is stated that with three convex lenses can
be shown objects enlarged and erect. This is the principle of the
terrestrial or erecting telescope.

Kepler first showed the real action of the eye as an optical instru-
ment, and that the retinal image must be inverted, and that unless
inverted, objects would appear wrong side up. Now we know that
is true, for it is an easy demonstration to show, as did Scheiner in
1619-1625, that the retinal image is actually inverted in the eye of
an animal or man.

As Kepler showed the actual dioptrics of the eye, he was the first
to explain the action of spectacles in correcting the defects of long
sight and short sight, viz., to aid the refracting surfaces of the eye to
make a sharp image of the object upon the retina.

While Kepler gave the optical demonstration for a microscope
or telescope with convex lenses, he, so far as known, did not actually
construct such a microscope or telescope. Christopher Scheiner,
while he lacked the original genius of Kepler for discovering and
expounding principles, had greater mechanical ability. He actually
constructed the Keplerian telescope and microscope and used them
both for observation and for projecting real images. On page 130
of the Rosa Ursinae (1626-1630) occurs this remarkable passage:
" In the same way (i.e., by two convex lenses) was produced that

562

BRIEF HISTORY OF LENSES AND MICROSCOPES fCu. XV

wonderful microscope by which a fly was made as large as an
elephant and a flea to the size of a camel. "

BINOCULAR MICROSCOPES

From the invention of the telescope-microscope there was dissatis-
faction that it was for but one eye, and before 1610 there were made
those for both eyes by putting two equal instruments side by side
the right distance apart for the eyes of the observer. That arrange-
ment of the Dutch telescope still holds in opera glasses.

One of the first examples shown in pictured form is that of the
Cherubin d'Orleans in 1677 (fig. 311). This, as seen from the
picture, is a binocular Keplerian microscope, or rather two of them,
as both objectives and oculars are of convex lenses. The objectives
needing to be close together makes a divergence of the tubes neces-
sary to get the right pupillary distance for the oculars. In general,

FIG. 311. BINOCULAR MICROSCOPE OF CHERUBIN D'ORLEANS.

A The binocular in its mounting.

B Sectional view showing the two objectives and two oculars.

this form of binocular has been recently revived for dissection, only
in the modern form achromatic objectives are used and Huygenian
oculars, and by means of prisms the image is made erect.

Only rather large objects can be studied with such binoculars, and
the effort to divide the light from a single objective reached success
only as late as 1851, when it was worked out by J. L. Riddell of
New Orleans. His description and a figure were published in the
Quarterly Journal of Microscopical Science in 1854. From that
time on successful binocular microscopes have been made. The one
of Wenham (fig. 28) in England (1860) enjoyed the greatest favor.

CH. XV] BRIEF HISTORY OF LENSES AND MICROSCOPES 563

Tolles in 1864-1865 produced his binocular eyepiece, and Nachet in
France and Zeiss in Germany produced binocular instruments, but
there were defects inherent in the construction of all forms, es-
pecially the defect that they could not be used very satisfactorily
with high powers, and they were expensive. Finally, in 1902, Mr.
F. E. Ives figured and described a form of binocular suitable for
all powers, including the highest oil immersion objectives (§ 49).
Several recent models have been produced in which the principles
he enunciated so clearly have been incorporated (figs. 30-35).

In the first binoculars of the Dutch form, the tubes were parallel,
as with opera glasses, but in many of the later forms the tubes have
been put at an angle (fig. 33).

MICROSCOPES FOR Two OR MORE OBSERVERS

The projection microscope with its real images on a screen has
been commended from the first invention of projection apparatus
because many can see the image at the same time, and the teacher
or exhibitor can be sure that the observers are seeing the special
things he wishes to show. But in looking into the microscope in
the ordinary way only one person can look at a time, even with the
ordinary binocular. Therefore there arose the effort to divide the
light from the object so that two or more could see the same image
at the same time. The use of prisms for dividing the light in the
binocular gave the hint, and in 1853 Nachet constructed a micro-
scope for two observers, and another for three observers (see figures
of these in Harting and in Robin's work on the microscope, also in
the original paper). Harting, 1858, also produced a microscope for
two observers. For this the tubes were parallel. By putting them
closer together they served for a binocular for one person.

Finally, in his enthusiasm for demonstration, he constructed a
microscope in which the beam was divided among four diverging
tubes so that four persons could see the same specimen at once.

Within recent years the demand for a way by which two ob-
servers could look at once has given rise to two very practical double
oculars which are far enough apart so that two can look into the

564 BRIEF HISTORY OF LENSES AND MICROSCOPES [Cii. XV

oculars conveniently. One was devised (1910) by Dr. Edinger of
Frankfurt and produced by Ernst Leitz in Germany, and the other
in 1916, by the Spencer Lens Company of Buffalo, New York. In
both these double oculars there is an adjustable pointer so that the ex-
act structure which is to be studied can be indicated ; then both teacher
and student can be sure that they are talking about the same thing.

OCULARS OR EYE PIECES TOR THE MICROSCOPE

As shown above, the first oculars were of single lenses, — for the
Dutch telescope-microscope a concave lens, and for the Keplerian

microscope a convex lens (figs.
309-310).

For the Keplerian microscope,
which soon became the only one
used for microscopic work, all
sorts of experiments were tried
both for oculars and for ob-
jectives. Finally, about 1660,
Huygens, the great Dutch
astronomer and physicist, de-
signed for the telescope the
ocular (figs. 24-25) which now
bears his name. It was soon
adopted for the microscope and
DUTCH COM- is to this day the most used of
any.

The Ramsden ocular was
devised by J. Ramsden (1782)

FIG. 312. DESCARTES'
POUND MICROSCOPE WITH A PARABOLIC
MIRROR AND A CONDENSING LENS.

abc, def Concave ocular (amplifier).

S T Stand and circle holding the mi-
croscope and pointing it toward the sun or
other light source.

N 0 P Convex objective.

C C Parabolic mirror for illuminating
opaque objects.

* * Condenser for illuminating trans- especially for the ocular micro-
parent objects. r ^

meter (figs. 22, 160).

The compensation oculars were invented by Abbe (1885-1886)
to go with the apochromatic objectives and to correct the residual
defects 'in the objectives (figs. 23, 114-115).

for the telescope and, like the
Huygenian, was adapted to the
microscope. It has been used

CH. xvi HKIEF I:II:TOUY OF LENSKS AND MICROSCOPES

565

Mirrors and condensers. — The first objects looked at through the
microscope, whether simple or compound, were opaque and were
illuminated by light falling upon their surface. For this were used
condensing lenses, and plane and concave mirrors. The origin of
the mirror is prehistoric. The first were of polished metal and of
dark minerals. Those with a metal backing have been known only
since about the i2th or i3th century, and those with silver only since
about 100 years ago. It is not to be forgot-
ten that still water and other smooth objects
in nature serve as mirrors, and have always
existed.

In Descartes' picture of the Dutch com-
pound microscope (fig. 312) there is a
parabolic mirror for lighting the object if
opaque, and a condensing lens for trans-
parent objects. Descartes also gives a
picture of a simple microscope with a
similar concave mirror for illuminating the
opaque object (fig. 313). In 1668 Hooke
speaks of looking-glasses for illuminating
transparent objects for projection. The first
pictures of compound microscopes with the
mirror, as at present under the stage, are by

Hertzel (1712) and Marshall (1718). Fl£- 313. DESCARTES'

, , . . . , , . SIMPLE MICROSCOPE.

A condenser of a single lens or of a combina- 7 7 Rays of light pass.

tion of lenses for transparent objects dates ing to the reflector,
from the earliest use of the compound micro-

scope, as shown by Descartes' figure. Its the opaque object.

importance for adequate lighting has never len^

been lost sight of, as indicated by Brewster nifier.

(§ »8) and by Nelson (see in collateral t

reading); and never so thoroughly appreciated // Crystalline lens of

as at the present day. The form most com- t e eye*

mon on microscopes is the uncorrected one of Abbe which was first

described in the Archiv fur Mikr. Anat., Vol. 9, 1873, p. 469.

Achromatism. As pointed out in §§ 257-258, white light, being

566 BRIEF HISTORY OF LENSES AND MICROSCOPES [Cn. XV

composed of different wave lengths (figs, in, 121), must be dif-
ferently refracted when passed through a prism or lens. To the
normal human eye the different waves when separated or dispersed
out into groups appear of different colors. Although the nomen-
clature used by Newton was somewhat different from that now
used, he supposed that the refraction of the different waves was
in exact accordance with their wave lengths, as is the case with
a diffraction grating, and hence there could be no achromatization
of dioptric instruments, for when the dispersion was overcome the
refraction must also be eliminated. The mistaken belief that the
human eye was achromatic, however, kept alive the hope of produc-
ing achromatic microscopes and telescopes. Experiments on a large
number of transparent substances showed that while all dispersed
the light, the dispersion was not the same in all, some affecting one
group out of proportion to another. This irregularity gave the clue
to the way to accomplish achromatism, for if two or more trans-
parent bodies could be combined to neutralize their dispersive
effect without overcoming the mean refraction, it would be possible
to make achromatic combinations. This is shown by the course
of the beam of white light traversing the two prisms (fig. 112). The
first to accomplish the feat in a way to make achromatic telescopes
possible was John Dollond (1757). Naturally, the telescope took the
lead in the improvement, as it at that time was by far the most
important optical instrument. Furthermore, the lenses were rela-
tively large; for in the differentiation of the telescope and micro-
scope the objective of the telescope became progressively larger and
that for the microscope progressively smaller. The smaller the
lenses the more perfect must be the grinding and polishing, for
slight imperfections in their small area introduce obscurations which
in the larger surface of the telescope lenses would be negligible (§272,
fig. 119). But the microscope makers undertook the task in several
different countries, — England, France, Russia, Holland, Germany
and Italy — and from 1759 to 1824 were tireless in their efforts.
Finally Selligue laid before the French Academy the result of his
efforts with the help of the practical opticians, Vincent and Charles
Chevalier. From that time on, achromatic objectives became more

CH. XVI BRIEF HISTORY OF LENSES AND MICROSCOPES 567

and more commcn for microscopes, although from their small aper-
ture they were not liked by some workers so well as the more
brilliant, uncorrected lenses.

In our own country, Charles A. Spencer took the lead in trying to
overcome the lack of brilliancy in achromatic objectives. He, too,
early realized and grasped the importance of aperture for the micro-
scopic objective. He realized also that for the balancing of the dis-
persions and refractions to make true achromatic combinations, it
was necessary to have materials for lenses with special properties.
He worked in two directions. One was the use of the natural min-
eral fluorite whose properties had been pointed out by Brewster
(§ 259a) and the other was the production of new forms of glass
with specially desired optical qualities.

It fills one with admiration to think of this genius with small
means working alone in his cramped quarters trying to make new
forms of glass, which with the old forms and with natural minerals
would enable him to produce the objectives of his dream with large
aperture and perfect color and spherical correction. While his suc-
cess, and that of his pupil Tolles, were certainly great in producing
the highest type of objective for the telescope and microscope with
the materials already to be had, his glass making did not bring him
all that he wanted. It was reserved for the optical works of Zeiss
and the genius of Abbe, with the help of the practical glass maker
Schott and the liberality of the German government, finally to
overcome the difficulties in making new forms of glass with specially
desired qualities of dispersion and refraction; and even then it was
necessary to go back to the natural mineral fluorite to make possible
the apochromatic objectives. Those interested are recommended to
read the work of Hovestadt on the new Jena glass.

Immersion objectives. In the development of any art the science
needed almost always lags behind, and is developed in most cases
to explain what has already been discovered by the hard and
roundabout method of " trial and error." This was the case with
immersion objectives. Amici in Italy and David Brewster in
Great Britain were busy in trying to improve microscope objectives
by any feasible method. They used all sorts of liquids for immersion.
Water was one of the most successful and still holds its own.

568 BRIEF HISTORY OF LENSES AX!) MICROSCOPES [Cn. XV

The advantage of the immersion principle gradually became
understood to be the possibility of increasing the aperture under
which the object could be viewed. The final step by which the
aperture could be pushed to the limit of human skill in figuring the
lenses came when Mr. Tolles (1871-1874) showed in the clearest
manner the possibility of making such objectives and increasing the
aperture by means of homogeneous contact between the condenser
and the slide or object and between the object or cover-glass and
the front lens of the objective. The matter is well stated by Hon.
J. D. Cox in his presidential address before the American Micro-
scopical Society for 1884 (pp. 5-39), and in Mr. Mayall's Cantor
Lectures on the History of the Microscope (1885). On p. 96 Mayall
says: " If priority of publication of the formula on which homo-
geneous immersion objectives could be produced carries with it the
title of inventor, then Mr. R. B. Tolles stands alone as inventor;
but he not only published the formula, he constructed objectives on
it." The formula was submitted with the objective in 1874. The
homogeneous immersion objectives of Zeiss came out in 1878.

Many substances have been tried for the homogeneous fluid.
Thickened cedar-wood oil has proved most satisfactory. Mr. Tolles
used Canada balsam; if one is out of cedar- wood oil and has
Canada balsam of moderate thickness, good results can be obtained
by using the balsam as an immersion liquid with ordinary light.
As shown above (§ 309), none of the regular homogeneous immersion
liquids will answer for the immersion medium with the ultra-violet
microscope. Petrolatum has nearly the right refractive index, and is
non-fluorescing, therefore it answers well for the immersing fluid
in ultra-violet work. It is also used by many for the usual routine
examinations with the oil immersion objectives, but one cannot get
the most perfect images when it is used (§ 269).

THE DARK-FIELD MICROSCOPE

In the earliest literature giving directions for the use of optical
instruments, there is made over and over again the statement that
for the clearest images no light should reach the eye except from the
object itself. But when the object is on a white background, or

Cn. XV] I5UIKF HISTORY OF LKNSKS AND MK'ROSCOPKS 569

when lighted by rays from behind and on all sides, filling the whole
field of view, it is evident that the light from the object is only a
small part of that which enters the eye, and the fine details are
wholly obliterated or only dimly seen. To overcome this difficulty
two means have been employed: — First, myriads of dyes have
been invented to stain the delicate parts of the microscopic objects
so that color images are given in the bright field. The second
method is an application to microscopy of the knowledge gained in
astronomy — that is, to view the objects only by the light which
they themselves send into the microscope. Of course, if the objects
are truly self-luminous, as are the fixed stars in astronomy, no ac-
cessory light is needed, but if, as with the planets in the sky, objects
without any intrinsic light, must in some way be illuminated brightly
by an outside source, and in that way objects become visible as if
self-luminous, by the extrinsic light which they reflect, refract or
diffract into the microscope, just as the planets deflect the light
from the sun to the earth.

With the sky at night, the back ground will be dark and appear
like empty space, and the whole attention can be given to the
shining objects. If the objects are too small to be resolved by the
microscope used, then they will appear simply as points of light, as
with the ultra-microscope, but if they come within the resolving
power of the microscope, all the finest details will be brought out
with striking clearness. Naturally, to get this dark-field illumina-
tion the light from the source must be of so great obliquity that
none of it can enter the microscope objective directly, and it must
be of sufficient brilliancy so that the objects to be studied will be
bright enough to be clearly visible. This dark-field microscopy
was begun by Lister in 1830, and by Reade in 1837, and made
available for the highest powers by Wenham in 1850-1856.

It seems to me after many years of experience with all the dyes
used in microscopy for bright-field work, and with all the dark-field
methods so far devised, that the future physicist, chemist and
biologist will feel as much handicapped without the ultra-microscope
and the dark-field microscope as would the astronomers if they had
no clear, dark nights, and could work only in the daytime.

570 BRIEF HISTORY OF LENSES AND MICROSCOPES [Cii. XV

OCULARS FOR USE WITH SPECTACLES

Wherever in the brain the final visual effects may be interpreted,
it has been recognized since the time of Kepler that for the clearest
vision there must first be formed a perfect image on the retina of
the eye, and that the entire optical and accommodating mechanism
of the eye exists for the sole purpose of producing a sharp retinal
image. Kepler, Young and Airy showed exactly what concave, con-
vex and cylindrical spectacles did to aid in giving a perfect retinal
image when there were defects of short sight, long sight or astigma-
tism. Now, as the optician strives to make his instruments capable
of giving, with a normal eye, a perfect retinal image, it follows
logically that with an imperfect eye in the optical train no perfect
retinal image is possible, no matter how good the optician's work
has been. If, then, the observer's eyes must be helped by spectacles
to get a perfect retinal image, the spectacles should be worn when
looking into an optical instrument as well as when reading or using
the eyes for any other accurate vision. Probably every optician
would agree to this as a general proposition, but strange to say,
until very recently, microscope makers, at least, have constructed
their oculars so that it is almost or wholly impossible to use one's
spectacles when looking into the microscope. That is, they have con-
structed them so that the eyepoint or exit-pupil is so near the eyelens
that spectacles, especially those of toric form, cannot be worn
because they keep the eye too far from the eyelens of the ocular.
Furthermore, some of the best makers, when the oculars were
constructed with high eyepoints to give the best effects, have added
a perforated tube to the top of the ocular so that the spectacled
user even than had to remove his spectacles.

One English optical house has listened to the appeals of their
toric-spectacled patrons, and has produced a full series of oculars
with eyepoints high enough so that toric spectacles can be worn with
comfort when using the microscope.

I have appealed to our American opticians to give the spectacle
users — and practically every one doing serious research must

Cn. XV] BRIEF HISTORY OF LENSES AND MICROSCOPES 571

wear spectacles — this measure of assistance, and I hope the mem-
bers of the Optical Society will add their influence.

ARTIFICIAL DAYLIGHT

I suppose that every one will agree that the human eye was
created or developed for daylight; and what it required untold ages
to evolve, naturally resists rapid change. In the short days of fall
and winter, and in the dimness of foggy weather in many regions,
the daylight is distressingly short or inadequate for the exacting
work of the modern world, hence the artificial lights to gain ex-
tention of time and efficiency. Even the best artificial lights are
so unlike sunlight that the eyes are put to a great strain.

To remedy this trouble many efforts have been made to give
daylight qualities to the artificial lights which must be used. For-
tunately, within the last few years such artificial daylight has been
made available at a very moderate cost. Certainly for users of the
microscope it is a great boon, and from much experience it is be-
lieved that with this help the eyes of the experts will be able to
serve their owners for a longer time to carry on their researches,
much to the advantage of the individual and of the community.

REAL IMAGES AND PROJECTION

The production of real images by means of a naked aperture and
by means of a lens were the beginnings of the magic lantern, the
photographic camera, the projection microscope and the drawing
camera.

As shown elsewhere (Optic Projection, p. 673), the production of
real images in dark places by means of an aperture or hole in the
wall is a purely natural phenomenon. The systematic utilization of
this phenomenon by man had its beginnings in the sixteenth and
seventeenth centuries. * The first certain statement of the use of a
lens in the aperture to make the picture clear and vivid occurs in
the work of Daniel Barbaro on perspective.

From this time on, a lens is always used for projection. At first
the images were smaller than the object, as naturally only the

572 BRIEF HISTORY OF LENSES ANJ) MICROSCOPES [Cn. XV

brightly lighted objects in the exterior world were projected, but as
artificial and natural light were used to illuminate smaller and
smaller objects, many of which were transparent, and the projection
lenses were made of shorter focus, the images became larger than
the object. Finally (1665), when the apparatus became small, and
only the object and lens and light were enclosed and the image was
on a screen outside, the magnifying action seemed like that of a
microscope, and Milliet de Chales, in speaking of the magic lantern
of Walgensten, says (Vol. II, p. 667): " In this machine you have a
kind of microscope," and Zahn, p. 255, in discussing the magic
lantern, says: " It is a kind of microscope." Both authors point
out the great advantage this kind of microscope has over the
ordinary one in that many persons can see the image at the same
time. Kepler (1611) showed that the Dutch telescope-microscope
and also his own combination of convex lenses, could be used for
projecting images. Schemer (1626-1630) used them for projecting
images of the sun so that he could draw the spots. See also Hooke,
Trans. Roy. Soc,, 1668, p. 741.

Naturally, with the perfecting of objectives (1824 and onward),
and the finding of more powerful artificial lights (lime light, 1824,
electric light, especially since 1880), the projection microscope is
coming to be used more and more.

