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ARTES
1837)
SCIENTIA
LIBRARY RITAS OF THE
UNIVERSITY OF MICHIGAN
*. PLURIBUS UNUM »
TUE BOR
SI-QUAERIS PENINSULAM AMOENAM
CIRCUMSPICE
THE GIFT OF
Prof. Henry Kraemer,
"THRIS
DR.HENRY KRAEMER.
CHEM. LI
LJ
QH
207
13358


MICROSCOPY
AND
MICRO-TECHNIQUE
BY
ALBERT SCHNEIDER, M. D., PH. D.
PROFESSOR OF BOTANY, PHARMACOGRAPHY AND MATERIA MEDICA,
NORTHWESTERN UNIVERSITY SCHOOL OF PHARMACY
ELEITZ WETZLARI
CHICAGO MEDICAL BOOK COMPANY
CHICAGO
1899

200000
3000se
COPYRIGHT, 1899, BY
ALBERT SCHNEIDER.
ALL RIGHTS RESERVED.

PREFACE.
This book is primarily intended for the use of students in
schools and colleges where the microscope is employed in
biological study. A somewhat comprehensive knowledge of
microscopy is necessary to enable the student to do the most
effective work with the compound microscope. Frequently it is
found that even advanced students of biology are not sufficiently
grounded in the optics, mechanism and manipulation of the
instruments placed at their disposal. Such knowledge, if ob-
tained early, will make the advanced work of the biologist more
fruitful of results.
Students not versed in geometry and trigonometry will of
necessity be obliged to omit the discussion of the mathematical
principles involved in the reflection and refraction of light.
While this is to be regretted it does not materially alter the
value of the other chapters of the book.
The methods of micro-technique are treated in a general way;
many minute of details are omitted. The student must acquire
the ability to modify the methods suitably to the special work
in hand. While most of the methods will apply to both animal
and vegetable biology it will be observed that the tendency of
the treatment of the subject matter in Part II is toward the side
of botany. The student of animal biology must employ many
special methods which it would be impracticable to mention or
describe in a small work.
The special chapter on the eye is introduced to enable stu-
dents to have a more correct idea of the relation of that organ
to the work with the compound microscope. A prevailing
notion exists that working with the microscope is injurious to
the eye. It is hoped that this chapter will tend to dispel such
erroneous conceptions.
In conclusion I wish to acknowledge my indebtedness to the
Bausch & Lomb Optical Company of New York and the Leitz
Optical Company of Germany for the loan of electros.
Chicago, September, 1899.
315262
ALBERT SCHNEIDER.

TABLE OF CONTENTS.
Preface
Introduction
PART I.
MICROSCOPY.
CHAPTER I.
HISTORICAL.
I. The History of the Microscope.....
II. The History and Manufacture of Glass.
iii

I
CHAPTER II.
OPTICAL PRINCIPLES OF MIRRORS AND LENSES.
I. Reflection of Light.....
I.
Mirrors....
..
39
12
2.
a.
Plane Mirrors. . . .
b. Spherical Mirrors.
c. Images Formed by Mirrors.
Diffuse Light....
II. Refraction of Light..
1. Medium with Parallel Surfaces.
2. Prisms
3. Lenses
III. Intensity of Light.
...12
.... 13
.
...
..14
.
15
21
.22
..23
...25
..26
..35

vi
TABLE OF CONTENTS.
CHAPTER III.
MICROSCOPES.
I. Simple Microscopes..
1. General Construction.
2. Use of the Simple Microscope.
3. Forms of Simple Microscopes.
II. Compound Microscopes.
1. Formation of Images.
2. The Parts of a Compound Microscope.
a. Mechanical Parts or Stand..
b. Optical Parts...
3.
·
37
•
37
.40
42
..50
51
52
.53
..65
Different Forms of Compound Microscopes...73
CHAPTER IV.
IMPORTANT ACCESSORIES.
I.
Micrometers
II. Camera Lucida.
.77
.80
III. Micro-Photographic Apparatus.
IV. Test Objects...
...84
Fo o
.87
CHAPTER V.
OPTICAL AND WORKING PROPERTIES OF THE COMPOUND
MICROSCOPE.
I.
Aberration
1. Spherical Aberration.
2. Chromatic Aberration.
.
TI. Aperture
III. Focal Distance and Working Distance.
IV. Penetrating Power.
•
89
89
.
91
885258
.
97
·
98


TABLE OF CONTENTS.
vii
V. Definition
VI. Resolving Power.
VII. Magnifying Power.
99
.
99
.100
CHAPTER VI.