The use of real-image forming optical appliances is increasing
with ever accelerated velocity in our own time. To realize this one
has only to think of the photographic cameras in the hands, not
only of experts, but of old and young everywhere, and to think of
the moving picture machines in every village and in many private
homes. So, too, the magic lantern is a part of the regular outfit
of many churches and societies and practically every high school
and college in the land; and the projection microscope for showing
the minute details of the objects to be studied finds an honorable
place in laboratories of all universities.

Recently projection apparatus has found a welcome in testing
laboratories, much to the advantage and comfort of those making
the tests, for the real images can be seen with both eyes and the
head and shoulders held in a natural, unstrained position (§ 444).

Cn. XVI BRTKF HISTORY OF LENSES AND MICROSCOPES 57^

Its aid in getting accurate drawings is now more appreciated than
ever. It also serves to obtain photographs of microscopic objects
which give the minutest details with a delicacy and accuracy that
the human artist finds difficult to approach.

The first drawings made by the aid of the microscope were free-
hand. Examples of the drawings may be seen in the work of
Borellus, and in facsimiles shown in the Journal of the Royal
Microscopical Society, 1915, pp. 317-340. The desire for accuracy
and ease in tracing outlines of microscopic images comparable with
those so easily attained with the real images of the projection
microscope led to the invention of the camera lucida, by which the
microscopic field and the drawing field, pencil, etc., can be super-
posed. The first one invented is still used. It is the Wollaston form
(fig. 1 68), and was described by Wollaston in Nicholson's Journal,
1807, pp. 1-5. The other form shown in fig. 169 was described in
principle by G. Burch, Jour. Quek. Micr. Club, 1878, p. 47; and by
Dippel in the Bot. Centrlbl., 1882, pp. 242-3.

Drawing with the projection apparatus has been practised from its
first invention. Indeed, in all those who described such apparatus,
the great help that was to be gained in drawing was emphasized.
Both eyes can be used, and perfect freedom of the artist is enjoyed,
which is in marked contrast with camera lucida drawing. For the
early appreciation of projection apparatus and the camera obscura
for drawing see: Barbaro, 1568; Kepler, 1611; Scheiner, 1626-1630;
Robert Hooke, 1668; Henry Baker, 1742; G. Adams, 1746; Goring
and Pritchard, 1837; Chevalier, 1839.

Daniel Barbaro. — In his work, La pratica della perspettiva, Venice, 1568,
Ch. V, p. 192, Barbaro says: "Take an old man's glass, convex on both sides,
not concave like the glasses of youths of short sight, fix the convex glass in a
hole, close all the windows so that no light may enter except through the lens.
Now take a sheet of white paper and bring it toward the lens until all outside
the house is clearly seen. When the proper position .is found you will see the
images on the paper as they are, and the gradations in colors, shadows, move-
ments, clouds, the rippling of waters, birds flying, and everything that can be
seen. For this experiment the sun must be clear and bright, for the sunlight has
great power in bringing out the images. You can draw on the paper with a pen-
cil all the perspective, and the shading and coloring according to nature/'

Johannes Kepler. — In Reliquiae Wottonianae, edited by Izaak Walton,
London, 1672, pp. 298-300. In a letter to his kinsman, Francis Bacon: "I have
your Lordship's letters dated the 2oth of October (1620). I lay a night at"

574 BRIEF HISTORY OF LENSES AND MICROSCOPES [Cn. XV

Lintz. . . . There I found Keplar, a Man Famous in the Sciences, as your Lordship
knows, to whom I purpose to convey from hence one of your Books, that he may
see we have some of our own that can Honor our King, as well as he hath done with
his Harmanica. In this Mans study I was much taken with the draught of a Land-
skip on a piece of Paper, methoughts Masterly done: whereof inquiring the Author,
he bewrayed with a smile, it was himself; adding, he had done it, Non tanquam
Pietor, sed tanquam Mathematicus. This set me on Fire: At last he told me how.
He hath a little black Tent (of what stuff is not much importing) which he can
suddenly set up where he will in a Field, and it is convertible (like a Wind-mill)
to all Quarters at Pleasure, capable of not much more than one Man, as I con-
ceive, and perhaps at no great ease; exactly close and dark, save at one hole,
about an Inch and a half in the Diameter, to which he applies a long perspec-
tive Trunk, with a Convex glass fitted to the said hole, and the concave taken
out at the other end, which extendeth to about the middle of this erected Tent,
through which the visible Radiations of all the Objects without, are intromitted,
falling upon a Paper, which is accommodated to receive them, and so he
traceth them with his Pen in their natural Appearance, turning his little Tent
round by Degrees, till he hath designed the whole Aspect of the Field. This I
have described to your Lordship, because I think there might be good use made
of it for Chorography: for otherwise, to make Landskips by it were illiberal;
though surely no Painter can do them so precisely."

Henry Baker. — The Microscope Made Easy, 1742. On page 25 occurs this
"Such too as have no skill in drawing may, by this contrivance [projection mi-
croscope], easily sketch out the exact figure of an object they have a mind to
preserve a picture of; since they need only fasten a paper upon a screen and trace
it out thereon either with a pen or pencil as it appears before them." This old book
has an abundance of illustrations and contains a mine of good suggestions.

SPECTROSCOPE, POLARIZING MICROSCOPE,
ULTRA-VIOLET MICROSCOPE

Spectroscope for use with a microscope. Since the fundamental
studies by Newton on the colors in white light by the aid of a
prism in 1666, all kinds of light have been subjected to spectral
analysis and many important facts concerning the physical world
have been discovered. For the investigation of minute objects, a
special form of spectroscope has been devised for use with tht
microscope. It is of the direct- vision form (§§ 274, fig. 120) and
was devised and perfected by Sorby and Huggings (1865) and per-
fected later by Browning, Swift, Ward and Abbe, etc. Spectroscopes
for the microscope have been named: Micro-spectroscopes, spectral
oculars, and Mr. Dallinger suggests that the name spectro-micro-
scope be used. This would bring the name in harmony with polar-
izing microscope, ultra-violet microscope, etc.

CH. XV] BRIEF HISTORY OF LENSES AND MICROSCOPES 575

Polarizing Microscope. Since 1808-10, when Malus found that
light reflected from glass surfaces had peculiar properties which he
named polarization, an immense amount of investigation has been
undertaken to find out the meaning of polarization, and the phe-
nomena which polarized light produces. Much study and many
investigations have been applied to the means of polarizing light,
and many phenomena when first discovered have been brought in
line with this peculiarity, for example, the double refraction in
calcite discovered and described by Bartholinus in 1669. The
investigations have been greatly simplified and made exact by the
invention of the polarizing and analyzing prisms of Wm. Nicol,
described by him in 1828.

Sir David Brewster sought to discover the effects of polarized
light upon small objects, and devised a method of doing so with a
simple microscope in 1816. It was not, however, until after the
Nicol Prism was invented in 1828 that a completely successful ap-
plication of polarized light was made to the compound microscope.
This was accomplished by Henry Fox Talbot in 1834. First he
used tourmaline, but the color was objectionable. Then he used
Nicol prisms with complete success. One prism was put under the
stage to polarize the light and a similar one over the ocular to
analyze it. He called the two prisms (figs. 91-92) polarizers. In a
paper in 1836 Talbot gave the true explanation of the single beam
shown by the Nicol prism, asserting that one of the beams was wholly
removed by total internal reflection (§ 216).

The information concerning the physical character of objects
revealed by the use of polarized light was early appreciated, and its
use advocated with great earnestness by the early workers, Brewster,
Talbot, Quekett, etc. Lately there has appeared an entire volume of
over 500 pages dealing with animal tissues and organs (W. J.
Schmidt). Drs. Chamot and Mason show in their Micro-Chemistry
what an indispensable aid the polarizing microscope is in chemistry.

INVISIBLE RADIATION

The discovery that outside the band of visible light were similar
radiations which were invisible, was epoch making. The first of

576 BRIEF HISTORY OF LENSES AND MICROSCOPES fCn. XV

these discoveries was by William Herschel in 1801, when he found
the infra-red of the solar light, or the invisible heat rays of sunlight.
The second, stimulated by Herschel's work, was the discovery by
Johann Wilhelm Ritter in 1801 to 1803 of the ultra-violet radiations
beyond the violet end of the spectrum. As the infra-red radiations
were found by their heating effect, and still are called heat rays, the
ultra-violet was discovered from the action on chlorid of silver, and
was called actinic or chemical radiation.

These heat rays and chemical rays have, since their discovery,
played a great role in physics, and are destined to play an equally
great r61e in physiology.

Ritter says that he found the chemical action, the 22d of Febru-
ary, 1801, by the use of one of Newton's prisms in a region below
the violet (ausserhalb des Violet). (Annalen der Physik, Bd. 7, p.
527 and p. 409.)

It is a long road leading to the present activity in experimenting
with ultra-violet radiation, and now the activity is at a highly
increased velocity. For the scores of papers dealing with the
physiological side, one can find full information in the Quarterly
Cumulative Index Medicus.

In 1833, David Brews ter found that certain substances glowed
with visible light when acted upon by ultra-violet. He called the
effect " internal dispersion." In 1852 came the fundamental work
of George Gilbert Stokes, " On the Change of Refrangibility of
Light, " and he proposed to call the change by which objects gave
out light visible to the eye when acted on by the invisible radiation,
" Fluorescence " and that name is now universally employed.

The ultra-violet or fluorescence microscope utilizing radiation of
250 m/x to 410 mju wave length can "now be used in every laboratory.
The capillary mercury arcs (fig. 129 A) are as simple to use as any arc
or mazda light. For elimination of visible light from the mercury
light there must be an ultra-violet filter like the red-purple corex A
(fig. 129 B). For photo-micrography there are other filters to transmit
only "special mercury spectral lines. The condenser must be of quartz
and for most purposes must have a dark-field element at the top
as illustrated in figures 126, 280. The slips on which the objects are

CH. XV] BRIEF HISTORY OF LENSKS AND MICROSCOPES 577

mounted must be of quartz or of corex or other ultra-violet trans-
mitting glass.

The cover-glasses, objectives and oculars ordinarily used with a
microscope are employed, (fig. 125, 128).

The objects to be studied are either fluorescent and thus send
their visible light into the microscope, or, if non-fluorescent, they
are observed by ordinary light to see if any changes have taken
place due to the ultra-violet irradiation.

Ultra-Violet for Photography. It was thoroughly appreciated by
the leading microscopists of the igth century that the resolution of
the microscope was bound up with the wave length of the light used.
That being assumed, it was felt certain that if one could make use of
the short ultra-violet radiation, finer and finer details could be
brought out. As these radiations are invisible to the eye, recourse
was had to photography, for the salts of silver were sensitive to
these shorter waves. Hence arose ultra-violet photography. But
it is only for the longer ultra-violet waves that glass is transparent.
For still shorter waves it is necessary to use quartz. Then a source
of radiation had to be found which produces the maximum number
of ultra-violet waves. The mercury arc is good for many purposes
where the longer waves are desired. For still shorter ones the arc
light with cadmium electrodes gives abundant waves down as short
as 0.275/1.

To return to the microscope, in 1903-1904 Kohler described and
the Zeiss Works produced a microscope with quartz lenses by which
photo-micrographs could be made with ultra-violet. In America
Drs. Ernst and Wolbach published an account of their work and
results in this ultra-violet photo-micrography, and quite recently
Dr. Francis F. Lucas has described an apparatus for photo-microg-
raphy and has produced many wonderful photographs of living
cells using the ultra-violet.

As a final word in the history and future promise of the services
the microscope has given and it is believed will give, there are two
reasons for astonishment. The first is that mankind was so late in
discovering the laws of refraction, and the possibilities which it
might lead to in the production of lenses and optical instruments.

578 BRIEF HISTORY OF LENSES AND MICROSCOPES [Cn. XV

Secondly, it is astonishing to think of the rapid progress that has
been made since the possibilities of lenses were discovered some six
hundred years ago, and especially during the last three hundred
years, since the compound microscope, the telescope and achromatic
instruments have been invented.

And finally, with the abundance of stains and the newer methods
of physical analysis of the structure and action pi living, fresh and
fixed material with the spectroscope, the dark-fieH microscope, the
polarizing and the ultra-violet microscope, one tan look forward
with confidence to still greater discoveries, and with a corresponding
deeper insight into the complex structure and the marvelous func-
tions of living things.

(In the accompanying references to the history of optics and the microscope,
one will find the sources of information on which this brief history is founded).

COLLATERAL READING FOR CHAPTER XV

For further and more extended information on the History of the Microscope,

see, "Origin and Development of the Microscope," and the bibliographies of

original authorities, by Disney, Hill and Baker with an historical survey of the early

progress of optical science by the editor of the Journal of the Royal Microscopical

Society. Published by the Royal Microscopical Society, 20 Hanover Square,

London, 1928. See also the works in the general Bibliography in this book.

AIRY. — On astigmatism, 1827, Young, 1800.

ALHAZEN. — The Opticae Thesaurus, 1572.

BACON, ROGER. — Opus majus and lesser writings, 1266-1267.

BARBARO. — Perspettiva, Venice, 1568. One of first references to concave spec-
tacles.

BREWSTER, SIR DAVID. — Discoverer of fluorescence, 1833.

BARTHO LINUS. — Discoverer of double refraction, 1669.

CUSA, CARDINAL DE. — Opera theologica, methematica, etc., Paris, 1514, first
reference found which refers to concave spectacles.

DESCARTES. — Dioptrique, 1637.

DOLLOND. — Achromatic telescopes, 1758.

ERNST AND WOHLBACH. Ultra- Violet photography, 1906.

GALEN. — 131-201 A.D.

HERSCHEL, SIR WM. — Discovery of infra red, 1801.

HIPPOCRATES. — 460-375 B.C., Brain is the final organ of vision.

IVES, F. E. — Inventor of the modern binocular with one objective, in which
each eye receives the full aperture. Jour. Franklin Inst., 1902.

KEPLER. — Explained the use of spectacles and inverted image on the retina;
inventor of the Keplerian microscope. Opera Omnia, 1604-1611, pp. 232-
234; 255-256; 54Q-S50. t

KC)HLER. — Mikrophotographische Untersuchungen mit ultraviolettem Licht,
1904.

CH. XV] BRIEF HISTORY OF LENSES AND MICROSCOPES 579

LUCAS. — Ultra-Violet photography, 1930, 1931.

MALUS. — Polarized light, 1808-1810.

NEWTON, SIR ISAAC. — Optics.

NICOL, WM. — Invented the Nicol prism for use with polarized light, Edin. New

Philos. Jour., Vol. VI., 1829, pp. 83-84; 1839, Vol. xxvii, pp. 332-333.
PTOLEMAEUS. — Optics. (70-147 A.D.)
REDI, FRANCESCO. — Lettera interno all'invenzione degli occhiale, 1299. First

statement found as to the actual use of spectacles by an old man.
RIDDELL. — First one to produce a practical, single objective, binocular micro-
scope, 1853.

RITTER, J. W. — Disco^Ted ultra-violet, 1801.
SCHEINER. CHR. — The .first to make a Keplerian compound microscope. 1619-

1626.

SORBY. — Application of a direct vision spectroscope to the microscope.
STOKES. — Fundamental article on ultra-violet and fluorescence. Gave the name

fluorescence. 1852.

FOX-TALBOT. — First to produce a micro-polariscope. 1834.
THOMAS, EDWARD. — On Improvements in the Microscope. American Journal of

Science and the Arts, Vol. XIX, 1831, pp. 57-60.
THOMAS, EDWARD. — On the Achromatic Microscope. American Journal of

Science and the Arts, Vol. XX, 1831, pp. 265-269.

These two articles, prepared in 1830 and 1831 by the assistant engineer of

the Cayuga-Seneca Canal, with illustrations of the combinations, and formulae,

are the first scientific discussions of objectives that have been found in American

scientific literature.
WENHAM. — Inventor of means for dark-field microscopy with all powers. 1850-

1856.
YOUNG, THOMAS. — The first to point out astigmatism in the eye, and gave a

means of correcting it (1800). See also Airy.

See the brief statements concerning the portraits p. 556. See also col-
lateral reading, pp. 50, 168, 257.

BIBLIOGRAPHY

For new information the reader is advised to consult the Journal of the Royal
Microscopical Society, The Quarterly Cumulative Index Medicus and the Wistar

Catalogue of the Surgeon General's Office, the Catalogue of Scientific Papers
published by the Royal Society of London, the larger works on the microscope,
and the microscopical journals.

ADAMS, GEORGE, 1720-1773. — Micrographia illustrata; or, the microscope ex-
plained, in several new inventions; likewise a natural history of aerial, ter-
restial, and aquatic animals, etc., considered as microscopic objects, lix -h
325 pp. 72 plates. 4th ed. Published for the author, London, 1771.

ADAMS, GEORGE, 1750-1795. — Essays on the microscope, containing a descrip-
tion of the most improved microscopes, a history of insects, their transforma-
tions, peculiar habits, and oeconomy, with a catalogue of interesting objects,
xiii + 724 pp. 31 plates. Published for the author, London, 1787.

AIRY, G. B. — On a peculiar defect in the eye and a mode of correcting it. Cam
bridge Phiios. Trans., Vol. II, 1827, pp. 267-271. Here is discussed astig-
matism and its correction by cylindrical glasses. See Young.

ALHAZEN. — Opticae thesaurus Alhazeni Arabis, libri septem, nunc primum editi,
ejusdem liber de crepusculis et nubium ascension ibus, item Vitellionis Thu-
ringopoloni, libri X. omnes instaurati, figuris illustrati et aucti, adjectis etiam
in Alhazenum commentariis. A Frederico Risnero. Folio, many figures,
Basileae, per Episcopios. 1572.

AMERICAN ACADEMY OF PHYSICAL THERAPY. Papers published by American
Medicine, Burlington, Vt.

AMERICAN JOURNAL OF PHYSICAL THERAPY. Chicago, 1924 -+-

ARCHIVES OF PHYSICAL THERAPY . . . WITH INTERNATIONAL ABSTRACTS. Ra-
diological Publishing Co., Omaha, Nebraska. 1920 +

BACON, ROGER. — Opus Majus, edited with introduction and analytical table by
John Henry Bridges. 2 vols. and supplementary vol. Vol. I, clxxxvii +
440 pp. 23 fig. Vol. II, 568pp., 187 figs. Supplement, xv + 187 pp. Williams
& Norgate, London, 1897-1900. 315. 6d. For modern optics the part desig-
nated De Scientia Perspectiva is "most important. For use of convex lenses
to aid the sight of old men, see vol. ii, p. 157, and for burning flasks, p. 471.

BACON, ROGER. — Opus Majus, 1266-1267. Combach's edition, 1614. On p. 159
occurs the description of a convex glass to aid old men in reading.
Bridges edition, 1897-1900. Vol. II, p. 157* spectacle for old men.
Opus tertium, opus minus. Brewer, in Compendium studii philosophiae,
1859.

Part of the Opus tertium, including a fragment now printed for the first
time. Edited by A. G. Little, 1912. On p. 40 is repeated the statement
about the convex lens for old men, and much other optics.

BACON, ROGER. — Essays contributed by various writers on the occasion of the
commemoration of the seventh centenary of his birth, Collected and edited

$81

582 BIBLIOGRAPHY

by A. G. Little. 426 pp. Clarendon Press, Oxford, England, 1914. Price,
$5.25. Biography of Bacon and essays upon his work in various fields. List
of Bacon's writings.

BACON, ROGER. — Opus majus. Translation of Robert B. Burke. Univ. of
Penn. Press, 1928. Translation based on the corrected text of Bridges edi-
tion of 1900.

BAKER, HENRY, F. R. S. — Of Microscopes and Observations made thereby.
2. Vols. New edition. Vol. I, The Microscope made easy. Projection micro-
scope. Vol. II, Employment for the Microscope. London, 1785.

BARBARO, DANIEL. — La pratica della perspettiva di Monsignor Daniel Barbaro,
eletto patriarca d'Aquileia. Opera molto utile a pittori, a scultori & ad
architetti. Con privilegio. 208 pp. Many figures. In Venetia, appresso
Camillo & Rutilio Borgominieri fratelli, al segno di S. Giorgio MDLXVIII
(1568). First known user of a lens in the camera. Cap. V, p. 192.

BARTHOLINUS, ERASMUS. Experimenta cristelli Islandici quibus mira et insolata
refractio detegitur. Hasniae, 1699 (Discovery of double refraction).

BAUSCH, E. — Manipulation of the Microscope. A manual for the work table
and a textbook for beginners in the use of the microscope. 200 pp. Illust.
New edition. Rochester, N. Y., 1906.

BAUSCH & LOMB OPTICAL COMPANY. — Lenses, their History, Theory and Manu-
facture. Published in honor of the ninth annual convention of the Ameri-
can Association of Opticians. Rochester, 1906. 47 pages, many figures.

BAUSCH & LOMB OPTICAL COMPANY. — Use and Care of the Microscope; Use
and Care of the Microtome. Rochester, N. Y. These little booklets are of
great value to the young worker with the microscope and microtome.

BEALE, L. S. — How to Work with the Microscope, sth edition, 1880, 518 pages,
99 plates with 500 figures. Harrison, London.

BECK, CONRAD. — the Theory of the Microscope. Cantor Lectures delivered
before the Royal Society of Arts, Nov.-Dec., 1907. 59 pp. Illust. London,
1908.

BECK, CONRAD, and ANDREWS, HERBERT. — Photographic Lenses. 7th edition,
completely revised. 287 pp., 163 figs., 44 plates. R. & J. Beck, Limited.
68 Cornhill, London, England. Full discussion of modern objectives for
photography and for projection.

BECK, CONRAD. — The Microscope, a simple handbook. 144 pages, 131 figures.
London, 1921. Published by R. & J. Beck, Ltd.

BECK, CONRAD. — The Microscope, Part II. An advanced handbook. 231 pp.
170 figs. London, 1924. Published by R. & J, Beck, Ltd.

BELLING, JOHN. — The Use of the Microscope; a handbook for routine and re-
search work. 315 pages, 28 figures. McGraw-Hill Book Co., N. Y.

BOCK, DR. EMIL. — Die Brille und ihre Geschichte. 62 pages, frontispiece, and
32 text figures. Wien, 1903. Verlag von Josef Safar.

BORELLUS, PETRUS. — De vero Telescopii inventore, cum brevi omnium Con-
spiciliorum historia. Ubi de eorum confectione, ac usu, seu de effectibus
agitur, novaque quaedam circa ca proponuntur. Accessit etiam centuria
observationum microscopicarum. Authore Petro Borello, regis christianis-
simi consiliario, et medico ordinario. Hagse-Comitum, ex typographia
Adriani Vlacq, MDCLV (1655). Important for the history of optic instru-
ments. See esoecially pp. 25-26.

BOYER, CHARLES S. — The Diatomaceae of Philadelphia and Vicinity. Quarto,
143 pages, 40 plates (700 drawings by the author at a scale of 800 diameters).
Philadelphia, 1916. Press of J. B. Lippincott Company, East Washington
Square.

BREWSTER, SIR DAVID. — A Treatise on the Mikroscope. From the yth edition

BIBLIOGRAPHY 583

of the Encyclopaedia Britannica, with additions. Illust. 1837. P. in for
fluorite in objectives.

B RE WSTER, SIR DAVID. — The Edinburgh Encyclopaedia. Optics, Vol. 14. Joseph
and Edward Parker, Philadelphia, 1832. On page 764, 2d column, near
middle, is described the use of the amalgam on the back of looking-glasses
as a screen. 1 have tried this and found it wonderfully efficient.

B RE WSTER, STR DAVID. — Discovered fluorescence (internal dispersion) in an
alcoholic solution of chlorophyll. 1833. Edinb. Trans. Vol. xii, 463-464.

BRITISH JOURNAL OF PHYSICAL MEDICINE. — Incorporating the British Journal
of Actinotherapy, and Physiotherapy. London, England. 1926 +

BROADHURST, DR. JEAN. — Bacteria in Relation to Man, a. study-text in general
micro-biology. J. B. Lippincott Company, Philadelphia and London, 1925.
306 pages, 146 figures. Gives information that every one should have. The
book has an excellent glossary.

BURNETT, SAMUEL HOWARD. — The Clinical Pathology of the Blood of Domesti-
cated Animals. 156 pp. Ithaca, 1908. 24 figures, 4 colored plates.

CARNOY, CHANOINE J. B. — La Biologic Cellulaire, e"tude compare de la cellule
dans les deux regncs. Paris, 1884. Much good biological history.

CARPENTER-DALLINGER. — The Microscope and its Revelations, by the late
Wm. B. Carpenter. Seventh edition, in which the first seven chapters have
been entirely rewritten. London & Philadelphia. 1891. References mostly
to the 7th edition.

CARPENTER-DALLINGER. — The Microscope and its Revelations, by the late
William B. Carpenter. 8th edition, in which the ist seven and the 23rd
chapters have been entirely rewritten, and the text throughout reconstructed,
enlarged, and revised by the Rev. W. H. Dallinger. 22 plates and nearly
900 wood engravings. n8r pp. London and Philadelphia, 1901. P. Blaki-
ston's Son & Co.

CHAMBERLAIN, C. J. — Methods in Plant Histology. 3d edition, 314 pages, 107
figures. The University of Chicago Press, 1916.

CHAMOT, SMILE MONNIN. — Elementary Chemical Microscopy. 410 pages, i
plate, 139 text figures. John Wiley & Sons, N. Y., 1915.

CHAMOT, E. M. — Elementary Chemical Microscopy. 2d ed., 1921. 479 pages,
162 figures. John Wiley and Sons, Inc. New York.

CHAMOT and MASON. — Handbook of Chemical Microscopy. 2 vols., I, 474 pages,
162 figures; II, 411 pages, 181 figures. John Wiley and Sons, Inc. New
York, 1930-1931. These works invaluable to the microscopist.

CHEVALIER, CHARLES. — Des Microscopes et de leur usage. Illust. Paris, 1839.

CLARK, C. H. — Practical methods in microscopy. 2d ed. Illust. Boston,
1896.

COLES, ALFRED C. — Critical Microscopy: How to get the best out of the micro-
scope. TOO pages, 8 illustrations. New York, D. Van Nostrand Co., 1922.

COMSTOCK, ANNA BOTSFORD. — Handbook of Nature Study. 90x3 pages, 1000
illustrations. Comstock Publishing Co. Ithaca, N. Y., New, 220! edition
(1931), from new type, many new figures and a portrait of the author.

Cox, HON. J. D. — Robert B. Tolles and the angular aperture question. Deals
with the origin of the homogeneous immersion objective also. Transactions
of the Amer. Micr. Soc., 1884, PP- 5~39-

CROSS, M. I. and COLE, MARTIN J. — Modern Microscopy. A handbook for
beginners and students. Fifth edition revised and rearranged by Herbert
F. Angus, with chapters on special subjects by various writers. Chicago
Medical Book Company, 1922. 315 pages, 114 figures and 12 plates.

CONN, H. J. — Biological Stains, a handbook on the nature and uses of the dyes
employed in the biological laboratory. 2d edition, 1929. 224 pages, graphs

584 BIBLIOGRAPHY

and formulae. Geneva, N. Y. Published by the commission on standardiza-
tion of stains.
CUSA, NICOLAUS DE, CARDiNALis. — Opera Thcologica et Mathematica, etc. Paris,

1514. Folio clxxxiiii. "Beryllus lapis est lucidus, albus & transparens cui

datur forma concava pariter & convexa, & pipm. vides attingit pri invisible."

(First reference found to concave spectacles.) See also Barbaro,
CZAPSKI, S. — Grundziige der Theorie der optischen Instrumente nach Abbe.

2 Aufl., unter Mitwirkung des Verfassers und mit Beitragen von M. v. Rohr.

490 pp. Illust. Leipzig, 1904.
DANCKWORTT, P. W. — Lumineszenz-Analyse im filtrierten ultra violetten Licht.

ad ed. 50 figs., 2p plates, 147 pp. Leipzig, 1929.
DESCARTES (Lat. Cartesius), RENE. — CEuvres, publiees par C. Adam et P.

Tannery sous les auspices ministere de 1'instruction publique, Vols. i-xii.

Dioptrique, Vol. 6, pp. 87-228, 73 figs. Leopold Cerf, 12 Rue Sainte Anne,

Pans, 1902.
DESCARTES (Lat. Cartesius), RENE". — (Euvres, publie*es par V. Cousin. Paris,

1824-26. ii vols. Dioptrique, Vol. 5. pp. 1-153, 5 plates, including 66

figs.
DIPPEL, L. — Das Mikroskop und seine Anwendung. Illust. Braunschweig,

1898,
DOLLOND, J. — Philos. Trans. Roy. Soc., London, 1758, pp. 733-743. An account

of some experiments concerning the different refrangibility of light. Every

one interested in optical instruments ought to read this paper.
EALAND, C. A. — The Romance of the Microscope. 3 14 pages, 39 illustrations

and numerous diagrams. London, Seeley, Service & Co., Lt'd, 1921.

This book is full of interesting information, and especially adapted to the

amateur.
EDINGER. — See for description of the double ocular, Jour. Roy. Micr. Soc., 1911,

p. 252.
EHRLTCH. — Enzyklopadie der Mikroskopischen Technik. Herausgegeben von:

Ehrlich, Krause, Moose, Rosin and Weigert. 2d edition, 2 Vol., 800 + 680 pages,

56 4- in figures. Urban & Schwarzenberg, Berlin and Vienna, IQIO.
ENZYKLOPAEDIE DER MIKROSKOPISCHEN TECHNIK. Ed. Dr. Rudolph Krause.

3d ed. Berlin, Urban & Schwarzenberg. 3 vols. 2444 pages, 350 figures,

48 plates.
ERNST, HAROLD C, and WOLBACH, S. B. — Ultra-Violet Photomicrography.

Journal of Medical Research, Vol. XIV, pp. 463-469, 7 plates. See also

Kohler and Lucas.
FERRY, ERVIN S. — General Physics and its application to industry and to

everyday life. New York. John Wiley & Sons, Inc. 1921.
GAGE, S. H. and FISH, P. A. — Fat digestion, absorption and assimilation in man

and animals as determined by the dark-field microscope and a fat soluble dye

(Sudan III). American Journal of Anatomy, Sept., 1924. pp. 1-85, 25 text

figures, 4 colored plates.
GAGE, S. H. and H. P. — Optic Projection. Principles, installation and use of

the magic lantern, projection microscope, reflecting lantern and moving pic-
ture machine. 731 pages, 413 figures. Comstock Publishing Co. Ithaca,

N. Y. 1914.
FaX^vos, KXauSto?. — Hfpl ypeJas r&v kv avOp&irov aw/mri poplwv (Galenus,

Claudius. — De usu partium corporis humani, 131-201, A.D.).
GARRISON, FIELDING H. — An introduction to the History of Medicine, with

medical chronology, suggestions for study, and bibliographic data. W. B.

Saunders Co., Philadelphia and London. 4th edition revised and enlarged,

1929. 996 pages, many portraits.

BIBLIOGRAPHY 585

GATES, FREDERICK L. — A study of the bacteriacidal action of ultra-violet.
Studies from the Rockefeller Institute, Vol. 73, pp. 9-26.

GORING and PRITCHARD. — Micrographia, containing practical essays on reflect-
ing, solar, oxy-hydrogen gas microscopes, micrometers, eyepieces, etc., etc.
231 pp. Many figures in the text, one plate Whittaker & Co., Ave-Maria
Lane, London, England, 1837.

Govi, GILBERTO. — Galileo, the inventor of the compound microscope, — Journal
of the Royal Microscopical Society, 1889, pp. 574-598. Discussion of the
earliest discoveries and inventions in optics. The compound microscope here
referred to as the invention of Galileo is the Dutch telescope used as a micro-
scope, i.e., an instrument like the ordinary opera glass with a longer tube for
the convex objective and concave ocular.

GUYER, MICHAEL F. — Animal Micrology. Practical exercises in Zoological
micro-technique. 289 pages, 74 figures. Revised ed. The University of
Chicago Press, 1917.

HALL, CHARLES A., F. R. M. S. — How to Use the Microscope. A guide for the
novice. 2d edition. 16 plates, 24 line cuts; 90 pages of text. London, A.
& C. Black, Lt'd, 1925.

HARDESTY, IRVING. — A Laboratory Guide for Histology, with a chapter on
laboratory drawing, by A. W. Lee. 193 pages, 30 figures. P. Blakiston's
Son & Co. Philadelphia, 1908.

HARTING, P. — De Nieuwste Verbeteringen van het Mikroskoop en Zijn Gebruik,
Sedert 1850, 176 pages, two triple plates. Te Tiel. bij H. C. A. Campagne,
1858.

HARTING, P. — Das Mikroskop. Theorie, Gebrauch, Geschichte und gegenwar-
tiger Zustand desselben. Deutsche Originalausgabe, von Verfasser revidirt
und vervollstandigt. Herausgegeben von Dr. Fr. Wilh. Theile. In drei
Banden. Bd. I, Theorie; Bd. II, Gebrauch; Bd. Ill, Geschichte. Bd. I,
pp. 346, 134 text figures, i plate. Bd. II, pp. 310; 104 text figures. Bd.
Ill, pp. 452, 231 text ^figures. Druck und Verlag von Friedrich Vieweg und
Sohn, Braunschweig, Zweite wesentlich verbesserte und vermehrte Aurlage,
1866.

HERSCHEL, WILLIAM. — Untersuchungen tiber die warmende und die erleuchtende
Kraft der farbigen Sonnenstrahlen; Versuch iiber die nichtsichtbaren Strahlen
der Sonne und deren Brechbarkeit und Einrichtung grosser Teleskope zu
Sonnenbeobachtungen. Ann. der Physik, Bd. 7, 1801, pp. 137-156. Dis-
covery of infra red by its heating effect.

HIPPOCRATES. — 460-375, B.C. Adams, Francis, surgeon. The genuine works of
Hippocrates, translated from the Greek (Sydenham Society), London, 1849
(2 vols).

LITTRE, E. — (Euvres complete d'Hippocrate. Ten vols. Paris, 1839-1861.

JONES, W. H. S. — Hippocrates with an English translation. Two vols. Greek
and English text. Loeb Classical Library. London and N. Y., 1923.

HOGG, J. — The Microscope, its History, Construction and Application. i5th ed.
704 pp. 900 Illust. Rutledge, London and New York, 1898. Much atten-
tion paid to the polariscope.

HOOKE, ROBERT. — Animadversions on the Machina Coelestis of Hevelius. p. 8.
Published in 1674. It is in this place that Hooke states that for two points
, to be seen as two the visual angle must be one minute.

HOVESTADT, H. — Translated by J. D. and A. Everett. Jena Glass and its scien-
tific and industrial applications. London, Macmillan & Co. Limited.
N. Y. The Macmillan Company. 1902. 419 pages, 29 figures.

HOWELL, WILLIAM H. — A Text-book of Physiology for Medical Students and
Physicians, nth Ed., 1930. Philadelphia, Pa.

586 BIBLIOGRAPHY

HUYGENS, CHR. — For Huygens' ocular see Nelson. Jour. Roy. Micr. Soc., 1900,
pp. 162-169.

IVES, FREDERICK E. — A New Binocular Microscope. Journal of the Franklin
Institute, vol. 154, Dec. 1902, pp. 441-445. Jour. Roy. Micr. Soc. 1914, p.
488. See also Conrad Beck, Jour. Roy. Micr. Soc., 1914, pp. 17-23-

INDEX Catalogue of the Library of the Surgeon General's Office of the United
States Army. Government Printing Office, Washington, I). C. First series,
vols. i-xvi, 1880-1895. Second series, vols. i-xxi, 1896-1916. Book and peri-
odical literature; subjects and authors in one continuous alphabetical list.
Full lists of current literature. 3d ser., Q-Sy; Vol. ix, 1931, already published.

JOHNSON, B. K. — Practical Optics for the laboratory and workshop. London,
Benn Bro's Ltd., 1922. 189 pages, 140 figures.

JANSEN, ZACHARTAS. — See in Borellus, Petrus. De vero telescopii inventore,
1655. Singer, Studies in the History and Method of Science, Oxford, 1921,
pp. 408-413.

JORDAN, HARVEY ERNEST. — A Text-Book of Histology. 857 pages, 593 figures.
D. Appleton & Co., N. Y. and London, 1931. There is a chapter on technic
and 54 pages of directions for laboratory work.

JOURNAL OF APPLIED MICROSCOPY. — Vols. I to VI, 1898 to 1903. Published by
the Bausch & Lomb Optical Company. Rochester, N. Y.

JOURNAL OF THE FRANKLIN INSTITUTE, devoted to science and the mechanic arts.
1826 + Philadelphia, Pa.

JOURNAL OF THE OPTICAL SOCIETY OF AMERICA. — Published by the Society;
Review of Scientific Instruments, a late division of the Journal began in
1930. The Journal now in its 2ist volume. As the name implies, this jour-
nal and the Review contain many articles of vital interest to the worker with
the microscope. Geo. Banta Publishing Co., Menasha, Wisconsin.

JOURNAL OF THE ROYAL MICROSCOPIC \L SOCIETY. 1878 -f . Published by the
Society at 20 Hanover Square, London W., England. 6 numbers per year.
In nearly every number is the announcement of some new thing pertaining
to the microscope. For the special purposes of this chapter attention is
called especially to the volume of 1886, pp. 849-856, for the apochromatic
objectives.

In 1891, pn. 90-105, Mr. Nelson deals with the substage condenser, and in
1900, pp. 162-169, with the history of the Huygenian ocular. In 1902,
pp. 20-23, Mr. Nelson gives a bibliography of works (dated not later than
1700) dealing with the microscope and other optical matters. In 1914 Dr.
Jentzsch, pp. 1-16, and Conrad Beck, pp. 17-23, 205-210, deal with binocu-
lar microscopes, past and present.

In 1915 Charles Singer, pp. 317-340. deals especially with early drawings
made by the aid of the microscope; and in 1916, Mr. Heron- Allen and Ch.
F. Rousselet give a summary of the progress of knowledge of vision and the
microscope from 1673-1848. Attention is especially called to the volume on
"The Origin and Development of the Microscope" published by the Society
in 1928.

JOURNAL OF THE ROYAL SOCIETY OF ARTS. London, England. The 79th volume
of the Journal is now being published (1931).

KEPLER, JOHANNES. — Opera Omnia, Vol. II. Ad Vitellionem Paralipomena,
(De modo visionis et humorum oculi usu.) 1604. pp. 226-269. n figs.
Correct dioptrics of the eye here given, and also the explanation of the effect
of convex and concave spectacles. Dioptrica. Demonstratio eorum qua? visui
et visibilibus propter conspicilla non ita pridem inventa accidunt. pp. 519-
567. 35 figs. 1611. The amplifier, real images, and erect images. The
Keplerian microscope (modern microscope).

BIBLIOGRAPHY 587

KINGSBURY, B. F. — Laboratory Directions in Histology and Histological Tech-
nique, pp. 1-60 and 1-95. The Macmillan Co., N.Y. 1916.

KINGSBURY, B. F. and JOHANNSEN, O. A. — Histological, Technique, a guide for
use in a laboratory course in histology. N. Y. J. Wiley & Sons; London.
Chapman & Hall, Ltd., 1927.

KOHLER, DR. AUGUST. — Mikrophotographische Untersuchungen mit ultravio-
lettem Licht. Zeit. wiss. Mikroskopie, Bd. xxl, 1904, pp. 129-165 and 273-
304. 8 text figs, and 6 plates. See also Ernst and Lucas.

LAMBERT, AVERY E., PH. D. — Guide to Study of Histology and Microscopic
Anatomy. 262 pages, 152 figures. 1930. P. Blakiston's Son & Co. Phila-
delphia.

LANGERON, MAURICE. — Precis de Microscopic. Technique, experimentation,
diagnostic. 4th ed. 1034 pages, 315 figures. Paris, Masson et Cie. 1925.

LEE, A. B. — The Microtomist's Vade-mecum. A handbook of the methods of
microscopic anatomy. 9th ed. 710 pp. 1928. P. Blakiston's Son & Co.,
Philadelphia, Penn.

LEEUWENHOEK, ANTONY VAN. — Opera omnia, seu arcana naturae; ope exac-
tissimorum microscopiorum detecta, experimentes variis comprobata, epistolis
ad varies illustres viros. Lugduni Batavorum, 1722, 4 vols. (Delphis Apud
Adrianum Beman, 1719.) 1514 4- pages, 124 figs.

LEITZ, ERNST. — The Microscope and its Application. Description and guide to
the use of Leitz Microscopes. On p. 19 the mechanical tube-length for
Leitz microscopes is given as 170 mm. Most manufacturers have adopted
1 60 mm. as the mechanical tube-length. Wetzlar, 1929.