THE MANIPULATION AND CARE OF THE COMPOUND MICRO-
SCOPE.
I. Manipulation of the Instrument..
I. Position of Instrument and Worker..
2. Illumination
3. Focussing
II. How to Use the Eyes..
III. Care of the Instrument.
IV.
Hints on the Purchase of a Microscope.
. 104
.. 104
·
105
. 107
·
109
..IIO
.I12
PART II.
MICRO-TECHNIQUE.
CHAPTER I.
MATERIAL.
I. Microscope Desks, Tables and Chairs...
II.
Microtomes and Section Knives.
1. Simple Microtomes..
2. Automatic Microtomes.
III. Glass Slides, Object Slides.
IV.
Cover-glasses
..
•
•
•
.
.115
• ·
117
..117
.119
120
121

viii
TABLE OF CONTENTS.
V. Turn Table-Slide Centerer.
VI. Accessory Material....
1. Watch Glasses.
2. Section Lifters.
3. Dissecting Needles...
4. Slide Boxes.
5. Other Accessory Material....
CHAPTER II.
REAGENTS.
I. Reagents in Alphabetical Order...
II. Reagents Arranged According to Properties.
..124
..125
.125
. 126
.127
127
..127
...
..129
134
CHAPTER III.
I. Temporary Mounts.
METHODS.
1. Mounts of Entire Objects...
2. Crushed and Powdered Substances.
3. Needle Preparations. . . . .
4. Hand Sectioned Specimens.
II. Permanent Mounts..
1. Direct Method.
2. Celloidin Method...
3. Paraffin Method..
III. Special Methods.....
·
1. Examination of Bacteria.
2. Examination of Meat for Trichinæ.
3. Mounting Sections of Bone..
IV. General Directions...
..138
138
. 140
.142
..143
·
.
.148
.
.148
.
.150
.
•
.
151
.
154
6
·
154
..
·
..156
•
158
159

TABLE OF CONTENTS.
ix
THE NORMAL AND ABNORMAL EYE.
I. The Optical Properties of the Eye...
1. Refraction
2. Accommodation
3. Aberration
4. Entopic Phenomena.
.161
162
..164
..167
...167
.168
5. Special Optical Phenomena.
II. Defective Sight.....
1. Diplopia or Double Vision...
2. Achromatopsy or Color Blindness.
3. Weak Eyes (Amblyopia).
4. Squinting (Strabismus)
5. Near-sight (Myopia)....
..169
. 169
·
170
.
171
.
·
·
171
•
174
.
175
....
•
·
177
177
.178
·
.178
185
6. Far-sight (Hypermetropia)
7. Senile Far-sight (Presbyopia)
8. Astigmatism
9. Inflammatory Conditions of the Eye.
III. Hints on the Care of the Eyes....
General Index..
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MICROSCOPY
AND
MICRO-TECHNIQUE.
INTRODUCTION.
Microscopy and micro-technique treat of the mechanical aids
involved in biological study. They are therefore not sciences in
the true sense of the word. The majority would at once rele-
gate these branches of knowledge to art, of course using the
term in its broader sense. It is, however, a difficult matter in
many instances to draw the line between art and science. In
fact, it is customary to speak of reducing any subject to a
science when it has been thoroughly and systematically in-
vestigated.
Microscopy treats of the mechanism and use of the simple
and compound microscopes. By some scientists it is considered
as belonging wholly within the realm of pure mechanics and as
a result is greatly neglected. This is much to be regretted
since the improvement of the microscope depends primarily
upon those who use it in their investigations. Or, to be more
accurate, the scientist and mechanic must combine their energies
in the preparation of the most efficient scientific apparatus; the
former points out defects and suggests improvements, the lat-
ter remedies the defects and adds the improvements in so far as
mechanical skill will permit. Both must adhere strictly to that
which seems reasonable and practicable.
Micro-technique treats of the preparation of substances for
examination with the microscope so that advancement in this
branch depends essentially upon the improvements of the
microscope. Within the past decade microscopical methods
have reached a high degree of perfection and complexity.
1