LENSES, their History, Theory and Manufacture. Bausch & Lomb Optical Co.,
Rochester, N. Y., 1906. 47 pp., 34 figs.

LISTER, JOSEPH JACKSON. — On some properties in achromatic object glasses
applicable to the improvement of the microscope. Philos. Trans. R. S.,
Vol. 120 (1830), pp. 187-200. Dark field effects by retro-objective stop, p.

19 f<
LUCAS, FRANCIS F. — The Architecture of Living Cells; a discussion of recent

advances in methods of biological research by means of optical sectioning
with the ultra-violet microscope. Bell Telephone System, Monograph B 514,
October, 1930. Also in the Journal of Morpholoprv, Vol. 52, 1931, pp. 91-
107. Text figures and plates in both. See also Kohler, and Ernst.

LUCKIESH, M. — Artificial Sunlight combining radiation for health with light
for vision. D. Van Nostrand Co., Inc., N. Y. 1930.

MAcMtiNN, C. A. — The Spectroscope in Medicine. 325 pp. Illust. London,
1885.

MALLORY, FRANK BURR and WRIGHT, JAMES HOMER. — Pathological Tech-
nique. A practical manual for work in pathological histology and bacteriol-
ogy. 8th edition, 666 pages, 180 figures. \V. B, Saunders Co., Philadelphia,
Pa. 1924.

MALUS, ETTENNE LOUTS. — The"orie de la double refraction de la lumiere dans
les substances cristallis6es. Memoire couronn6 par ITnstitute, Jan., 1810.
302 pages, two plates. Polarisee, etc., on pp. 239-241.

MAYALL, JOHN, JR. — Cantor Lectures on the Microscope, delivered before the
Royal Society for the encouragement of arts, manufactures, and commerce.
Five lectures, Nov.-Dec. 1885; 97 pp., 103 figs, and two additional lectures
in 1888; 1 8 pp., 26 figs. Published by the Society at John Street, Adelphi,
London, W. C., England.

MCCLUNG, C. E. — Handbook of Microscopical Technique. For workers in both
animal and plant tissues. 495 pages, 43 figures. P. B. Hoeber, N. Y. 1929.
24 collaborators.

588 BIBLIOGRAPHY

McKENziE. T., and KING, A. — Practical ultra-violet light therapy. 108 pages.
Wm. Wood & Co., N. Y., 1926.

METZNER and ZIMMERMANN. — Das Mikroskop, ein Leitfaden der wissenschaft-
lichen Mikroskopie. Leipzig and Wien. Franz Deuticke, 1928.

MILLIET DE CHALES, CLAUDE FRANCOIS. — Cursus seu mundus mathematicus;
nunc primum in lucem prodit. Ex officina Anissoniana, Lugduni (Lyons).
1674. 3 vols. Folio. The second edition is dated 1690 and has 4 folio vols.

MOLYNEUX, WILLIAM. — Dioptrica nova, a treatise of Dioptricks, in two parts,
wherein the various effects and appearances of spheric glasses, both convex
and concave, single and combined, in telescopes and microscopes, together
with the usefulness in many concerns of human life are explained. By Wil-
liam Molyneux of Dublin, Esq. Fellow of the Royal Society. Presented to
the R. S. 1690, printed 1692. Much history and translations of Latin ex-
tracts. The first figure of a magic lantern with condenser lens.

MOORE, V. A. — Principles of micro-biology; a treatise on bacteria, fungi, and
protozoa pathogenic for domesticated animals, 19 r 2. $06 pages. ior figures.
The Macmillan Co., N. Y.

NACHET. — Sur un nouveau microscope approprie* aux besoins des demonstrations
anatomique, et permettant a plusieurs personnes d'observer ensemble.
Compte rendu des stances de la Societe* de Biologic, Oct. 1853, pp. 141-145.
In this article is described and illustrated forms of microscopes with two
tubes for two observers, and with three tubes for three observers. See also
Harting, Vol. IIT, pp. 24^-248. See also plates T-II in the Dutch edition of
1858.

NEEDHAM, J. G. and LLOYD, J. T. — - The Life of Inland Waters; an elementary
textbook of fresh-water biology for American students. 438 pages, 2 plates
and 244 text figures. Comstock Publishing Company. Ithaca, N. Y., igi6.

NEWTON, SIR ISAAC. — Optics, or a treatise of the reflections, refractions, inflec-
tions and colours of light, 3d edition, 1721. 382 pages, many diagrams.
London, England.

NICHOLS, EDWARD L. and MERRITT. ERNEST. — Studies in Luminescence. Physi-
cal Review, Vol. XIX, 1904, pp. 18-36.

NICHOLS, EDWARD L. and HOWES, HORACE L. — In collaboration with Ernest
Merritt, D. T. Wilber and Frances G. Wick. Fluorescence of the Uranyl
Salts. 241 pages, n figs. Carnegie Institution of Washington, 1919.

NICHOLS, EDWARD L., HOWES, H. L. and WILBUR, D. T. — Cathode-Lumines-
cence and the Luminescence of Incandescent Solids. 350 pages. 176 figs.
Carnegie Institution of Washington. 1928.

NICHOLS, E. L. — The Outlines of Physics. 452 pp., 414 figs. The Macmillan
Co., New York, 1897.

NICHOLS, E. L. and FRANKLIN, W. S. — The Elements of Physics; Light and
Sound. 201 pages, 182 figures. The Macmillan Company, New York, 1897.

NICOL, WILLIAM. — On a method of so far increasing divergence of two rays in
calcareus spar that only one image may be seen at a time. Edinburgh New
Philos. Journal, VI. 1829, pp. 83-84. See also 1833, p. 372, 1839, pp.
332~333- Invention of Nicol prism.

PANSIER, P. — Histoire des Lunettes par le Docteur P. Pansier, d'Avignon. 137
pages, 19 figures, 6 plates. 1901. Paris, A. Maloine, fiditeur.

PETPI, R. J. — Das Mikroskop von seinen Anfangen bis zur jetzigen Vervoll-
kommung far alle Freunde dieses Instruments. 248 pp., 191 figs., 2 plates.
R. Schoetz, Berlin, Germany, 1896.

PIERSOL, GEORGE A. — Normal Histology, with special reference to the structure
of the human body. 8th edition, 418 pages, 438 illustrations, colored frontis-
piece. J. B. Lippincott Co., Philadelphia, Pa., 1910.

BIBLIOGRAPHY 589

POGGENDORFF, J. C. — Geschlchte der Physik, 1879. History.

POLICARD, A. — Emploi de la fluoroscopie dans I'Stude des tissus. Bulletin
d'histologie apphque'e. Vol. II, 1925, pp. 167-180. Gives history and tech-
nique, and an excellent historical bibliography. Policard and his pupils
have made many contributions to the subject.

PRIESTLEY, JOSEPH. — The History and Present State of Discoveries relating to
Vision, Light and Colours. 812 pages, 24 plates. Five pages of references
to 288 other works. London, 1772.

PTOLEM^EUS, CLAUDIUS. — L'Ottica di Claudio Tolomeo da Eugenic Ammiraglio
di Sicilia-Scrittore del Secelo XII. Ridotta in Latino sovra la traduzione
Araba di un testo Greco imperfetto. Edited by Gilberto Govi. Torino,
1885. 171 pages, 9 double plates.

QUEKETT, JOHN. — Practical treatise on the use of the microscope. Editions of
1848, 1852, 1855-

RAMSDEN, J. — Philos. Trans. Roy. Soc., 1782. A description of a new construc-
tion of eye-glass (oculars) for such telescopes as may be applied to mathe-
matical instruments. (Pp. 94-98, i plate. In Vol. LXXIII, 1783.)

RANVIER, L. — Trait6 Technique D'Histologie. 1109 pages, 379 text figures,
and one colored plate. Librairie F. Savy, Paris, France. 1875-1888.

READE, REV. J. B. — On a new method of illuminating microscopic objects, pp.
227-231 of Goring and Pritchard's Micrographia, London, 1837.

REDI, FRANCESCO. — Opere Classic! italiani. Vol. ii "Lettera interno all' in-
venzione degli occhiali di naso." Written in 1299.

RIDDELL, PROF. J. L. — University of La., New Orleans. On the Binocular
Microscope. Read before the American Association for the Advancement of
Science, July 30, 1853. Devised 1851, constructed 1852. American Journal
of Science (Silliman's Journal), Vol. 65, 1853, p. 68. Quarterly Jour. Micro-
scopical Science, vol. ii, 1854, pp. 18-24, four figures. Single objective bin-
ocular to use with or without oculars.

RICHTMYER, F. K. — Introduction to Modern Physics. McGraw-Hill Book Co.,
Inc., N. Y. 1928.

RITTER, JOHANN WM. — Versuche iiber das Sonnenlicht. Annalen der Physik.
Bd. XII, 1803, pp. 409-415. R. says he found the blackening of silver salts
beyond the violet (ausserhalb des Violet). Used Newton's prism to get the
proper position. See also Herschel's discovery of infra red.

ROBIN, CH. — Traite du microscope et des injections, not pages, 336 figures.
Bailliere, Paris. 2d edition, 1877.

ROSENHAIN, DR. WALTER. — Cantor Lectures on optical glass. 1916. Royal
Society of Arts, London, England.

RUSBY, H. H. and JELLIFFE, S. E. — Morphology and Histology of Plants de-
signed especially as a guide to plant analysis and classification, and an intro-
duction to pharmacognosy and vegetable physiol. 378 pp., 693 figures. New
York, 1899.

RUSSELL, ELEANOR H. and W. KERR. — Ultra-violet Radiation and Actino-
therapy. 30! edition. 1928. 648 pages, many text figures. Edinburgh,
E. & S. Livingstone.

SCHEINER, CHRISTOPHORUS. — Oculus hoc est fundamentum opticum, etc. Oeni-
ponti, 1619.

SCHEINER, CHRISTOPHORUS. — Rosa Ursina sive Sol, ex admirando facularum et
macularum suarum phoenomeno varius. A Christophoro Schemer, Germane
sueuo e societate Jesu. Ad Paulum Jordanum II, ursinum Bracciani ducem.
784 folio pages, many plates. Apud Andream Phajum Typographum Duca-
~lem. Bracciani, 1626-1630.

SCHMIDT, DR. J. W. — Die Bausteine des Tierkdrpers in polarisiertem Lichte.
230 text figs., 528 pages. Fr. Cohen, Bonn, Germany. 1924.

590 BIBLIOGRAPHY

SCIENCE, a weekly journal devoted to the advancement of science. N. S., 1895 +.
Science Press, Garrison. N. Y.

SCIENTIFIC AMERICAN, 1845. — Now a monthly publication. 24 West 4oth St.
N. Y. Publishers of Amateur Telescope Making, Albert G. Ingalls, as-
sociate editor Many experts added information.

SEARS, J. E. — Precise length measurements. Cantor Lectures, Journal of the
Royal Society of Arts. Vol. LXXI, Oct., 1923, No's 3698, 3699, 3700.

SEDGWICK, W. T. and TYLER, H. W. — A short history of science. New York,
1917; 474 pages, many portraits.

SHADBOLDT, G. — Trans. Micr. Soc. London, Vol. Ill (1851), pp. 132, 154.

SHIPLEY, SIR ARTHUR E. — Edited by C. F. A. Pantin. Hunting under the Mi-
croscope, 184 pages, 34 figures. The Macmillan Co., N. Y. up28. This little
work is on pond life by one who knew. Jt also has a chapter on Sir
Ronald Ross and the Malarial problem.

SIEDKNTOPF, H. — Vorgesichte der Spiegelkondensoren, Zeit. wiss., Mikr., Vol.
XXIV (1907), pp. 382-395-

SMITH, ROBERT (LL.D.). — A Compleat system of opticks. pp. 458 + 171 of
remarks. 63 plates in the text, 20 plates in the remarks. Cambridge, Eng-
land, 1738.

SOUTHALL, JAMES P. C. — Mirrors, Prisms and Lenses. A textbook of geometri-
cal optics. Enlarged and revised edition. New York, the Macmillan Co.,
1923. 657 pages, 287 figures.

SPENCER, CHARLES A. — Use of Eluorite in Objectives as early as 1851. Also
used by his son, H. R. Spencer in 1864-5. Sir David Brewster used it in
1837. See § 259a and Vol. XII, 1890, Proc. Amer. Soc. Microscopists, pp.
248-249. 1901, p. 23.

SPENCER LENS Co — The Microscope, Construction, Use and Care. The
Spencer Lens Co., IQ Doat St., Buffalo, N. Y.

SPITTA, E. J. — Microscopy, the construction, theory and use of the micro-
scope. 468 pages, 241 text figures and 47 half-tone reproductions of original
negatives. John Murray, London, 1907.

STAIN TECHNOLOGY. — Published by the Commission on Standardization of
Biological Stains. Editor and business manager, H. J. Conn, Geneva, N. Y.
1926 +.

STEVENS, WM. C. — Plant Anatomy — and hand-book of micro-technic. 399
pages, 155 illustrations. P. Blakiston's Son & Co., Philadelphia, Pa. 3d
edition, 1916.

STITT, E. R. — Practical bacteriology, blood work and animal parasitology in-
cluding bacteriological keys, zoological tables and explanatory clinical notes.
8th edition revised and enlarged with one plate, 211 other illustrations con-
taining 683 figures. 837 pages. Philadelphia, P. Blakiston's Son & Co.,
1927.

STOKES, GEORGE GABRIEL. — On the Change of Refrangibility of Light. Philos.
Trans. R. S., Vol. 142, pp 463-479. and Vol. 143, (1853), PP- 385-396 (1852).
In a note, p. 479 of vol. 142 Stokes proposed the word ''Fluorescence".
This has become universally recognized. These two articles are the most
fundamental ones ever published on fluorescence. The names of many ob-
jects are given which show the fluorescence well. There is much good history
given also.

STRAHLENTHERAPTE — Berlin, 1912+.

TALBOT, HENRY Fox. — Experiments on Light. Philos. Mag. Vol. V, 1834, pp.
331-334; Philos. T^ans., 1837, pp. 25-28; Philos. Trans., 1837, pp. 29-36.
Applied polarizer tr > the Microscope.

THRO, WILLIAM CROOKS. — Clinical Laboratory Methods. The methods used in

BIBLIOGRAPHY 591

the laboratory of clinical pathology, Cornell University, Medical CoHege,
New York City. 2d edition, 1926. 215 pages.

TRANSACTIONS OF THE AMERICAN MICROSCOPICAL SOCIETY. 1878 +. Published
by the Secretary of the Society, and hence the place of publication varies.

WALLIS, T. E — Analytical Microscopy, its aims and methods. 45 text figs.
149 pages. Edward Arnold & Co, London, 1923.

WATERHOUSE, J. — Notes on the Early History of the Camera Obscura. The
Photographic Journal, including the transactions of the Royal Photographic
Society of Great Britain, Vol. XXV, May 31, 1901, pp. 270-290. This is
the best statement of the case found. Many extracts from original sources
are given See also the last edition of the Encyclopaedia Britannica under
Camera Obscura, written by General Waterhouse.

WATERHOUSK, J. — Notes on Early Tele-dioptric Lens-systems, and the Genesis
of Telephotography. The Photographic Journal, including the transactions
of the Royal Photographic Society of Great Britain. Vol. XLIT, Jan. 31,
1902, pp. 4-21, one pi. This paper gives a good account of the introduction
of the combination of a convex and a concave lens for projection, i.e., the
use of an amplifier.

WATSON, WM. — A Text-book of Physics. 4th edition, 927 pages, 579 figures.
Longmans, London, 1907.

WATSON'S MICROSCOPE RECORD. — Published by W. Watson & Sons, Limited,
313 High Holborn, London, England. This record contains many excellent
articles for both amateur and professional worker. 1924+.

WENHAM, F. H. — Reflecting condensers, Trans. Micr. Soc., London, Vol. Ill,
1850, pp. 83-90. Quart. Jour. Micr. Sci., Vol. T£, 18^4, pp. 145-158. Trans.
Micr. Soc., London, in Vol. IV, 1856, Quart/Jour. Micr. Sci., pp. 55-60.

WHTPPLE, G. C. — The Microscopy of Drinking Water. 3d ed. 409 pages, 20
colored plates. John Wiley & Sons, New York, 1914.

WHITE, ANDREW DICKSON. — - The Warfare of Science with Theology. 2 vols.
D. Appleton & Co., N. Y., 1910.

WINSLOW, CHARLES-EDWARD AMORV. — Elements of Applied Microscopy. A
textbook for beginners. 183 pages, 60 figures. John Wiley & Sons, New
York, 1905.

WINTON, ANDREW L., in collaboration with DR. J. MOELLER. — The Microscopy
of Vegetable Eoods. 2d edition. 701 pp. Illust. John Wiley & Sons, New
York, 1916.

WISTAR INSTITUTE OF ANATOMY AND BIOLOGY, Woodland Ave. and 36th St., Phil-
adelphia, Penn : Style Brief giving methods of preparing manuscript for
publication; Publishers of scientific journals* Journal of Morphology;
Journal of Comparative Neurology; American Journal of Anatomy; Ana-
tomical Record; Journal of Experimental Zoology.

WRIGHT, SIR A. E. — Principles of Microscopy, being a hand-book to The Mi-
croscope. 250 pages, 97 text figures, and 18 plates. The Macmillan Com-
pany, New York, 1907.

WRIGHT, LEWIS, and DREW, A. H. — The Microscope, a practical handbook.
287 pages, 195 figures. Colored frontispiece. London, The Religious Tract
Society 1922.

YOUNG, THOMAS. — On the mechanism of the eye, 1800, in Philos. Trans. Roy.
Soc., London, 1801, pp. 23-88. On pp. 39-40 he describes astigmatism, and
shows that it can be corrected by tilting the spectacles. See Airy.

ZAHN, JOANNES. — Oculus artificialis teledioptricus, sive telescopium ndva
methode explicatum ac comprismis e triplici fundamento physico sen naturali,
mathematico dioptrico et mechanico, sen practico stabilitum; opus curiosum
theorico-practicum magna rerum varietate adornatum. 2d edition. 50 4-

592 BIBLIOGRAPHY

t>45 "H *5 PP- Over 600 figs. Johannis Christophorilochneri Norimberga e,

1702.
ZEISS. — The Use and Care of your Carl Zeiss Microscope, by W. Marquette.

Carl Zeiss, Jena, 1929.
ZEITSCHRIFT FtfR WISSENSCHAFTLICHE MIKROSKOPIE UNO FUR MIKROSKOPTSCHE

TECHNIK. Illust. Methods, bibliography and original papers. 1884-!-.

Verlag von S. Firzel, Leipzig, Germany. Published quarterly.

ADDITIONAL BIBLIOGRAPHY

Several of the more recent works whose titles are given in the preceding list have
reached new editions. The following, most of them recent works, are so pertinent
for the microscopical worker that it seems worth while to add them to the list.
In addition to the works mentioned in this list there are many smaller works for
beginners and others which furnish valuable hints for every one using the micro-
scope.

It is most earnestly recommended that those using the microscope pay particular
attention to the booklets and the catalogs put out by the manufacturers of mi-
croscopes. They know what they are talking about.

ALLEN, R. M. — The Microscope, D. Van Nostrand Co., Inc., New York, 1940.
This volume with its clear statements and abundant illustrations will be of
real help to microscopists.

BARNARD, J. E. and WELCH, F. V. — Fluorescence Microscopy with High Powers
and a Simplification of Methods. Journal of the Royal Microscopical
Society of London, 1936, pp. 361-371. See also the new edition of their
work, Practical Photomicrography, Edward Arnold & Co., London.
BECK, CONRAD. — The Microscope, Theory and Practice. 264 pages, 217 figures.
The two volumes published earlier have been combined in one. A most
valuable work by one who knows both from practice and from theory. R. & J.
Beck, London, 1938.

BUKDON, KENNETH L. — Medical Microbiology. An able treatise giving historical
and technical information as well as a knowledge of microbes.

The frontispiece is an excellent portrait of Pasteur. There are also portraits
of Lister, Koch, Theobald Smith and Ehrlich. 763 pages and 1 20 text figures.
The MacMilian Co., New York, 1939.

BUTTOLPH, L. J. — High Efficiency Mercury and Sodium Vapor Lamps. Journal
of the Optical Society of America. Vol. 29, No. 3. March, 1939, pp. 124-130.
Description of the lamps giving high efficiency for illumination and pho-
tography.

CHAMBERLAIN, C. J. — Methods in Plant Histology. 5th revised edition, 416
pages, 140 figures. The University of Chicago Press, 1932. Second impres-
sion, 1933. The book has been entirely rewritten and contains the wisdom
and discrimination that long, varied and intense personal work have brought
to the author. All biologists are urged to read and take to heart the conclusions
in the introduction.

CHAMOT, EMILE MONNIN and MASON, CLYDE WALTER. — Handbook of Chemical
Microscopy. Second ed., 1938, two volumes. John Wiley and Sons, Inc.
Dr. Chamot was one of the first and foremost in advocating, teaching and
practicing chemical microscopy in America.

CLARK, WALTER. — Photography by Infrared. 397 pages, 103 figures. Excellent
directions; comparison with visible light photography. John Wiley and
Sons, Inc., New York, 1939.
CORRINGTON, JULIAN D. — Adventures with the Microscope. 410 pages, 352

BIBLIOGRAPHY 593

figures, mostly original. Colored frontispiece of Leeuwenhoek and the dis-
covery of bacteria. This is a charming book covering a wide range and is
especially designed for the amateur, but the researcher will find many valuable
suggestions, and much useful information. Published by the Bausch & Lomb
Optical Co., Rochester, N. Y., 1934.

COWDRY, E. V. — A Textbook of Histology, Functional significance of cells and
intercellular substances, 2d edition, 1938. Lea and Febiger, Philadelphia.
This work presents histology from the physiological aspect and is to be highly
recommended.

COWDRY, E. V., Editor. — General Cytology. University of Chicago Press, 1924.

COWDRY, E. V., Editor. — Special Cytology, 2d ed., 3 vols. P. B. Hoeber, Inc.,
New York, 1932.

DOWNEY, HAL, Editor. — Handbook of Hematology, 4 vols. P. B. Hoeber, Inc.,
New York, 1938.

EAMES, A. J. and L. H. MCDANIELS. — Introduction to Plant Anatomy. 364
pages, 146 illustrations with a full-page frontispiece. This work will be
found helpful to those especially interested in plant structure. McGraw-Hill
Book Co., N. Y., 1925.

EASTMAN KODAK Co. — Photomicrography. An introduction to photography
with the microscope. i3th edition, 1935, 122 pages, some in color. This
book gives valuable information on photomicrography with all" powers and
with different sources of illumination, the principles and practice of the use
of light screens or filters. There is also a helpful bibliography. See also the
following.

EASTMAN KODAK COMPANY. — The Photography of Colored Objects. i4th edition,
1938. 124 pages, 70 figures, colored frontispiece, and insert. This little
treatise with the preceding will be found most useful to any one interested in
making photomicrographs.

GALIGHER, ALBERT E. — The Essentials of Practical Microtechnique in Animal
Biology. 288 pages and 58 original photomicrographs and drawings. An
excellent guide, and up to date Published by Albert E. Galigher, Inc.,
Berkeley, California, 1934.

GREAVES, R. H. and H. WRIGHTON. — Practical Microscopical Metallography.
2d edition, 416 pages, 311 text figures, 54 plates. Chapman and Hall, London,

1933-

GOODSIR, JOHN, F.R.S. — The Anatomical Memoirs of John Goodsir, edited by
William Turner. 2 vols., 469 & 524 pages, illustrations and portrait.

Goodsir was so highly thought of by Virchow that in 1858 he dedicated his
Cellular Pathology to him in these words: " To John Goodsir, F.R.S., Professor
of Anatomy in the University of Edinburgh, as one of the earliest and most
acute observers of CELL LIFE both physiological and pathological, this work
on Cellular Pathology is dedicated as a slight testimony of his deep respect
and sincere admiration by the author (Rudolph Virchow)."

GOODSTR, JOHN and A. HANNOVER. — On the Construction and Use of the Micro-
scope. 100 pages with two plates of 21 figures. The 20 figures in the text
are mostly with dark field. It is an excellent little book for the time. Edin-
burgh and London, 1853.

HAITINGER, MAX. — Fluorescenz Mikroskopie ihre Anwendung in der Histologie
und Chemie. 108 pages, 32 figures in the text and four colored plates. The
special point of this work is the coloration of materials by fluorescing dyes.
Akademische Verlagsgesellschaft, M.B.H. Leipzig, 1938.

HIRSCHLAFF, E. — Fluorescence and Phosphorescence in Methunes Monographs
on Physical Subjects. 130 pages, 52 diagrams. Methuen & Co., Ltd., London,
1938. This deals especially with the theory of fluorescence.

594 BIBLIOGRAPHY

LEE, A. B. — The Microtomist's Vade-Mecum. Edited by J. B. Gatenby and
T. S. Painter. Tenth edition, 784 pages, n illustrations. P. Blakiston's
Son & Co., Inc., Philadelphia, Pa., 1937. This has served the workers in
histology and embryology from its first edition in 1885 to the present and
is always referred to as a final authority.

MALLORY, FRANK BURR. — Pathological Technique. A practical manual for
workers in pathological histology including directions for the performance of
autopsies and for photomicrography. W. B. Saunders Company, Philadelphia,
1938. This work is also a practical and invaluable manual for microscopy in
general. It is dedicated to his former co-author, James Homer Wright.

MATHESON, ROBERT. — Medical Entomology. 489 pages, 211 figures with portraits
of the great leaders, Sir Patrick Manson, Sir Ronald Ross, Major Walter
Reed, Dr. Howard T. Ricketts, Dr. Leland Ossian Howard and Professor
John Henry Comstock. This book contains many references to original papers
and is indispensable for the investigator in the transmission of disease by insects.
Published by Charles Thomas, Springfield, 111., and Baltimore, Md., 1932.

McCuiNG, C. E., Editor. — Handbook of Microscopical Technique. Thirty-four
contributors. Second edition revised. Paul B. Hoeber, Inc., New York,
1937, This work has proved of great value to workers in microscopy.

The Microscope. — The British Journal of Microscopy and Photomicrography.
Vols. 1-2, 1937-38. In 1939 the name changed to The Microscope and En-
tomologicel Monthly. London.

NEEDHAM, JAMES G., J. TRAVER, YiN-Cm Hsu, ANNA H. MORGAN, etc. — The
Biology of May Flies. 800 pages, 42 plates, 165 text figures and a colored
frontispiece. Comstock Publishing Co., Inc., Ithaca, N. Y., 1935. This
volume is of interest not only to entomologists, but to fishermen. Mayfly
larvae and pupae supply much food for fishes, and to the microscopist the book
suggests a world of interesting material for study.

POLICARD, A. — Precis d'Histologie Physiologique. 3d ed. entierement remanie'e.
895 pages, 350 figures. G. Doiri & Cie, editeurs, Paris, 1934. An excellent
work by the inventor of the micro-incinerator.

PRESTON, J. M. — Modern Textile Microscopy. 315 pages, 134 text figures.
Emmot & Co., Ltd., London, England.

RADLEY, J. A. and J. GRANT. — Fluorescence Analysis in Ultra- Violet Light.
Being vol. 7 of a series of monographs in applied chemistry, edited by E.
Howard Tripp. 219 pages, text figures, frontispiece and photomicrographs.
D. Van Nostrand Co., Inc., N. Y., 1933. A valuable book for those interested
in the effect of ultra-violet light in all fields. An excellent feature is reference
to original sources.

RASPAIL, FRANCOIS-VINCENT. — Nouveau Systeme de Chimie Organique Tond£ sur
des Methode Nouvelles D' Observation. Chez J. B. Bailliere, Libraire de
L' Academic Royale de MSdecine. 1833 Incineration, § 1390, p. 529.

Nouveau Systeme de Chimie Organique Fond£ sur des Nouvelles Methodes

D'Observation et Pr£ced6 d'un Traite" Complet de 1'Art d'Observer et de
Manipuler en Grand et en Petit dans le Laboratoire et sur le Porte-Objet du
Microscope. Deuxieme fidition Entierement Refondue, Accompagne"e d'un
Atlas in 4° de vingt Planches de Figures Dessinees d'Apres Nature et Gravies
avec le plus grand soin. 3 volumes.
Chez J. B. Bailliere, Paris 1838.

Incineration same as in frst edition, tome 3, pp. 610-11, § 4277.
Raspail has been called the father of micro-chemistry. His work certainly
gave the study a tremendous forward push. His discussion of the theory
and use of the microscope in tome i, pp. 174-281, is a masterpiece for the
time (1838).

INDEX

Abbe apertometer, 211-212

Abbe camera lucida, 320-324

Abbe condenser, 93-94

Abbe, importance of diffracted light in

microscopy, 218

Abbe, numerical aperture, 82, 209
Abbe, portrait, 55O~557
Abbe test-plate, 106
Abbe-Zeiss micro-spectroscope, 574
Aberration, chromatic, 203
Aberration, corrections for, 202-207
Aberration of cover-glass, no
Aberration of lenses, 198-199
Absolute alcohol, 441-442
Absolute index of refraction, 186-187
Absolute temperature, 56, 616
Absorbent gauze and lintless towels

for wiping slips, 406
Absorption bands, 222-236
Absorption spectra, Angstrom and

Stokes law, 228
Absorption spectrum, 226-239
Absorption spectrum of blood, 234-235
Absorption spectrum of colored bodies,

226-227

Absorption spectrum of didymium, 228
Absorption spectrum of terbium, 228
Absorption spectrum, permanganate,

234
Acetate of copper for pleochroism, 181-

182

Achromatic-aplanatic condenser, 95
Achromatic combinations, 205
Achromatic objectives, 21
Achromatism by different kinds of glass,

204
Achromatism, history of, 565-566

Acid balsam, 444

Adams, George, projection image, 286
Adjustable objectives, 22, 109-110, 199
Aerial image, demonstration, 68
Air and oil by reflected light, 266
Air bubbles and oil globules, 264
Airy, George B., astigmatism and cy-
lindrical spectacles, 558
Albumen fixative, 441
Alcohol, absolute, 441-442
Alcohol, denatured, 442
Alcohol, ethyl, 441-442
Alcohol lamp, 479
Alcohol, methyl, 442
Alcohol, normal propyl, 443
Alcohol, percentages, 440-441
Alhazen and the optics of the Arabs, 55 1
Ailing and Ward, book catalogue, 431
Aluminum for reflecting ultra-violet, 244
Aluminum vapor mirror, 242-243
American manufacturers of micro-
scopes, 42

Amici, immersion objectives, 567-568
Amici prism, 223
Ammonium sulphide for venous blood,

235, 239

Amplification, 282-283
Amplifier of Tolles, 291
Analyzer of polariscope, 170-1 71
Anchoring the cover-glass, 418
Angstrom unit (A) definition, 302
Angular aperture, 81, 208-221
Animal tissues with the polarizing mi-
croscope, 179-180
Aniso tropic objects, 172
Anthracene for fluorescence, 244
Aperture and diffracted light, 93
Aperture and intensity for micro-
incinerations, 536-538

595

596

INDEX

Aperture, angular and numerical, 81-

104, 208-221
Aperture, centering the condenser, 85-

Aperture, critical illumination, 99-100
Aperture, depth of focus, 218
Aperture, determination with a thick

glass plate, 213-215
Aperture diagrams, 83
Aperture, effect of opacities, 221
Aperture for dark-field microscopy,

132-143
Aperture from an immersion condenser,

90-91

Aperture from a white surface, 87-88
Aperture from the mirror, 88
Aperture from the sky, 81, 87
Aperture from translucent objects, from

a mirror, from a condenser, 89-90
Aperture of condenser and objective, 84
Aperture of objectives, table, 210
Aperture, Spencer and Tolles, 567-568
Aplanatic objectives, 21
Apochromatic objectives, 22
Apparatus for micro-incinerations, cost,

544-546
Appearances due to difference of focus,

273

Arc lamp for dark-field work, 145-146
Archer and Diamond, photography, 374
Aristotle, theory of vision, 551
Arrangement of serial sections, 501
Arranging diatoms, etc., 427
Artificial daylight, 55~56, 57 1
Artificial illumination, 54-55
Atkinson, photographing bacterial cul-
tures, 37°~37I

Autochrome color photography, 401
Avoidance of dense shadows in photog-
raphy, 367-368
Avoidance of distortion in camera lucida

drawings, 322-323

Avoidance of inversion in drawings, 514
Axial and oblique light with a conden-
ser, 78, 97-98
Axial or central light, 53
Axis, principal and secondary, 12, 195,
198

Back combination, 21
Bacon, Roger, 551-554
Baker, H., on projection, 574
Balsam, acid, 444
Balsam, Canada, 443
Balsam, filtering, 443
Balsam, neutral, 443
Balsam, xylene, 443
Barbaro, projection, concave and con-
vex spectacles, 555, 573
Bausch, Edward, 49
Bausch & Lomb's comparison ocular,

274
Bausch & Lomb's special dark-field oil

immersions, 135
Bausch & Lomb's student and research

microscopes, 36, 40
Bausch & Lomb's tube-length, 200
Beale, need of actual experiments, 2
Beale, objects for interpretation, 275
Beck, Conrad, 34-35, 92
Beck, Conrad, dark-field, 125
Beck, Conrad, fu'l aperture if proper

lighting, 104

Beck, Conrad, lighting, 100-102
Beck, lens holder for eyepoint, 85, 107-

108
Beck's focusing dark- field condenser for

different thickness of slips, 137-

138
Behrens, celluloid slips, 404

Bent-neck vials, 422
Bernhard's drawing board, 324
Bibliography for the whole book, 581-

5Q3

Binocular microscope, Ives form, 32
Binocular microscope, mon-objective

type of Riddell, 29

Binocular microscope, original form, 562
Binocular microscope, Wenham type,

29, 3*

Binocular microscopes, 28-42, 562-563
Binocular microscopes, advantages and

disadvantages, 30-32
Binocular microscopes, diverging and

parallel tubes, 34-35

INDEX

597

Binocular microscopes, experiments,
115-118

Binocular microscopes, Greenough type,
29

Binocular microscopes with unlikeness
of the observer's eyes, 117-118

Bleaching blue prints, 330

Bleile on use of pocket spectroscope, 234

Blood for Brownian movement, 273

Blood for dark-field, 159-163

Blood, spectrum, 234-235

Blood-dust, chylomicrons, 163

Blueprints, drawings on, 329-330

Bon ami for cleaning slips and cover-
glasses, 406-409

Borax carmine, 444

Born, wax models, 509-510

Bottles or jars for histology, 455

Boyer, diatoms, 427

Brain, the final organ of vision, 5, 10,

54Q
Brewster David, 92, 94, 100, 206, 565,

567-568

Brewster, David, condensers, 94, 565
Brewster, David, immersion objectives,

567-568

Brewster, David, lighting, 100
Brewster, David, optical qualities of

fluorite, 206

Bright- and dark-field microscope, 121
Bright-field microscope, 51
Brightness for best acuity, 101-102
Brislee on high aperture condensers for

dark-field, 136
Brown & Sharpe's micrometer calipers,

409
Brownian movement, how stopped, 270-

271
Brownian movement, pedesis, 161-162,

270-272

Brownian movement under the polar-
izing microscope, 271-272
Browning, the micro-spectroscope, 574

Cabinets for slides, 432-437
Calcite, Iceland spar, 1 70

Calipers for measuring the thickness of
slips and covers, 409

Camera for photo-micrography, 374-375

Camera lucida, Abbe's, 320

Camera lucida for drawing, 317-328

Camera lucida, Wollaston's, 288-289

Camera obscura, drawings with, 329

Canada balsam, 443

Cap, pinhole for centering, 86-87

Carbol-xylene clearer, 445

Carbon-monoxide hemoglobin, spec-
trum, 237

Cardioid dark-field condenser, 142

Care of the eyes in microscopy, 48

Care of the microscope, 46

Carmine, borax, 444

Carmine for mucus, 444-445

Carmine, spectrum of, 237

Carpenter, binoculars, 31

Carpenter, direct sunlight for dark-field
microscopy, I44~r45

Carpenter-Dallinger, 7, 92

Castor-xylene clarifier, 446, 487

Catalogues to guide in purchasing a mi-
croscope, 42

Cedar-wood oil, 445

Cells, isolation of, 422

Cells, mounting, 415-416

Cells, preparation of, 415-416

Center of lens, 12

Centering by the aid of a pinhole cap,
86-87

Centering dark-field condenser, i53-IS5

Centering, experiments, 86-87, 108

Centering light for the dark-field mi-
croscope, 154-155

Centering the microscope stage, 176

Centering the ocular, 108

Centering with nose-pieces, 108-109

Central, axial light, 53

Central stop, size of for dark-field, 120-
130

Chalet lamp, 58-59

Chalet lamp for demonstrations, 362

Chalet lamp in section, 58, 362

Chalet microscope lamp for dark-field,

150, 541
Chalet microscope lamp, new, 59

INDEX

Chamberlain's plant histology, 478
Chamot, E. M., comparison ocular, 274
Chamot, E. M,, glass slips for micro-
chemistry, and for polarized light,

403

Chamot, E. M., micrometry by the con-
denser image, 308-310

Chamot, E. M., usefulness of dark-
field microscopy, 156

Chamot, spectrum analysis, 23g

Chamot's wire gauze experiment, 275-
276

Chamot and Mason, for index of refrac-
tion by the use of liquids, 268

Changing alcohols, 492; objectives, 108;
dark to light field, 540-541

Chemical constituents of animals and
plants, 522-523

Cherubin d'Orleans binocular, 562

Chevalier, constructed Selligue's achro-
matic objectives, 566

Chloral hematoxylin, 454

Chloroform, 445

Choice of plates and color screens, 400

Chromatic aberration, 202-203

Chylomicron, derivation of the term,
162

Chylomicrons under the dark-field mi-
croscope, 152, 162, 164

Clarifier, castor xylene, 446

Cleaning diatoms, 427

Cleaning glass slips, 405-407

Cleaning mixture, dichromate, 411

Cleaning mixture for slips and cover-
glasses, 411

Cleaning slips and covers for dark-field
microscopy, 141, 525

Cleaning used slips or slides, 406

Cleaning with bon ami, 407-409, 525

Clearers, 445-446, 524

Clearing sections, 492

Coles, 92

Collateral reading for the different chap-
ters, 6c, 50, 58, 1 20, 168-169, 182,
221, 257-258, 278, 316, 363, 402,
463, 520, 546, 578-579

Collection of material from brooks,
425

Collodion, celloidin, parlodion, pyroxy-
lin, 446-447

Collodion, infiltration with, 484
Collodion parlodion, pyroxylin, 269
Collodion or parlodion sections, 483-488
Collodion sections, fastening to the

glass slide, 487
Collodionizing sections, 481
Colophonium for methylene blue stain,

45i
Color screens and plates in photography,

390-397, 400-401
Color screens, exposure with, 397
Combinations of objectives, 20-21
Combined dark- and bright-field con-
densers, 136-137, 1 68
Comparison of electron and projection

microscopes, 6-6&
Comparison oculars fo\^ interpretation,

273-274

Comparison spectrum, 23^
Compensating ray filters, 394 '
Compensation oculars, 25-26, 205-206
Complementary spectra, 228
Compound microscope, 4
Compound microscope, definition, 8
Compound microscope and parts, 17
Compound microscope, magnification,

definition of, 287
Compound microscopes, Dutch and

Keplerian forms, 18, 559-560
Comstock bent-neck vials, 422
Concave spectacles, 555
Condenser, aperture of, 214
Condenser, centering by eyepoint, 85
Condenser, corrections of, 93
Condenser, dark- and bright-field, 136,

168, 540
Condenser, dark-field, cardioid form,

151
Condenser, dark-field, centering, 140,

153-155
Condenser, dark-field, selection of, 166-

167

Condenser for student microscope, 95
Condenser, lighting entire field, 96
Condenser, mirror and light for, 96
Condenser, quartz bull's eye, 244

INDEX

599

Condenser, quartz for ultra-violet, 243
Condenser, why immersion necessary,

210-211, 539

Condensers, 80-104, 127-142, 166-167,
210-211, 243-244, 387, 531-533,
556

Condensers and dark-stops for micro-
incinerations, 53J-533
Condensers and mirrors, history of, 565
Condensers, dark-field, 127-140, 531-