2
MICROSCOPY.
Many of the minutæ of detail are, however, of little or no prac-
tical value.
From what has been said it must not be supposed that the
quality and quantity of work done by a biologist is directly pro-
portional to the perfection and excellency of the microscopes
and methods at his command. Excellent work has been done
by scientists with very inefficient mechanical means, but other
things being equal the best equipped worker will accomplish
the highest results.

PART I.
MICROSCOPY.
CHAPTER I.
HISTORICAL.
I. THE HISTORY OF THE MICROSCOPE.
There seems to be no comprehensive historical record of the
invention and evolution of the microscope, simple and com-
pound. Within comparatively recent years a host of authors
have written about the various improvements of the compound
microscope. But no one has as yet sought to bring together
the most reliable data regarding the earlier microscopes, par-
ticularly the earlier compound microscopes. The records thus
far are scattered and fragmentary.
The microscope in its simplest form consists of a convex
lens of some transparent substance. As such it was no doubt
known long before the discovery of glass. Lenses of rock-
crystals were known in very ancient times. Such a lens was
found in the ruins of Nineveh. According to some authorities,
Layard among others, the use of the lens was known in the
palace of Nimrod about 1500 B. C. Tradition has it that
Archimedes during the siege of Syracuse (212 B. C.) set fire
to the Roman fleet by means of a lens. According to another
tradition it was not a lens which the noted physicist employed,
but a series of plane mirrors so arranged as to concentrate the
rays of the sun upon the Roman ships. There is, however, per-
haps no truth in these traditions, as they are not confirmed by
such noted historians as Polybius or Plutarch.
Early Greek and Arabian physicists seem to have been well
versed in mathematical optics, but their discussions were con-
3

4
MICROSCOPY.
fined chiefly to the reflection of light from plane and curved
mirrors, from which it would appear that lenses were rare or at
least not considered of importance. Aristophanes (5 B. C.)
refers to lenses as "burning spheres," and the philosophical
Seneca (first century) found time to comment upon the nature
of lenses.
Thus far no special use seems to have been made of lenses,
except that they were used by a few physicists to illustrate
principles in optics. The use of the simple microscope
or lens as an instrument in scientific investigations was not
known until the latter part of the sixteenth century. Robert
Hooke of England (1656) constructed lenses of high magni-
fying power from minute spheres or globules of glass. Of
these he says: "If you are desirous of obtaining a microscope
with a single refraction, and consequently procuring the great-
est clearness and brightness any one kind of microscope is
capable of, spread a little of the fluid you intend to examine on
a glass plate, bring this under one of your globules, then move
it gently upward till the fluid touches the globule, to which it
will adhere so firmly as to bear being moved a little backward
or forward. By looking through the globule you will have
a perfect view of the animalcules in the drop." From this it is
evident that Hooke was the first to employ immersion lenses.
Anton van Leeuwenhoek, who made a great many important
discoveries, used only simple microscopes. The lenses were
made by himself and were not spherical or globular, as were
those of Hooke. They were double convex lenses of various
sizes and curvatures and were fixed between metal plates and
provided with arrangements for holding the object and regu-
lating its distance from the lens. Of these microscopes he
possessed a great many, using one for one or two objects only.
In 1677 he discovered a large number of fresh water animal-
culæ, such as rotifers, vorticellæ, etc. In human histology he
made a special study of nerve tissue and capillaries, which led
to his discovery of the red blood corpuscles in 1673. From his
most careful measurements he estimated the diameter of red
corpuscles at 1-1940 of an inch. The actual diameter is, how-
ever, 1-3200 of an inch, from which we conclude that Leeuwen-
hoek's lenses had great spherical aberration.
About this time the errors arising from spherical and chro-