533, 569

Condensers for high aperture in dark-
field work, 135-136; for drawing,
5i6

Condensers for photo-micrography, 387

Condensers, immersion for dark-field,
139-140, 539

Condensers, refracting for dark-field,
127-130, 531-540

Cones and rods of the retina, 280-281

Cones of light, solid and hollow, 536-538

Congo red, 447

Connective tissue stain, 456

Construction of real images, 14

Continuous spectrum, 225

Contrast ray filters, 394 t

Convex spectacles, 555, 573

Corex glass slips for ultra-violet work,
243, 246, 403-405

Corex slips for the polarizing and the
ultra-violet microscope, 178-179

Corning Glass Works, corex and ultra-
violet filters, 246-248, 403, 545

Correction of aberrations, 199, 202-
207

Corrections in achromatic and apochro-
matic objectives, 204-208

Correlation of aperture of objective and
condenser, 92

Cost of equipment for micro-incinera-
tion, 544-546

Counterstaining, 491-492

Cover-glass, aberration produced by, no

Cover-glass, anchoring, 418

Cover-glass, cleaning, 406-411

Cover-glass correction, 199

Cover-glass, determination of, thickness
of, 76-77, 410

Cox, J. D., homogeneous immersion ob-
jectives, 568
Cox, J. D., need of understanding the

microscope, 2-3
Critical angle and total reflection, 188-

189

Critical angle, definitions, 188-190
Critical illumination, 98-100
Crossed polarizer and analyzer, 171
Crystal violet for elastic tissue, 449
Currents in liquids under the micro-
scope, 269

Curve of eye sensitiveness, 392
Curve of ordinary plates, 393
Curve of orthochromatic plates, 393
Curve of panchromatic plates, 394
Curves to show ultra-violet transmis-
sion of filters, 248

Cusa, Cardinal, concave spectacles, 555
Cylindrical spectacles for astigmatism,
558

Daddi, use of Sudan for fat, 460
' Dark-field and bright-field microscope,

121

Dark-field and ultramicroscopy, 123-

124
Dark-field condenser, centering, 140,

I53-I55

Dark-field condensers, focusing for dif-
ferent slip thickness, 137-138

Dark-field condensers, refracting, para-
boloid, bispheric and cardioid, 134,
142, 151

Dark-field, determining thickness of slip
for, 141-142

Dark-field element for refracting con-
densers, 130-131, 532

Dark-field element for ultra-violet con-
densers, 243-246

Dark-field, focusing the microscope in,

Dark-field illumination, 127
Dark-field, lamp for, 145-150, 167, 541
Dark-field, light above the stage, 125-
126

6oo

INDEX

Dark-field, light below the stage, 126
Dark-field microscope, definition, 122
Dark-field microscope, experiments,

153-163

Dark-field microscope, history, 568-569
Dark-field microscope, interpretation

with, 272

Dark-field microscope for micro-in-
cinerations, 530-543
Dark-field microscope, troubles with,

163-166

Dark-field, mounting objects for, 156
Dark-field, naked-eye-demonstration ,

"5
Dark-field objectives and oculars, 23,

132-135, 535
Dark-field, polarizing and ultra-violet

demonstrations in a dimly lighted

room, 361-362
Dark-field, resolution and visibility

with, 124-125
Dark- field, slips and covers for, 140-

141, 524-525

Dark-field, test slides for, 141
Dark-field, visibility and resolution,

124-125
Dark-field with condensers, 127-140,

131, 134, 530-532
Dark-stops for micro-incinerations, 531,

535
Davy and Wedgewood, photography,

372

Daylight, artificial, 55~56, 57 l
Daylight glass filter, 55-56
Daylight-lamp or lantern, 5, 57~58
Decalcifier, 447
Dehydration and clearing, 492
Demonstration for classes, 350-363
Demonstration oculars for two, 28
Demonstration room for polarizing, and
ultra-violet microscopes and dark-
field^ 361-362

Demonstration table, 360-361
Demonstrations with euscope, 354-355
Denatured alcohol, 442
Denmark or table black, 461-462
Deparamning, 480
Pepth of focus and aperture, 218

Descartes' condenser and microscopes,

93, 564-565
Descartes and Snell, index of refraction,

185

Developing light, 397-398
Diagrams by projection, 335
Diaphragm ocular, 24
Diaphragms, size and position, 54
Diaphragms, use in microscopy, 54, 531
Diatoms, collecting and cleaning, 426-

427

Dichroic and trichroic bodies, 181
Bichromate cleaning mixture, 41 1
Didymium, erbium and terbium, ab-
sorption spectra of, 228
Difference in the diameter of the field
and the magnification of different
objectives with the same ocular, 67
Differences in magnification with dif-
ferent objectives with the same
designation, 67
Differential staining, 489
Diffracted light, 93, 218-220, 536
Diffracted light and aperture, 93
Diffraction, definition, 194
Direct vision spectroscope, 224
Directions for adjustment of objectives,

Dispersion, 193
Dispersion, table of, 217
Dissociating liquids, 422-424, 447-448
Distance of distinct vision, 282
Distance, working, with a microscope,

7i-77

Distinctness of outline, 266-269
Distortion, avoidance in drawing, 319
Dollond, achromatic instruments, 566
Donne & Foucault, photography, 374
Double imbedding in collodion and

paraffin, 488
Double microscope, 559
Doubly contoured, 268
Drawing board, Bernhard's, 324
Drawings, avoidance of inversion, 514
Drawings by the aid of a camera, 329
Drawings, determination of their mag-
nification, 315
Drawings for models, 514

INDEX

60 1

Drawings for publication, 340-345
Drawings, lettering, 343~344
Drawings, magnification of, 315
Drawings on photographs, 329-334
Drawings, reduction in engraving, 343
Drawings, scale of, 327
Drawings, size of, 342
Drawings with a camera lucida, "317-

328
Drawings with the microscope, and the

projection microscope, 317-350,

336-342, 515-517
Dry mounting, 414
Dry objectives, 21
Drying oven, 508
Drying spread sections, 480, 525
Dumond, M. A., 158
Dust, removal from lenses, 41
Dutch binoculars (opera glasses), 563
Dutch compound microscope, 559-560
D wight, papier machc models, 511

Kberbach, slide cabinet and trays, 437
Edinger's ocular for two observers, 564
Edmunds, blood under dark-field, 159,

167 '
Edmunds, direct sunlight for dark-field

microscopy, 144-145
Eikonometer of Sir A. E. Wright, 307
Elastic stain, hematoxylin and mucicar-

mine, 493

Elastic stains, 448-450
Electric furnace for micro-incineration,

522, 526
Electric oven and spreading plate, 473-

475
Electrification of paraffin ribbons, 477-

478

Electro-magnets, 6a
Electronic waves, 6a
Electron microscope, 6-6b
Emerton, paper models, 511
Enlarging small negatives, 388-389
Eosin, 450

Eosin in the clearer, 492
Eosin methylene blue, 450, 494

Equivalent focus, 18
Erbium didymium and terbium, spec-
tra, 228

Erect and inverted images, 71
Erect images, how to procure with the

microscope, 349

Erect images in drawing, 348-349
Erythrocytes under the dark-field mi-
croscope, 122, 161-162
Ether, ether-alcohol, 451
Euscope, 32, 354

Evaporated Films Co., for aluminum-
vapor mirrors, 244
Experiments, aid in understanding

principles, 3

Experiments in centering, 86-87
Experiments in interpretation of ap-
pearances, 259-278

Experiments in lighting and focusing, 59
Experiments in photo-micrography,

380-399

Experiments, need of, 2
Experiments with binoculars, 115-118
Experiments with condensers, 92
Experiments with fog and glare, 103
Experiments with objectives, 109-114
Experiments with simple and compound

microscopes, 59-81

Experiments with single- and with
double-objective binoculars, in,
117-118
Experiments with sky as light source, 81,

Experiments with the compound mi-
croscope, 61-77

Experiments with the micro-spectro-
scope, 233-239

Experiments with the polarizing micro-
scope, 177-182

Experiments with the ultra-violet mi-
croscope, 249-256

Exposure with color screens, 397

Extraordinary ray of polarizer, 171

Eye, makers of, 550

Eye, nodal point or center, 281

Eyelens, 24

Eyepieces, 24

Eyepoint, definition, 70

6O2

INDEX

Eyepoint, demonstration of, 70
Eyes, care of, in microscopy, 48
Eye-shade to use with the microscope,

Farrant's solution, 451

Fastening the sections to the slide with

series, 498

Fibrin network under the dark-field mi-
croscope, 122, 161-164
1'ield, definition, and how obtained, 66
Field in the microscope, diagram, 65
Field lens, 24

Field, lighting entire, 96-97
Field of dark-field microscope, 152
Filar ocular micrometer, 300-301
Filters for ultra-violet, 247-248
Filters or screens in photography, 390-

397

Fish, P. A., Sudan, table black, etc.,
461-462

Fixation for micro-incineration, 523, 528

Fixation of tissues, 464

Fixing for series, 498

Flemming's fluid, 451

Fluorescence, 241, 544

Fluorescence discovered by David
Brewster, 576

Fluorescence microscope, 241

Fluorescence of Stokes, 576

Fluorescence, spectroscope to analyze,
249

Fluorite for objectives, 205-206, 567

Fluorite objectives, 22, 567

Focus, equivalent, 18

Focus, principal, definition, 12, 13, 198
real and virtual, 194

Focusing dark-field condensers, Beck
and Zeiss forms, 137-138

Focusing experiments, 62-65

Focusing glass, 330, 368-369

Focusing in photo-micrography, 380

Focusing stand for photography, 330

Focusing stand for vertical camera, 368

Focusing the objective with the micro-
spectroscope, 232-233

Focusing the slit of the micro-spectro-
scope, 229-230

Focusing when objects scattered, 64

Focusing with the dark-field, 155

Focusing with parfocal oculars and ob-
jectives, 64-65

Fog and glare, 102-103

Foot candies and candle meters for
microscopic study, 102

Formaldehyde, 451

Formaldehyde dissociator, 422-423, 448

Formalin, 451

Fraunhofer lines, 225-227

Free-hand drawing with a microscope,

3i7

Free-hand sectioning, 470
Freezing microtome, 471
Front combination, 21
Function of an objective, 66-67
Function of an ocular, 69-70

Gage, Henry Phelps, Daylight glass,

58
Determination of aperture by the aid

of thick glass, 212-213
Method of getting the magnification
of the objective and of the ocular
by one process, 315
Quartz mirror for ultra-violet, 244
Water-cell with heat-absorbing win-
dows, 147, 358

Gage, Susanna Phelps, blotting paper
in place of wax for models, 51 1-514
Branched muscle fibers, 431
Compartments for slide trays, 435
Sudan for eggs and chicks, 461
Gage and Fish, Sudan for fat, 461
Galen, perfection of the eye, 550

vision and the chiasma, 549
Galileo, portrait, 556-557
Gauze for cleaning slips and cover-
glasses, 406-407
General or counter stains, 489
Geometrical construction of images, 13
Glare and fog with the microscope, 102-
103

INDEX

603

Glass, ground or frosted, how to pre-
pare, 68

Glass rods in liquids, 267-268

Glass slips, cleaning, 405-40?

Glass slips for incinerations, 524-525

Glycerin, glycerol, 452

Glycerin jelly, 452~453

Glycerol, glycerin, 452

Glycogen, iodin stain for, 455, 494-495

Green man, slide trays with compart-
ments, 435-436

Ground glass, how to prepare, 68

Ground glass with clear center, 369

Guide for mounting, 417

Gulliver's molecular base of the chyle,
chylomicrons, 163

Haemin for pleochroism, 181-182

Hard tissue, Land's method of soften-
ing, 478

llarting's microscope for two or more
observers, 28, 563

Heat-absorbing glass for water cell, 357

Heating plate, 479

Hclly's fluid, 462

Hematein, 454

Hematoxylin, chloral, 454

Hematoxylin, picro fuchsin, 493

Hematoxylin staining, 490

Hemoglobin, carbon-monoxide spec-
trum, 237
spectrum of, 236

Herschel, discovered infra-red, 576

High objectives, 23

Highly refractive, definition, 268

Hippocrates, on the brain, 548

Histology, physiological, 431

History of lenses and microscopes, 547-

57Q

Hitchcock, R., clarifying shellac, 460
Hodge, change in nerve cells, 431
Hollow light cones, 536-538
Homal lenses for photo-micrography,

378

Homogeneous immersion of condenser
and slide, 210

Homogeneous immersion objectives for

dark-field work, 21, 135, 544
Hones and honing, 469-470
Hood over the objective for projection

microscope, 360
Hooke, Robert, seeing two objects as

two, 279

Hovestadt, Jena glass, 567
HowelPs physiology for eye, 282
Huggings and micro-spectroscope, 574
Huygenian ocular showing ordinary and

compensation action, 25-26, 207
Huygens, 26, 556-55 7, 564

Illuminating objectives, 23
Illumination, artificial, 54
Illumination, critical, 98-100
Illumination for dark-field, 127
Image, aerial, how to demonstrate, 68
Image and object, relative position, 15
Image in the microscope or on the draw-
ing surface, 321
Image, real, 4, 8
Image, retinal, 4, 10
Image, swaying of, 98
Image, virtual, 8; and real, 8, 195
Image, virtual, construction, 14
Images, erect and inverted with a mi-
croscope, 71

Images, erect in drawings, 348
Images formed by lenses, 194
Images, geometrical construction of, 13
Images, real by projection, 5, 286
Images, real, object near and far from

the focus, 15

Images, refraction and color, 1 1 2-1 14
Imbedding box, 475
Imbedding for incineration, 523
Imbedding in collodion, 485
Imbedding in paraffin, 473-474, 523
Immersion condensers for dark-field

microscopy, 139

Immersion media for ultra-violet, 246
Immersion objectives, 21, 567-568
Incinerated and stained tissue com-
pared, 534

604

INDEX

Incineration, heat and time required,

527-528
Incinerations, microscopic study, 530-

544

Incinerations with bright- and with
dark-field, 534

Independent magnification of objectives
and oculars, 312-315

Index of refraction, 185-187

Index of refraction, absolute, 186

Index of refraction and dispersion,
table, 217

Index of refraction and wave length,
190

Index of refraction of air, water and
homogeneous immersion oil, 210

Indicator to aid in focusing the dark-
field objective, 155

Infiltrating box, 473-475

Infiltration with collodion, 484

Infiltration with paraffin, 472-473, 523

Infra-red light waves, 6

Infra-red radiation, discovered by
Herschel, 183, 222, 576

Infusoria, cultures of, 425
for dark-field, 159

Initial magnification with 250 and 160
tube-length, 19

Intensity of light and aperture of ob-
jective for incinerations, 537-541

Intermediate combination, 20-21

Interpolation with sines, 616

Interpretation, collateral reading for,
277-278

Interpretation, objects for, 275

Interpretation, summary of require-
ments, 277

Interval timer for micro-incineration,

527

In toto staining, 498
Inversion, avoidance in drawings, 346-

349, 5H

Inversion of microscopic image, 276
Invisible and visible radiation, 183
lodin in alcohol, 456
lodin stain for glycogen, 455, 494~495
Iris diaphragm in objective for micro-
incinerations, 535, 544

Iris diaphragm under condenser, 90-91,

536, 542

Iron hematoxylin, 454
Irrigating a specimen, 418
Isolation by formaldehyde, 422-423
Isolation of cells, 422
Isolation of muscle fibers, 424
Isolation of structural units, 422-424
Iso tropic objects, 172
Ives, Frederic E., binocular micro-
scopes, 32, 563

mon-objective binocular, 32-34
Ives, Herbert E., artificial daylight, 58

Jansen, Dutch microscope and tele-
scope; 556-557, 559"56o, 574
Japanese filter paper, lens paper, 39

Kepler, drawing by projection, 572-574
Kepler, microscope and telescope, 556-
557, 559-560

Labeling and cataloguing, 428-431
Labeling serial sections, 507
Laboratory lockers, 438
Laboratory stools and tables, 49, 80
Lagrange disc, eyepoint, 70
Lamp, alcohol, for heating, 479
Lamp, for dark-field work, 144-150,

541-543

Lamp for demonstration, 362
Lamp for photo-micrography, 376-377
Lamp for the microscope, 57-58, 145-

150, 541-545

Lamp for ultra-violet, 247-249
Lamp, 6- volt for dark-field, 146-150,

167, 541-545
Lamp-black for ingestion of leucocytes,

456

Land's method of sectioning hard

tissue, 478
Large covers, cleaning, 409

INDEX

605

Lateral swaying of image, 98

Leitz combined dark- and bright-field
condenser, 137

Leitz, tube-length, 170 mm., 19

Lens, definition, n, 194

Lens holder for eyepoint, 85

Lens holder with joints and adjust-
ments, 17

Lens paper for cleaning lenses, 47

Lens, parallelizing, 145, 148

Lens, principal focus on each side, 13

Lens, reducing, 196

Lenses, aberration of, 198-199

Lenses and images, 194

Lenses and microscopes, history, 547-

579

Lenses, concave and convex, 194
Lenses, converging, 197
Lenses, diagrams of different forms, 197
Lenses, diverging, 197
Lenses, removal of balsam, etc., 48
Lenses, removal of dust, etc., 47
Lenses, spherical, forms of, 195-107
Lettering drawings, 343~344
Leucocytes under the dark-field micro-
scope, 122, 161-164
Ligamentum nuchae, 450
Light, artificial, experiments, 79
Light, axial and oblique, 78
Light, axial and oblique with a con-
denser, 97-98, 536
Light, axial or central, 53
Light, characteristics of, 182-184
Light, diffracted, and aperture, 93
Light for dark-field microscopy, 131-

147, 162-163, 530-543
Light for photography, 247, 378
Light in the work room for dark-field

microscopy, 123

for ultra-violet microscopy, 247-249
Light, incident or direct, 52
Light, oblique, 53
Light, quality and amount, 5-6
Light, reflected, 52
Light, source and character, 100
Light, transmitted, 52
Light-excluding sleeve, 382-383
Lighting a simple microscope, 60

Lighting and lamps for dark-field mi-
croscopy, 144-150* 541-543
Lighting experiments, 77
Lighting field with condenser, 96
Lighting for photography, 366-367
Lighting for the spectroscope, 232
Lighting opaque objects, 23, 52, 60
Lighting the mercury lamp, 248-249
Lighting translucent objects, 60
Lighting with* daylight, 51
Line drawings on the back of photo-
graphs, 331-334
Line spectrum, 225
Lister, dark-field, 569
Lockers for laboratory, 438
Low objectives, 23
Luckiesh, M., artificial daylight, 58

MacMunn, on spectra, 239

Maddox, R. L., gelatin dry plates, 374

Magnetic condenser, 6b

Magnetic objective, 6b

Magnetic projector, 6b

Magnification and micrometry, 279-

316

Magnification and principal focus, 292-

293

Magnification by projection, 314-315
Magnification, definition, 282-283
Magnification distance, 291-292
Magnification expressed in diameters, or

times linear, 283
Magnification in photo-micrography,

384
Magnification of compound microscope,

287-291

Magnification of drawings, 315
Magnification of ocular and objective,

312-315

Magnification of real images, 283
Magnification of simple microscope,

285-286

Magnification of virtual images, 282
Magnification rod for vertical camera,

33o> 3^6
Magnification table, 294-295

6o6

INDEX

Magnification, varying, 290-291

Mall, F. P., wax models, 510

Mallory and Wright's connective tissue

stain, 450, 456

Malus and polarized light, 575
Manuscript for publication, 341-342
Mark, E. L., cutting out the wax

plates, 510

Mayall, on homogeneous immersion, 568
Measuring slips and covers, 409
Mega-Microscopes, 46
Mercer, A. C, photography, nomencla-
ture, 373
Mercuric chloric!, 457

eliminating, 498, 529
Mercury arc, source of ultra-violet, 24 1
Met-hemoglobin spectrum, 236-237
Method of double vision, 285, 549
Methyl alcohol, 442
Methylated spirits, 442
Methylene blue, alkaline, 457
Mcthylene blue and eosin stain, 450
Metric scale, metric system, 328, 616
Micro-incineration, 521-546
Micrometer calipers, Brown & Sharpens,

Starrett's, 409-411
Micrometer, filar ocular, 300-301
Micrometer, ocular, 295, 297, 311
Micrometer ocular with movable scale,

298-301

Micrometer, stage or object, 287
Micrometer to indicate scale of drawing,

327

Micrometry, definition, 301
Micrometry and magnification, 279-316
Micrometry with compound microscope,