HISTORICAL.
5
20
matic aberration were discovered and various methods were
employed to correct these defects. Sir Isaac Newton proposed
to remove chromatic aberration by using monochromatic illumi-
nation, at the same time making the statement that the con-
struction of achromatic lenses was a physical impossibility.
Euler, the great Swiss mathematician, demonstrated the incor-
rectness of Newton's statement that "all refracting substances
diverged the prismatic colors in a constant proportion to their
mean refraction" and consequently "refraction could not be
produced without color." He made an achromatic objective
in 1776. John Dolland, an English optician, pointed out some
of the defects in Euler's lenses and succeeded in constructing
an achromatic objective for the telescope by combining a con-
cave lens of flint glass with a convex lens of crown glass. It
may be observed that for years all of the corrected lenses were
made for telescopes, the minute size of microscope lenses being
supposed to present an unsurmountable mechanical obstacle.
About the year 1815 Wollaston first proposed the use of a cor-
rected doublet consisting of two plano-convex lenses, placed
with plane sides toward the object. The two lenses acted as one
and gave a very clear field.
Thus we find that up to the year 1800, or somewhat later, it
was the simple microscope which was almost entirely used
in scientific research. As late as 1821 we find such eminent
authorities as M. Biot of France and Dr. Wollaston of Eng-
land boldly declaring that "opticians regard the construction
of a good achromatic microscope as impossible" and that "the
compound microscope will never excel the simple one," and
this practically 200 years after the invention of the compound
microscope.
As to who should be credited with the construction of the
first compound microscope authorities differ; several claim-
ants for this honor have been brought forth. It is also a ques-
tion which was invented first, the compound microscope or the
telescope. There are two important reasons why opticians and
scientists of the middle ages should have exerted themselves
more in behalf of the telescope. I. Astronomy and astrology
were the important sciences of the time, and, 2, telescopes, be-
ing constructed on a larger scale, it was easier to make the re-
quired mechanical improvements.

6
MICROSCOPY.
Galileo is usually credited with having invented the telescope.
In 1609 he presented his first complete instrument to Leonardi
Deodati, the doge of Venice, who tested it in the tower of St.
Marks and expressed his surprise at its magnifying power.
It was, however, left to Kepler to give the true theory of
the telescope and to find the focal length of lenses
and apply it in determining the magnifying power of the tele-
scope. Some historians credit Roger Bacon (1214-1294), the
English monk, with the invention of the telescope. It would
not be surprising that this eminent scientist and mathema-
tician should have known the principles of the telescope, but
there does not seem to be any evidence on record that he had
actually constructed one.
It is quite generally believed that the first compound micro-
scope was made in the year 1590 by the Dutch optician, Zacha-
rias Jansens, and was exhibited to James I in London by his
astronomer, Cornelius Drebbel. This will perhaps explain why
Drebbel has been credited with its invention. This microscope
is said to have been an imposing affair, consisting of a copper
tube six feet long with the lenses mounted on three brass
dolphins, supported upon a base of ebony. It was very im-
perfect, distorting and coloring objects very much. For many
years it was kept on exhibition in London as a curiosity, no one
dreaming that the compound microscope would ever be of any
practical value.
The following are also said to have invented the compound
microscope: Galileo (1610) is said to have constructed a com-
pound microscope from a telescope which, he said, made a fly
seem as large as a hen. In 1839 Charles Chevalier, a French
optician, rediscovered the compound microscope of Galileo.
The same thing occurred in 1851, when Ernest Brücke, the
German physiologist, again gives a description of a compound
microscope identical with that of Galileo. Fontana of Naples
claims to have made the first compound microscopes in 1618,
and even as far back as 1592. However, it is generally con-
ceded that Jansens was the real discoverer. It is from 1590
that most microscopical societies date their anniversaries of the
discovery of the compound microscope.
The earlier compound microscopes were very clumsy and
simple in construction as compared with these of to-day. Those

HISTORICAL.
ry
used by Robert Hooke previous to 1656 consisted of a simple
objective and a simple eye-lens with an intermediate lens corre-
sponding to the field lens of our present eye-pieces. The in-
termediate lens was inserted when it was desired to have a
wider field of view. When he wished to examine any part of
the object with greater accuracy the middle lens was removed,
as he soon found that the fewer the refractions the brighter
the image of the object appeared. The microscope used by
Eustachio Divino (1664) consisted of an object lens, a middle
or field lens and a compound ocular of two plano-convex lenses
with their convex surfaces in contact. The tube in which these
lenses were placed is said to have been "as large as a man's leg
and the eye-lenses as broad as the palm of a man's hand." Al-
though these measurements are very crude, they are sufficient
to indicate that this microscope was rather formidable. Its
magnifying power ranged from 41 to 143 diameters.
In 1672 Sir Isaac Newton communicated to the Royal So-
ciety a design for compound microscope by reflection. This con-
sisted of a spherical concave metal mirror; the object to be ex-
amined was placed in the focus of the mirror and the reflected
and magnified image was further magnified by means of an eye-
lens. Other forms of reflecting microscopes were proposed from
time to time, but none have ever been used extensively. In
1690 Philip Bonani published a description of a microscope
made by himself which was supplied with a rack and pinion for
adjustment of focus and a substage condenser for illumination,
evidently the first with those improvements.
In 1738 Dr. Lieberkühn of Berlin devised and constructed a
solar projecting microscope which created considerable inter-
est at the time, but owing to the fact that it only revealed in-
distinct shadows of things its use was soon discontinued. It
was, however, the forerunner of our present stereopticons,
which certainly are extremely useful.
Since 1821 various opticians have exerted their skill in the
preparation of corrected lenses, that is, lenses free from spher-
ical and chromatic aberration and from that time dates the ex-
tensive use and improvement of the compound microscope. In
1827 Amici of Modena arranged combinations of several lenses
which were quite free from distortion. His plan was to use
uncorrected front lenses, the aberrations of which were cor-

8
MICROSCOPY.
со
rected more or less completely by the over-correction of the
back lenses. Amici also introduced the immersion system of
objectives by connecting the front lens and cover-glass by a
drop of some liquid having a higher refractive index than air.
The use of immersion systems, however, fell into disuse until
revived by Hartnack, a German optician (1854). Along with
the improvements new difficulties arose. It was found that
an objective which defined uncovered objects very well did not
do so when they were covered with thin glass; and, further, the
difference in thickness of the cover-glasses greatly affected the
defining power of the best objectives. In 1837 Andrew Ross
of London published a paper on the aberrations produced by
the cover-glass. He corrected these aberrations by varying
the distance of the front lens of an objective from the other
systems. The change in distance was effected by means of
a correction-collar, which is still used with the so-called ad-
justable objectives. Ross also succeeded in increasing the
angular aperture of objectives, attaining an angle of 135 de-
grees for his highest powers, which, he concluded, was the
"largest angle that can be passed through an objective," a
statement which may be compared to that made by Biot and
Wollaston. Spencer, a young American, proved the incorrect-
ness of Ross' statement by preparing a 1-12 inch dry objective
with an aperture of 146 degrees.
Cherubin (1677) was perhaps the inventor of the binocular
microscope, but it was left to Prof. Riddle (1853) of New Or-
leans to construct the first efficient stereoscopic binocular. This
instrument was supposed to be a decided improvement upon
the monocular instruments, but it was soon found that its ad-
vantages were mostly illusionary. At the present time they are
seldom used and are beginning to be looked upon as historical
curiosities.
We shall conclude this historical review with the statement
that since 1850 great improvements have been made in the
compound microscopes. The mechanical and optical parts have
been greatly improved. To note what these improvements are
one need only to inspect the catalogues of dealers in microscopes
and microscopical supplies.