301-312

Micrometry with simple microscope, 301
Micrometry, unit of measure in, 302
Micro-millimeter, 302
Micron (/A), definition, 302
Micro-photography, photo-microgra-

phy, 373-374
Micro-polar iscope, 575
Micro-spectroscope, 222-239, 574-575
Micro-spe troscope, -adjustment, 229
Micro-spectroscope, experiments with,

229-231, 233-239

Micro-spectroscope, lighting for, 232
in photography, 396

Microscope, and projection apparatus,
554

Microscope and telescope, names, 560

Microscope, binocular, 28-42, 562-563

Microscope, binocular, advantages and
disadvantages, 32

Microscope, binocular and two objec-
tives, 29

Microscope, binocular, modern form by
Tves, 32-34

Microscope, binocular, parallel and
converging tubes, 34-35

Microscope, bright- field, 51

Microscope, care of, 46

Microscope, combination monocular
and binocular, 37

Microscope, compound, 4, 8

Microscope, definition, 7

Microscope, Dutch compound, 559-560

Microscope, experiments with, 6t

Microscope field, 60, 66

Microscope for two or more observers,

563-5^4 m

Microscope, history of, 547-579
Microscope, Keplerian, 18, 559-560
Microscope, laboratory forms, 36-37
Microscope, laboratory compound, with

parts named, 27

Microscope lamp, 50, 55-59, 541-543
Microscope, magnification of, 282
Microscope, micrometry with, 301-312
Microscope, name given by Faber, 7
Microscope, parts of, 16, 27
Microscope, polarizing, 170-182
Microscope, projection, 4-5
Microscope, projection for class demon-
strations, 353
Microscope, projection for drawing and

demonstration, 336-342
Microscope, research forms, 39-41, 46
Microscope, simple, 8, 553*554, 558
Microscope, simple, as aid to eye, 3
Microscope, testing of, 118—120
Microscope, ultra-violet, 240-258
Microscope, ultra-violet, dark-field,
243-258, 530

INDEX

607

Microscope, with a polariscope, 170
Microscopic preparations, cabinets for,

432-437

Microscopic specimens, mounting, 412
Microtomes and section knives, 466-

468

Middle combination, 21
Milliet de Chales, 572
Milli-micron (mju), definition, 302
Milli-millimeter, 302
Mineral matter in animal and plant

tissue, 522, 530
Mineral oil, medicinal, as a mounting

medium for ultra-violet, 421, 457
Minot, C. S., 431

Minot slide cabinet and trays, 436-437
Mirror, aluminum vapor, 243
Mirror and condenser, 565
Mirror, use of plane and concave, 78
Mixtures of alcohol and water, 440-441
Models, drawings for, 514
Models from series, 507-520
Models, size of, 512
Moist chamber, 424
Moist preparations, 424
Moitessier on photo-micrography, 375
Monazite, spectrum of, 238-239
Monochromatic light, 222
Monocular-binocular arrangement, 118
Monocular microscope, 32
Moore, Dr. V. A., haemospast, 161
Moore laboratory desk, 50
Mounting and staining sections, 489-

495

Mounting cells, 415
Mounting dry or in air, 414
Mounting fluid preparations for the

dark-field, 156-157
Mounting guide, 417
Mounting in cells, 415
Mounting in glycerin, 416-417
Mounting in glycerin jelly, 417-418
Mounting in media miscible with water,

416-419
Mounting in petrolatum for ultra-violet,

42 1 ; and for incinerations, 5 29-530
Mounting in resinous media, 419-421
Mounting material for the polarizing

and the ultra-violet microscope,

179
Mounting media for ultra-violet, 179,

246, 256
Mounting microscopic specimens, 412-

421

Mounting serial sections, 502
Mounting, temporary and permanent,

412-453

Movement of specimen under an erect-
ing binocular, 116-117

Mt. Holyoke College, cleaning slips and
cover-glasses, 409

Mucicarmine, 444-445

Muller's fluid dissociator, 448, 457

Muscat volitantes in the eyes, 274-

275
Muscular fibers, isolation of, 424

Nachet, multiple oculars, 28

Nachet's microscope for two or more
observers, 563

Natural sines, table and interpolation,
616-617

Negative oculars, 24

Negatives, storing, 371

Nelson, E. M., best aperture in histol-
ogy, 104

Nelson, E. M., origin of the Royal
Micr. Soc.'s screw, 38-39

Nelson, E. M., on parfocal oculars, 64

Nelson's method of magnification, 314

Neutral balsam, 444

Neutral red, 426, 457-458

Newton and chromatic aberration, 203

Nichols, E. L., 223

Nichols, E. L., fluorescence of uranyl
salts, 249; cathode luminescence,

257
Nichols, Southall and Watson, index of

refraction, wave length, and speed,

192

Nicol prism, 171, 575
Nitric acid decalcifier, 447, 458
Nitric acid dissociator for muscle, 448
Nitric acid for muscle dissociation, 424

6o8

INDEX

Nodal point or optic center of eye, 10,
281

Non-adjustable objectives, 22

Normal liquids, 458

Normal spectrum, 225

Nose-piece for changing objectives, 61-
62

Numbering serial sections, 497, 507

Numerical aperture, 82-104, 208-221

Numerical aperture, determination of
in objectives and condensers, 211-
215

Numerical aperture of dark-field con-
densers, 134

Numerical aperture for micro-incinera-
tions, 535

Numerical aperture, significance of,
209-210

Numerical aperture, table of, 210

Object, putting under the microscope,

Objective, 17
Objective, achromatic, 21
Objective, adjustable, 22, 109-110
Objective, adjustable for cover-glass,

199

Objective, aperture of, 208-221
Objective and aperture of condenser, 92
Objective and ocular for dark-field,

132-135

Objective and ocular for photography,
377-378

Objective, aplanatic, 21

Objective, apochromatic, 22

Objective, combinations in, 20, 52-53

Objective for the micro-spectroscope,
232

Objective, magnetic, 6b

Objectives, adjustable for cover-glass,
22-23

Objectives, and centering when changed,
108-109

Objectives and dark-stops for micro-
incinerations, 531, 535

Objectives, changers for, 109

Objectives, cover-glass thickness for,

199-200
Objectives, dark-field, 23, 132, 135, 155,

535
Objectives, different colored mounts,

Objectives, dry, 21

Objectives, fluorite, 22

Objectives for the polarizing micro-
scope, 175

Objectives for research, 104-105

Objectives for ultra-violet, 23

Objectives, function of, 66-67

Objectives, high, 20, 23

Objectives, homogeneous immersion, 2 1

Objectives, illuminating, 23

Objectives, immersion, 53

Objectives, independent magnification
of, 19-20, 312-315

Objectives, kinds of, 21

Objectives, low, 20, 23

Objectives, magnifying power, 19-20,

. 312-315

Objectives, name of combinations, 20 -
21

Objectives, oil immersion, 21

Objectives, oil immersion for dark-field
work, 135

Objectives, optical designation, 18

Objectives, parfocal, 64-65

Objectives, putting in position, 61

Objectives, removing oil from, 114

Objectives, special oil immersions for
dark- field work, 135

Objectives, unadjustable, 22

Objectives, variable, 22

Objectives, water immersion, 21

Objectives with iris diaphragm for
dark-field, 531-535

Oblique and axial light with a con-
denser, 97-98

Oblique light, 53, 78

Ocular micrometer valuation, 299

Oculars, 24

Oculars and spectacles, 107-108, 570

Oculars, centering, 108

Oculars, compensation, 25-26, 205-206

Oculars, demonstration, 28

INDEX

609

Oculars, designation, 28

Oculars, diaphragm in, 24

Oculars, for binocular microscopes, 29

Oculars for binocular microscopes, ex-
periments with unlike, 118

Oculars for dark-field, 132, 135

Oculars for demonstrations, 352

Oculars for two observers, 563-564

Oculars, function of, 69-70

Oculars, history of, 564

Oculars, Huygenian, 25-26, 556-557

Oculars, inclined, 19, 242

Oculars, independent magnification of,
312-315

Oculars, micrometer, definition, 295

Oculars, micrometer valuation, 296-301

Oculars, micrometer valuation with
movable scale, 290-301

Oculars, negative, 24

Oculars, parfocal, 64

Oculars, pointer in, 39

Oculars, positive, 24

Oculars, projection, 6b, 27

Oculars, projection for photo-microg-
raphy, 386

Oculars, Ramsden's, 24, 26

Oculars, telaugic and spectacles, 27,
107-108

Oculars to use in research, 106-107

Oculars, trade names for, 27

Oculars, ultra-violet, 27

Oil and air by reflected light, 266

Oil globules and air bubbles, 264

Oil immersion objectives, 21

Opaque and transparent objects with
binoculars, 116

Opera glasses, Dutch form, 28

Optic center of a lens, 195-197

Optical center, definition, 12

Optical designation of objectives, 18

Optical glass, 42

Optical instruments, two groups, 554

Optical parts of microscope, 1 7

Optical path, method of shortening,

Optical principles in microscopy, 182
Optical section, 269
Orcein for elastic tissue, 448

Ordinary ray of polarizer, 171

Orientation for series, 500

Orndorff, W. R., 447

Ott, H. N., on condensers, 95

Oven, drying for series, 508

Over and under correction of aberration,

199
Oxyhemoglobin spectrum, 236

Paper box for imbedding, 475
Paraboloid dark-field condenser, 531
Paraffin method, 471-483
Paraffin method for incinerations, 523-

525
Paraffin method with propyi alcohol,

482-483

Paraffin ribbons, storing, 478
Paraffin wax, 458
Parallel beams, simple and compound

microscope, diagram of, 294
Parfocal oculars and objectives, 64-

Pedesis, Brownian movement, 270-272
Pennock, Edward, parfocal oculars, 64
Pennock, Edward, thickness of cover-
glasses of the same number, 409-

410

Percentage of solutions, 439-441
Permanent mounting of specimens, 413—

453
Permanent preparations of isolated

cells, 423-424

Permanganate, spectrum, 234
Petrolatum mineral oil, 457
Photographing bacterial cultures, 370
Photographing embryos, etc., 367-370
Photographing with an ocular, 383,

387
Photographing without an ocular, 382,

387
Photographs, drawing on the back,

329-334
Photographs, retouching for half-tones,

334

Photography, 364-400
Photography, color, 400-401

6io

INDEX

Photography with ultra-violet, 401, 577
Photography with vertical camera, 364-

365

Photo-micrographs, method of getting
any desired magnification, 384

Photo-micrography, micro-photogra-
phy, distinction, 373-374

Physical analysis, 177

Physical analysis for interpretation,
276-277

Physical analysis with the ultra-violet
microscope, 241

Physiological histology, 431

Picric alcohol, 459

Picro-fuchsin, 459

Picro-fuchsin and hematoxylin, 493

Pillsbury, W. B., position of the image
in drawing, 321

Pinhole cap for centering, 86-87

Pinhole card, 10-11

Plant material with the polariscope,
I78-I7Q; with the ultra-violet
microscope, 255

Plates and color screens, choice of, 400

Pleochroism, pleochromatism, 171, 181

Pocket spectroscope, 234

Pohlman, A. G., wax models, 510

Pointer in the ocular, 39

Pointer ocular for demonstrations, 351

Polariscope, 170-182

Polarization of incinerations, 543-544

Polarizer, 170-171

"Polarizing microscope, 170-182

Polarizing microscope, history of, 575

Polarizing microscope of Chamot and
Mason, 173

Polarizing microscope, physical analysis
with, 177, 254

Polarizing microscope, testing the dif-
ferent elements, 172-175

Polarizing microscope with pedesis,
271-272

Polaroid for the microscope, 172

Policard's micro-incineration method,
522, 526

Portraits of Jansen, Kepler, Galileo,
Huygens, C. A. and H. R. Spencer,
Tolles, Wenham and Abbe, 556-557

Positive oculars, 24

Powell, made parfocal oculars, 64

Power, magnifying of microscopes, 282-

283

Preservation of incinerations, 529
Principal axis of a lens or lens system,

12, I95"i97

Principal focus, 12, iq8
Principles, need of understanding, 3
Prism, Amici, for spectroscope, 223
Prism, half-reflecting, 33"34
Prism, Nicol, for polariscope, 171,

575

Prismatic spectrum, 225

Production of color with the polarizer,
182

Projection and real images, 571

Projection, condensers for, 357-359

Projection for diagrams, 335

Projection method for determining
magnification, 314-315

Projection microscope, 4-5

Projection microscope for demonstra-
tions, 353-356

Projection microscope for drawings, 336

Projection ocular, 6h, 27

Projection oculars for photomicrogra-
phy, 386

Projection table, 517

Propyl alcohol, 443

Propyl alcohol for paraffin method,
482-483

Ptolenwis, 550-55*

Publication, preparation of manuscript
for, Wistar Institute and Trelease
and Yule, 341-342

Pupil of the lens, eyepoint, 70

Pupillary separation, 116

Putting an object under the micro-
scope, 62

Pyrex microscope slides for micro-incin-
eration, 525

Quartz aluminum-vapor mirror, 242-

244, 532
Quartz hull's-eye condenser. 244

INDEX

611

Quartz dark-field condenser, 243
Quartz dark-field element, 245
Quartz prism for mirror, 242-243
Quartz slips, 403
Quartz slips for ultra-violet microscopy,

178
Quekett, direct sunlight for dark-field

work, 144-145

Radiation, diagram of, 183

Radiation, ultra-violet and infra-red,
uses in microscopy, 5-6

Radiation, visible and invisible, 183, 222

Ramsden disc, eyepoint, 70

Ramsden ocular, 26

Ranvier, pedesis of frog crystals, 271-
272

Raspail's incinerations, 522

Razor blades for sectioning, 478, 524

Reade, dark-field, 569

Reading, collateral, 6c, 50, 58, 120, 168-
169, 182, 221, 257-258, 278, 316,
363, 402, 463, 520, 546, 578-579

Reading glass, 8

Reagents for microscopy, 439-462

Real and virtual images, 24, 195

Real image, definition, 8

Real image, how to demonstrate, 67-
68

Real images by projection, 5, 286, 571

Real images, construction, 14

Real images with object near and far
from focus, 290-293

Redi manuscript on spectacles, 555

Reducing diaphragm for dark-field ob-
jectives, 132, 535/544

Reduction in engraving, 343~345

Reede, photography, 373

Reflecting dark-field condensers, 132

Reflection, regular or mirror and irregu-
lar, 184-185

Reflection, total and critical angle, 188-
190

Refracting condensers for bright- and
dark-field, 89, 106, 127-132, 530-
532

Refraction, 12, 185-193
Refraction and Ptolemaeus, 550
Refraction images, 113
Refraction, index of, 185-187
Refraction, Snell and Descartes law,

185-186
Refractometer tests of liquids and

solids, 215-217

Reichert's combined bright- and dark-
field condenser, 137

Relative position of object and image, 15
Removal of mercuric chlorid from sec-
tions, 498, 529

Research microscopes, 40-41, 44-45
Research, objectives for, 104-105
Research, oculars for, 106-107
Resinous media, mounting in, 419-421
Resolution and visibility, 279, 537
Resolution and visibility with dark-field,

124-125, 537-539

Resolution, visual angle for, 279, 282
Retina, rods and cones of, 280-281
Retinal image with the microscope, 4
Retinal sensory receptors, 279
Riddell's binocular, 29
Riddle, Dr. Oscar, and Sudan, 461
Ritter, J. W., discovered ultra-violet,

576

Rods and cones of retina, 280-281
Roger Bacon, and inversion of the

retinal image, 553
Roger Bacon and transformation of

energy, light becomes heat, 554
Roger Bacon, spectacles, 553
Rogers, W. A., 49
Rogers, W. A., limit of accuracy in

micrometry, 311

Royal Micr. Soc. standards, 38-39
Rule of thumb, 1-3
Rumsey, W. E., avoidance of dense

shadows in photography, 367
Rutherford on Brownian movement,

271

Sabine, G. B., efficiency of aluminum-
vapor mirror on quartz, for ultra-
violet reflection, 244

612

INDEX

Safety razor blades for sectioning, 468

Sagittal sections, 506

Saliva for Brownian movement, 273

Scale of drawings, how to indicate, 327

Scale of photographs, 365-366

Scale of wave lengths in spectroscope,

230

Scalpels for ribbons, 476
Scheiner and projection, 572
Scheiner, Christopher, vision and the

Keplerian microscope, 561-562
Scott, G. H., 522

Scott-Policard micro-incinerator, 526
Screen for microscope and eyes, 5 1
Screens for photography, 392-395
Sealing incinerations, 529
Sealing the cover-glass, 416, 418-419
Secondary axis, 12, 198
Section knives, sharpening, 469-470
Section lifter, 485
Sectioning by collodion method, 485-

488; paraffin method, 470, 476
Sectioning, freehand, 470
Sectioning with a microtome, 470-477
Sections for incineration, 524
Sections with freezing microtome, 471
Sections, serial, 496-509
Selligue, achromatic, microscopic objec-
tives, 566

Seneca, and water flask, 548
Sensory epithelium of retina, 279
Sensory receptors of retina, 279
Serial sections, 496-509
Serial sections, baskets for holding

slides, 481

Serial sections, labeling, 507
Serial sections, mounting on a glass slip,

502

Serial sections, order on the slide, 501
Serial sections, thickness of, 501
Series for small animals, 499
Series, modeling, 507-520
Series, staining, 499; mounting, 502
Shadboldt, direct sunlight for dark-field,

144-145

Shadboldt, G., photography, 373
Shading the microscope stage, 1 14
Shadows, avoidance in photography, 367

Sharpening section knives, 469-470
Shell vials, 422-423
Shellac cement, 459

Sheridan's crystal violet for elastic tis-
sue, 449
Sheridan, propyl alcohol for paraffin

method, 482-483
Significance of numerical aperture, 209-

210, 537

Silvering tissue, 460
Simple microscope, 3
Simple microscope, definition, 8
Simple microscope, history, 558
Simple microscopes, mounting of, 16-17
Sine law and velocity, 191-193
Sines, natural and interpolation with,

616-617

Single microscope, 559
Single vision, Galen, 549
Six-volt lamp for drawings, 514
Size of drawings for the engraver, 342
Size of paper models, 512
Size of slip and cover for series, 502-503
Size of the microscope field, 60-67
Sky as light source, 81, 87
Slide baskets for serial sections, 481
Slide tray for dark-field preparations,

1 60

Slide trays, 434-435
Slides and covers for series, 502-503
Slides, cabinets for, 432-437
Slides or slips for microscopy, 403-411
Slides or slips, thickness of, 405
Slips and covers for dark- field, 140
Slips for mounting, cleaning, 405-407
Slips of quartz and corex for ultra-violet

work, 243-246
Slips or glass slides in microscopy, 403-

4". 525

Slips, thickness for dark-field, 1 28
Smith, Theobald, dedication of thh

book to, iii
Snell and Descartes, index of refraction,

i8S

Society screw for objectives, 38-39
Sodium, incandescent, spectrum, 227
Solar spectrum, 226-227
Solid and hollow light cones, 536-538

INDEX

613

Solutions, percentage of, 439-441
Solutions, saturated, 439
Sorby and micro-spectroscope, 574
Source of light, need of adequate, 5
Southall, 192

Spectacles and oculars, 570
Spectacles, Barbaro, concave and con-
vex, 573

Spectacles, concave, Cardinal Cusa, 555
Spectacles, convex, Roger Bacon, 553
Spectacles, cylindrical for astigmatism,

55«
Spectacles, explanation of their aid,

Kepler, 561
Spectacles, first reference in literature,

Redi manuscript, 555
Spectra, complementary, 228
Spectra, law of color, 228
Spectra of minerals, 238-239
Spectroscope, direct vision, 224
Spectroscope with fluorescence, 249
Spectroscope with the microscope, 574-

575

Spectrum, absorption, 226-239
Spectrum, comparison, 230
Spectrum, definition, 225
Spectrum, line and absorption, 225-239
Spectrum, normal, 225; prismatic, 225
Spectrum of blood, 234-237
Spectrum of colored bodies, 237-238
Spectrum of colorless bodies, 238
Spectrum of met- hemoglobin, 227, 236
Spectrum of permanganate of potash,

227

Spectrum of sodium, 227
Spectrum, prismatic, 225
Spencer, Charles A., 556-557, 567
Spencer, Charles A., fluorite in objec-
tives, 206