HISTORICAL.
9
II.
THE HISTORY AND MANUFACTURE OF GLASS.
As with other old and extensive industries nothing definite is
known as to the time when glass first came into use. It is fre-
quently mentioned in the Old Testament. Recently it has been
maintained that glass was manufactured in Egypt before the
exodus. The excavation of ancient royal sepulchers in Thebes
and Beni Hassan have revealed paintings representing glass-
blowers at work. The hieroglyphics upon these paintings proye
that they date from the year 1800 B., C. Large quantities of
glassware were manufactured and exported from Sydon, Tyrus
and Alexandria. It is supposed that this industry was intro-
duced into Rome during the time of Cicero. During the reign
of Tiberius several large factories were active. Plate glass
for windows is first mentioned by ecclesiastical writers of the
third century of our era. In the sixteenth century Venice
was renowned for its manufacture of glass and excellent mir-
From Venice these industries spread rapidly to France,
England, Germany, Bohemia and other countries.
rors.
Glass is a mixture of silica and metallic oxides and chem-
ically may be classified as follows: 1. Potassium-calcium
glass, which is entirely colorless, hard and very resisting to
chemicals. German crown glass and Bohemian crystal glass
are examples. 2. Sodium-calcium glass has a bluish tint and
is harder than the preceding; as English crown-glass, French
glass, window glass and glass for making chemical utensils.
3. Potassium-lead glass is readily fusible, comparatively soft,
but of high specific gravity and high index of refraction. It is
much used for optical purposes and forms the basis of artificial
diamonds. 4. Aluminium-calcium-alkali glass, used chiefly for
making bottles.
The raw material from which glass is made consists of silica
in the form of sand or quartz. Various deposits of sandstone
have been extensively used in the manufacture of glass. Some
of the deposits belonging to the St. Peter formation are highly
prized, while others are practically worthless, that, apparently,
depending upon the perfection of the crystals. Calcium, potas-
sium, lead and sodium are obtained from various mineral sub-
stances holding these metals in combination. In order to pre-

10
MICROSCOPY.
pare a pure quality of glass the ingredients must be pure. Το
color glass various substances are added. Oxide of iron colors
glass green; oxide of cobalt gives a blue color; oxide of cop-
per, red; antimony, yellow; etc.
Bohemian crystal giass is carefully prepared from the purest
materials mixed in the following proportion: Sand, 100; pure
potash, 60; carbonate of lime, 18. This glass has a world-
wide reputation and is used in making lenses to counteract the
chromatic aberration of flint glass lenses. English flint glass
contains lead, and for optical purposes pure materials are used.
The following are the proportions: Sand, 100; oxide of lead
(yellow), 100; calcium carbonate, 30. The ingredients are
pulverized, mixed and melted in an earthen vessel. After cool-
ing, the vessel is broken and the mass reduced to the desired
form by grinding. Paste diamonds or false diamonds are made
of glass rich in lead and hence of high refraction, but soft and
lacking in durability.
The durability of glass depends largely upon its composition.
In order that it may resist atmospheric conditions and chemicals
the oxide of silicon must be combined with the oxides of two
metals; one univalent, the other bivalent. Lead glass, while
highly refractive, is more readily attacked by chemicals than
glass free from lead. As a rule alkalies act upon glass more
readily than acids. Many glasses contain more or less arsenic,
which has proved very annoying in the chemical laboratory.
Under the influence of warmth, moisture and CO, a portion of
the glass is dissolved and washed away, while a fine deposit of
SiO, is formed on the surface, causing iridescence and dimness
of the glass. This is very often an unlooked-for and unaccount-
able nuisance to housewives and even to scientists. Frequent
polishing and protection against moisture will prevent this
condition.
The behavior of glass with varying temperatures is peculiar
and depends upon the rate of cooling. If the molten glass is
cooled suddenly there is produced a very high surface tension,
so that very frequently a very slight jar or shock is sufficient to
break the glass to atoms (example, Satan's tears). If the cool-
ing process is too slow numerous crystals of SiO, are deposited
on the surface. Rapidly cooled glass has a refractive power
nearly twice that of gradually cooled glass.
HISTORICAL.
11
Sunlight has a peculiar effect upon glass. White lead glass
and optical flint glass, as well as all glass with a bluish tint, re-
mains unaffected; all others are variously colored after long
exposure to sunlight.
This brief reference to the history and physical nature of
glass will enable the student to better understand the optical
properties and care of mirrors and lenses used in microscopical
work.