Spencer, Herbert R., 556-557
Spencer and Tolles, aperture, 208-209
Spencer Lens Co., 42, 545
Spencer Lens Co.'s ocular for two ob-
servers, 564
Spencer Lens Co.'s oil immersion for

dark-field work, 135
Spencer Lens Co.'s research micro-
scopes, 41, 45

Spencer Lens Co.'s tube-length, 200

Spencer Lens Co.'s ultra-violet micro-
scope, 242-243

Spherical aberration, 198

Spirochaetes for dark-field, 157-158

Spirochaetes, preparation for the dark-
field. Stitt, Thro, Dumond, 157-
158

Spitta, photographing bacterial cultures
in test tubes, 371

Spot lens for dark-field, 127

Spreading paraffin sections, 479-480,

525

Stage micrometer, 287
Stage micrometer with ring on the lines

for locating them, 288
Staining and mounting sections, 489-

495

Staining for series, 499

Staining, general and differential, 489

Staining in toto, 498

Staining isolated cells, 423

Staining isolation preparations, 423-
424

Stains for elastic tissue, 450

Standard image distance for magnifica-
tion, 291-292

Standards, Royal Micr. Soc., 38-39

Starrett's micrometer calipers for meas-
uring slips and covers, 409

Stephanson, direct sunlight for dark-
field work, 144-145

Sternberg on photo-micrography, 374

Stitt's method of cleaning slips and
covers by the aid of bon ami, 141,
158, 405-409, 525

Stokes, G. G., and fluorescence, 576

Storing paraffin ribbons, 478

Strops for sharpening knives, 470

Substage condenser, optical corrections
of, 93

Sudan, 460-461

Sunlight for dark-field microscopy, 144-
150

Superstage dark-field condenser, 1*38-

139

Swift and the micro-spectroscope, 574
Swift's telaugic oculars, 107-108

6i4

INDEX

Table-black, Fish, 461-462
Table for micro-incineration, 528
Table for projection drawing, 338-339
Table for steps in paraffin method, 482
Table of centigrade and Fahrenheit, 616
Table of dry, water and homogeneous

immersion objectives of 0.50 N.A.,

210

Table of height of eyepoint, 108
Table of illumination, by Beck, 102
Table of magnifications, 294-295
Table of metric and English measures,

616

Table of negative record, 388
Table of numerical apertures, 84, 210
Table of objectives and apertures for

micro-incinerations, 535
fable of refractive indices from Chamot,

190
Table of steps in the collodion method,

488
Table of the usual group of objectives

for microscopic work with the N.A.,

210

Table showing cause of spherical aberra-
tion, and remedy, 201
Table showing index of refraction and

dispersion, 217

Table showing size of field, 67
Talbot, Henry Fox, applied- the polari-

scope to the microscope, 575
Telaugic oculars, 27, 107-108
Telescope and microscope, names, 560
Temporary mounting, 412
Test preparations for dark-field, 141
Testing for binocular vision, 115
Testing glass slips for the polarizing

microscope, 175-176
Testing the microscope, n8-r20
Testing the polarizing microscope, 172-

i7S

Theories of vision, 548
Thickness of blotting paper, 511
Thickness of cover-glasses, 410
Thickness of serial sections, 501
Thickness of slips, 405

Thickness of slips for dark-field con-
densers, 141-142
Thomas, Arthur H., 44
Thro, William C., 158
Time development of photographs,

399

Tissues, fixing of, 464
Tolles' amplifier, 291
Tolles, Robert, homogeneous immersion

objectives, 556, 568
Tolles and Spencer, aperture, 208-209
Transections, definition, 503
Transformer for 6-volt lamp, 514-515;

for dark-field lamp, 146-149, 545
Trays for microscopic preparations,

434-437

Trelease and Yule, guide for the prepa-
ration of manuscript, 342

Trichroic and bichroic bodies, 181

Troubles with the dark-field micro-
scope, 163-166

Tube-length of the microscope, 18, 200

Turn- table, 415

Tyndall effect and dark-field, 124

Ultra-violet, discovered by Ritter, 576
Ultra-violet filters, curves of transmis-
sion, 248

Ultra-violet, filter in carrier, 245, 247
Ultra-violet, immersion media for, 246
Ultra-violet in photography, 577
Ultra-violet microscope, 240-258
Ultra-violet microscope, arrangement of

parts, 245
Ultra-violet microscope, diagram, 242-

243
Ultra-violet, mounting media for, 246,

421

Ultra-violet objectives, 23
Ultra-violet ocular, 27
Ultra-violet radiation, 183, 222
Ultra-violet radiation, photography

with, 401, 577
Ultra-violet radiation, transmitting

slips, quartz and corex, 246
Ultra-violet reflectors, 242-245

INDEX

615

Ultra-violet screens or filters, 247-248
Ultra-violet, source of radiation, 241
Uniaxial and biaxial crystals, 1 70
Unit of measure in micrometry, 302
Unna's orcein stain, 448
Uranium or fluorescent canary glass

for showing light beams, 533, 536-

539, 545

Valuation of ocular micrometer with

movable scale, 299-301
Valuation of the ocular micrometer,

296-301

Variable objectives, 22
Varley, suggested parfocal oculars, 64
Vegetable material with the polarizing

microscope, 1 78- 1 79
Velocity and index of refraction, 192
Velocity under the microscope, 269-270
Verhoeff's elastic stain, 449
Vertical camera, 330-33 1
Vertical camera for photography, 364-

?65
Vertical projection microscope, 359-360

Virtual foci, 12

Virtual image, definition, 8

Virtual images, 14-15, 195

Virtual images, construction, 14

Visible and invisible radiation, 183

Visibility and resolution, 279, 537

Visibility and resolution with dark-field,

124-125

Vision, ancient theories, 548
Vision by aid of a magnifier, 9
Vision by unaided eye, 9
Visual and actinic foci, 378
Visual angle, Hooke's angle for resolu-
tion, 279

Visual angle required for resolution, 282
Visual angle, ways to increase, 7

Ward and the micro-spectroscope, 574

Washing boxes, 464-465

Water cell for absorbing heat, 357

Water cell with heat-absorbing glass for

dark-field work, 147
Watson & Sons, Cassegrain condenser
by E. M. Nelson for high aperture,
dark-field work, 136, 192
Wave-length designation, 231-232
Wax models, 509
Wedgewood & Davy, photography,

372

Weigert's elastic stain, 448-449
Wenham and direct sunlight, 144-

H5
Wenham, dark-field microscopes, 556-

557, 568-569

Wenham's binocular, 29, 31, 562
Wenham's directions for adjustment of

objectives, iio-m

White letters and figures for black draw-
ings, 346

Wilson, atlas of fertilization, 373
Wire gauze experiment for interpreta-
tion, 275-276

Wistar Institute publications, 341
W7istar Institute slide trays, 436
Wistar Institute, style brief, 341
Wolbach, S. B., resin for eosin methyl-

ene blue stain, 451
Wollaston's camera lucida, 188, 288-

289, 318-319, 573
"Woodward, Col., photo-micrography,

373

Working distance, 71-77
Working distance and cover-glass,

72-75
Working distance, determination of,

75-77

Workroom for photography, 375

Work-tables, 49

Wright, A. E., and diffracted light,
218, 221

Wright, A. E., and eyepoint, 85

Wright, A. E., lighting, 100, 103

Wright, A. E., need of understanding
principles, 3

Wright, A. E., specimen to show ap-
pearance due to focus, 273

Wright, Lewis, achromatic combina-
tions, 205

616 INDEX

Xylene, xylene balsam, 443 Zahn, 572

Xylol, xylene, 443 Zeiss combined bright- and dark-field

condenser, 137

v Zeiss light-excluding sleeve for the mi-

croscope, 382-383

Young, Thomas, astigmatism, 555 Zeiss's special oil immersions for dark-

field work, 135
Zenker's fluid, 462

Interpolation with Natural Sines: — If one cannot find a sine exactly corresponding with an
angle in the table, or an angle corresponding with a sine found in solving a problem, the sine or
angle can be closely approximated by the method of Interpolation: Find the sine in the table nearest
the sine whose angle is to be determined. Get the difference of the sines of the angles greater and
less than the sine whose angle is to be determined. That will give the increase of sine for that
region of the arc for 15 minutes. Divide this increase by 15 and it will give with approximate accu-
racy the increase for 1 minute. Now get the difference between the sine whose angle is to be
determined and the sine just below it in value. Divide this difference by the amount found neces-
sary for an increase in angle of 1 minute and the quotient will give the number of minutes the
sine is greater than the next lower sine whose angle is known. Add this number of minutes to the
angle of the next lower sine and the sum will represent the desired angle. Or if the sine whose
angle is to be found is nearer in size to the sine just greater, proceed exactly as before, getting the
difference in the sines, but subtract the number of minutes of difference and the result will give the
angle sought. I('or example, take the case in Section 108 where the sine of the angle of 28° 54' is
given as 0.48327. If one consults the table the nearest sines found arc 0.48099, the sine of 28° 45',
and 0.48481, the sine of 29°. Evidently then the angle sought must lie between 28° 45', and 29°.
If the difference between 0.48481 and 0.48099 is obtained, 0.48481 - 0.48099 = 0.00382, and if this
increase for 15' be divided by 15 it will give the increase for 1 minute; 0.00382 + 15 = 0.000254.
Now the difference between the sine whose angle is to be found and the next lower sine is 0.48327
-0.48099 = 0.00382. If this difference be divHed by the amount found necessary for 1 minute it
will give the total minutes above 28° 45', 0.00228 ~ 0.000254 = 9. That is, the angle sought is 9
minutes greater than 28° 45' = 28° 54'.

Table of Metric and English Measures : —

Meter (unit of length) - 100 centimeters; 1000 Kilogram = 1000 grams; 2.2046 (2 1/5 Ibs.).

millimeters; 1,000,000 microns GU); 39.3700 Yard, 3 feet, 36 inches; 0.9144 meter; 91.4399cm.

inches; 3.2808 feet. Foot=\/3 yard; 12 inches; 0.3048 meter;
Centimeter (cm.) « 10 millimeters; 10,000 mi- 30.48 cm.

crons; 0.01 meter; 0.3937(2/5) inch. Inch = 1/36 yard; 1/12 foot; 2.54 cm.; 25.4mm.

Millimeter, (mm.) - 1,000 microns (/*); O.I cm.; Mile = 1760 yards; 5280 feet; 1.61 kilometers.

0.001 meter; 0.03937 (1/25 inch). , —

M icron (unit of length in micrometry) (/*) (§246) Quart = 1/4 gallon; 2 pints; 32 fluid ounces;

-0.001, one thousandth of a millimeter; 0.947 liter (947 cc.). (U. S. liquid).

0.000001, one millionth of a meter; 0.00003937 Mud ounce = 8 fluidrachms; 1/32 of a quart;

(1/25000) inch. 1/16 pint; 29.574 cubic centimeters (30 cc.

Kilometer* 1000 'meters; 0.621 or 5/8 mile. approximately).

— - Ounce avoirdupois = 43/ 1/2 grains; 28.349
Liter (unit of capacity) = 1000 cubic centimeters grams.

(or milliliters); 1 quart approximately. Ounce apothecaries or Troy = 480 grains; 31.103
Gram (unit of weight) = 1 cc. of water; 15.432 grams.

grains. Pound (avoirdupois) = 16 ounces, 453.6 grams.

To Change from Centigrade to Fahrenheit and the Reverse : —

From centigrade to Fahrenheit: Multiply the degrees centigrade by 9/5 and add 32. Exam-
ple: 20° C. - 20 x 9/5 + 32 or 68° F.

From Fahrenheit to centigrade: Subtract 32 and multiply by 5/9. Example: 77° F. - 77 - 32
X 5/9 or 25° C.

To change from centigrade to absolute temperature and the reverse: Add 273 to the degrees in
centigrade and the sum will be the absolute temperature. Example. Ice melts at 0° C. or 0° -f
273° - 273° absolute, and water boils at 100° C. or 100° 4- 273° - 373° absolute. If the abso-
lute temperature is given subtract 273 and the result will be the temperature on the centigrade
scale. Example: Ice melts at 273° absolute, 273° - 273° - 0°, that is, ice melts at 0° C. See Fig
45, where absolute temperature is given.

TABLE OF NATURAL SINES

Compiled from Prof. G. W. Jones' Logarithmic Tables

MINUTES

DEGREES AND QUARTER DEGREES UP TO 90°

1 '0.00029

1° 0.01745

16°, 0.27564

31°.. 0.51504

46°, 0.71934

61°. 0.87462

76°, 0.97030

2 0.00058! 1°,15'0.02181

160,15'0.27983'31°,'i5'0.51877

46°,15'0.72236i61°;i5'0.87673

76°,15'0.97134

3 0.00087

1,30 0.02618

16,30 0.2840231,30 0.52250

46,30 0.7253761,30 0.87882

76,30 0.97237

40.00116

1,45 0.03054

16,45 0.2882031,45 0.52621

46,45 0.7283761,45 0.88089

76,45 0.97338

5 0.00145

2 0.03490

17 0.29237132 0.52992

47 0.73135 62 0.88295

77 0.97437

6 0.00175

2,15 0.03926

17,15 0.29654

32,15 0.53361

47,15 0.73432:62,15 0.88499

77,15 0.97534

7 0.00204

2,30 0.04362

17,30 0.30071

32,30 0.53730 47^0 0.73 7 28; 62^0 0.88701

77,30 0.97630

8 0.00233

2,45 0.04798

17,45 0.30486

32,45 0.54097 47,45 0.74022 62,45 0.88902

77,45 0.97723

9 0.00262

3 0.05234

18 0.30902

33 0.5446448 0.7431463 0.89101

78 0.97815

100.00291

3,15 0.05669

18,15 0.31316

33,15 0.54829

48,15 0.7460663,15 0.89298

78,15 0.97905

110.00320

3,30 0.06105

18,30 0.31730

33,30 0.55194

48,30 0.74896

63,30 0.89493

78,30 0.97992

12 0.00349

3,45 0.06540

18,45 0.32144

33,45 0.55557

48,45 0.75184

63,45 0.89687

78,45 0.98079

13 0.00378

4 0.06976

19 0.32557

34 0.55919

49 0.75471

64 0.89879

79 0.98163

14 0.00407

4,15 OM7411

19,15 0.32969

34,15 0.56280

49,15 0.75756

64,15 0.90070

79,15 0.98245

150.00436 4,30 0.07846

19,30 0.33381

34,30 0.56641

49,30 0.76041

64,30 0.90259

79,30 0.98325

160.00465] 4,45 0.08281

19,45 0.33792

34,45 0.57000

49,45 0.76323

64,45 0.90446

79,45 0.98404

17 0.00495

5 0.08716

20 0.34202

35 0.57358

50 0.76604

65 0.90631

80 0.98481

18 0.00524

5,15 0.09150

20,15 0.34612

35,15 0.57715

SO 15 0.76884

65.15 0.90814

80,15 0.98556

19 0.00553

5,30 0.09585

20,30 0.35021

35,30 0.58070

50,30 0.77162:65,30 0.90996

80,30 0.98629

20 0.00582

5,45 0.10019

20,45 0.35429

35,45 0.58425

50,45 0.7743965,45 0.91176

80,45 0.98700

21 0.00611

6 0.10453

21 0.35837

36 0.58779

51 0.7771566 0.91355

81 0.98769

220.00640 6,15 0.10887

21,15 0.36244

36,15 0.59131

51,15 0.7798866,15 0.91531

81,15 0.98836

23000669 6,30 0.11320

21,30 0.36650

36,30 0.59482 51,30 0.78261 66,30 0.91706

81,30 0.98902

24 0.00698

6.45 0.11754

21,45 0.37056

36,45 0.59832 '5 1,45 0.7853266,45 0.91879

81,45 0.98965

25 0.00727

7 0.12187

22 0.37461

37 0.60182| 52 0.78801 67 0.92050

82 0.99027

26 0.00756

7,15 0.12620

22,15 0.37865

37.15 0.6052952.15 0.79069

67,15 0.92220

82,15 0.99087

27 0.00785

7,30 0.13053

22,30 0.3826837^30 0.60876!52;30 0.79335

67,30 0.92388

82,30 0.99144

28 0.00814

7,45 0.13485

22,45 0.38671

37,45 0.6122252,45 0.7960067,45 0.92554

82,45 0.99200

29 0.00844

8 0.13917

23 0.39073

38 0.6156653 0.7986468 0.92718

83 0.99255

300.00873 8,15 0.14349

23,15 0.3947438,15 0.6190953,15 0.80125|68,15 0.92881

83,15 0.99307

31 0.00902

8,30 0.14781 23,30 0.39875 38,30 0.62251 53,30 0.80386

68,30 0.9304283,30 0.99357

32 0.00931

8,45 0.1521223,45 0.40275

38,45 0.6259253,45 0.80644

68,45 0.93201 83,45 0.99406

33 0.00960

9 0.15643 24 0.40674

39 0.62932 54 0.80902

69 0.93358 84 0.99452

34 0.00989

9,15 0.1607424,15 0.4107239,15 0.6327154,15 0.81157

69,15 0.9351484,15 0.99497

350.01018

9,30 0.1650524,30 0.4146939,30 0.6360854,30 0.81412

69',30 0.9366784,30 0.99540

36 0.01047

9,45 0.1693524,45 0.4186639,45 0.6394454,45 0.81664

69,45 0.9381984,45 0.99580

37 0.01076

10 0.17365'25 0.4226240 0.6427955 0.81915

70 0.9396985 0.99619

380.01105

10,15 0.1779425,15 0.4265740,15 0.6461255,15 0.82165

70,15 0.9411885,15 0.99657

390.01134

10,30 0.1822425,30 0.43051

40,30 0.6494555,30 0.82413

70,30 0.9426485,30 0.99692

400.01164
41 0.01193

10,45 0.18652(25,45 0.43445
11 0.19081! 26 0.43837

40,45 0.6527655,45 0.82659
41 0.6560656 0.82904

70,45 0.9440985,45 0.99725
71 0.9455286 0.99756

42 0.01222
43 0.01251

11,15 0.1950926,15 0.44229
11,30 0.1993726,30 0.44620

41,15 0.65935156,15 0,83147
41,30 0.66262156,30 0.83389

71,15 0.9469386,15 0.99786
71,30 0.94832186,30 0.99813

440.01280

11,45 0.20364*26,45 0.4501041,45 0.66588

56,45 0.83629

71,45 0.9497086,45 0.99839

45 0.01309

12 0.2079L27 0.4539942 0.66913

57 0.83867

72 0.9510687 0.99863

46 0.01338

12,15 0.21218^27,15 0.4578742,15 0.67237

57,15 0.84104

72,15 0.9524087,15 0.99885

470.01367112,30 0.2164427,30 0.4617542,30 0.67559

57,30 0.84339

72,30 0.9537287,30 0.99905

480.0139612,45 0.2207027,45 0.4656142,45 0.67880

57,45 0.84573

72,45 0.9550287,45 0.99923

490.0142513 0.22495)28 0.4694743 0.68200

58 0.84805

73 0.95630

88 0.99939

500.01454113,15 0.22920^28,15 0.47332

43,15 0.6851858,15 0.8503573,15 0.95757

88,15 0.99953

51 0.01483J 13,30 0.23345|28,30 0.47716

43,30 0.68835

58,30 0.85264

73,30 0.95882

88,30 0.99966

520.0151313,45 0.23769!28,45 0.48099

43,45 0.6915158,45 0.85491

73,45 0.96005

88,45 0.99976

530.01542'14 0.24192|29 0.48481

44 0.6946659 0.85717

74 0.96126

89 0.99985

540.01571 14,15 0.24615:29,15 0.48862

44,15 0.6977959,15 0.85941

74,15 0.96246

89,15 0.99991

55 0.01600' 14,30 0.2503829,30 0.49242

44,30 0.70091 59,30 0.86163

74,30 0.96363

89,30 0.99996

560.0162914,45 0.2546029,45 0.49622

44,45 0.70401159,45 0.86384

74,45 0.96479

89,45 0.99999

57 0.01658 15 0.25882!30 0.50000

45 0.707 11 '60 0.86603

75 0.96593

90 1.00000

580j01687,15,15 0.2630330,15 0.50377

45,15 0.71019

60,15 0.86820

75,15 0.96705

590.0171615,30 0.26724;30,30 0.50754

45,30 0.71325

60,30 0.87036

75,30 0.96815

600.0174515,45 0.2714430,45 0.51129

45,45 0.71630

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617

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