12
MICROSCOPY.
CHAPTER II.
OPTICAL PRINCIPLES OF MIRRORS AND LENSES.
Before any one can use a microscope intelligently it is neces-
sary to have an understanding of the optical principles involved
in the reflection of light and in the formation of real and virtual
images by means of mirrors and lenses. It is true one may be-
come quite skilled in the manipulation of the microscope with-
out knowing anything about optics, but such a deficiency will
always be a hindrance to the performance of the best work and
will at once stamp one as "amateur" in the eyes of the knowing.
The optical principles here considered are such as are almost
continually involved in the manipulation of the compound
microscope. An effort has been made to simplify the subject as
much as possible, yet the introduction of geometrical formulæ
was found to be unavoidable.
I. REFLECTION OF LIGHT.
By way of introduction we shall state the two important laws
of the reflection of light. When a ray of light meets a polished
surface it is found that,
(1) the angle of reflection is equal to the angle of incidence,
and
(2) the incident and reflected ray are in the same plane and
perpendicular to the reflecting surface.
It is highly important to have a correct understanding of the
above laws, as they are especially involved in the use of mirrors
for illumining objects for microscopical examination.
I. MIRRORS.
Mirrors are bodies with polished surfaces, which show by
reflection objects presented to them. According to their form
mirrors may be divided into plane, concave, convex, parabolic,
conical, pyramidal, etc. It will not be necessary to define


MIRRORS AND LENSES.
13
these in particular, as the terms are sufficient explanation.
Futhermore, the plane and concave mirrors are the only ones
extensively employed in microscopy.
Mechanically it has been found impossible to prepare a
polished surface which is absolutely smooth or which will reflect
all of the incident rays. For this reason the reflected light is
always less than the incident light.
a. Plane Mirrors.
In a plane mirror all of the reflecting surface lies in the same
plane. All mirrors or objects that reflect light are opaque; that
is, they do not transmit light. Incidentally, it should, however,
be remembered that all transparent substances reflect some light.
The light which is incident upon an opaque reflecting surface
or mirror is separated into three parts. One is regularly re-
flected; that is, it is light whose rays, in reference to the plane
of the mirror, form angles equal to those of the incident rays.
They are therefore reflected from surface areas of the mirror
lying in the general plane of the mirror. A second part of the
incident light is irregularly reflected; that is, it is reflected from
surface areas of the mirror lying in some plane other than the
general plane of the mirror. This irregularly reflected light is
also spoken of as scattered light and is that which makes bodies
visible from any position. Regularly reflected light does not
make objects visible, it only represents the light-emitting ob-
ject. A perfect reflecting surface would, therefore, be invisible.
A third part of the incident light is extinguished or absorbed
by the reflecting body; that is, it is wholly lost, being neither
regularly nor irregularly reflected.
From the above it is evident that the reflected light is al-
ways less intense than the incident light, since some of the
vibrations of the incident rays are transformed into vibrations
of the reflecting surface, that is, they are lost or absorbed;
furthermore, most of the irregularly reflected rays become use-
less because they are too much scattered. Other things being
equal, it has been found that the intensity of the reflected light
increases with the obliquity of the incident rays.
The intensity of reflection also varies greatly with different
bodies, even when the degree of polish and angle of incidence


14
MICROSCOPY.
are the same. In a metal mirror the reflected light is about 3-5
of the perpendicular incident rays, 3-4 in the case of mercury,
1-25 in glass, and 1-50 in water.
As has been indicated, the irregularly reflected light repre-
sents the lustre or brightness of the reflecting surface and varies
greatly with the nature of the surface. In the case of freshly
fallen snow about 78 per cent of the reflected light is scattered;
white paper about 70 per cent, and in the case of ordinary soil,
perhaps 7 per cent.
Optically the best mirrors are those made from metal, be-
cause they have only one reflecting surface. Glass mirrors.
have two reflecting surfaces; first, a comparatively feeble re-
flection from the upper or outer surface of the glass, and sec-
ondly, the strong reflection from the surface of the metal behind
the glass. This accounts for the multiple images formed by
glass mirrors. Ordinarily we take no notice of the image
formed by the glass surface, because it is so indistinct as to be
scarcely perceptible.
b. Spherical Mirrors.
As has already been indicated, the concave mirror is most
frequently used in connection with microscopy. If we take a
sheet of metal enclosing one-half or less than one-half of a
sphere and polish it on both sides, it would serve to illustrate
both kinds of spherical mirrors. The outer convex surface
would form the convex mirror, the inner surface the concave
mirror.
Since the concave mirror can be applied to the outer sur-
face of a sphere it may be said to be formed by the rotation of an
arc equal in length to the diameter of the mirror, about a radius
of a sphere to which the mirror fits, the radius being attached to
the center of the mirror and the center of the circle.
The reflection of light from curved mirrors is readily under-
stood if one considers its surface as being made up of an in-
finite number of very small plane mirrors, all being so placed
that perpendiculars erected from the middle point of each
separate plane surface will pass to the center of curvature; or
in other words, each plane mirror is a tangent plane to its per-
pendicular.


MIRRORS AND LENSES.
15
c. Images Formed by Mirrors.
An optical image consists of a collection of focal points from
which light either really or apparently radiates. We have, ac-
cordingly, real images and apparent, imaginary or virtual
images. In the first case the reflected rays actually meet, while
in the second case they do not.
The following are facts in optics which should be kept in
mind:
I. In the case of plane mirrors: a. Rays diverging be-
fore reflection will diverge at the same angle after reflection.
b. Convergent incident rays will converge at the same angle
after reflection. c. Rays parallel before reflection will be
parallel after reflection. d. Images are only apparent and will
appear behind the mirror and equal in size to the object.

772
m
--
-n
FIG. I.
REFLECTION OF LIGHT BY PLANE MIRRORS.
Let m n in Fig. I represent a plane mirror; a b and a c
are diverging rays of light meeting the mirror at the points
b and c respectively. If we erect the perpendiculars b g and
cf at these points we must construct the paths of the reflected
rays b d and c e so that the angle a b g the angle g b d and
the angle a c f = the angle f ce; that is, the angle of incidence
must equal the angle of reflection; a b and a c diverge at an
angle ba c; b d and c e, the corresponding reflected rays, also
diverge, as will be seen upon constructing a line c e' parallel
to b d; e' c and e c diverge, therefore b d and c e also di-
verge. e' c e represents the diverging angle of reflection and
is equal to bac because they are angles on the same side of two
parallel lines cutting a third line.

16
MICROSCOPY.
To prove that converging incident rays also converge
after reflection we need only to reverse the proposition; that
is assume db and ec to be the incident rays and ba and
ca the reflected rays.
That parallel incident rays will also be parallel after reflec-
tion is sufficiently evident to require no further discussion.
m
AK
n
FIG. 2.
IMAGE FORMED BY A PLANE MIRROR.
Let m n in Fig. 2 represent a plane mirror and A B an object
in such a position that the reflected rays will enter the eye at
H. A ray of light incident upon the mirror from the point A
will be reflected in the direction X H, making the angle of in-
cidence A X R equal to the angle of reflection R X H. From A
let fall a perpendicular upon the mirror at E, prolong the line
H X below the mirror until it meets the prolonged perpendicu-
lar at a. We have then two equal triangles, A X E and a X E,
because they have the side E X in common and the angles
AEX, EX A are equal to angles a E X, a X E because the
angles at E are right angles and the angles A X E, a X E are
each equal to the angle H X n. From the consideration of these
equal triangles it follows that A E is equal to E a; that is, a
ray of light A X is reflected in such a manner than its pro-
longation below the mirror will cut the perpendicular A a at
the point a, which is the same distance from the reflecting sur-
face of the mirror as A. The same argument would apply to

MIRRORS AND LENSES.
17
rays of light emanating from the point B or any ray from points
intermediate between A and B.
If we now construct the perpendiculars A C and a c we find
that they are equal because they are perpendiculars between
two parallel lines. We have already learned that B G G b
and AE= E a. By substitution and subtraction we find that
B Cb c; the angles A C B and a c b are equal because by
construction they are both right angles, hence A B = a b.
We also know that the angles B A C and b a c, which are the
angles of inclination of object and image respectively, are equal.
From the above geometrical data we must therefore conclude
that the apparent image formed by a plane mirror is equal in
size, equidistant and equally inclined with the object.
2. Concave mirrors: Reflection of light and the formation
of images by spherical mirrors is generally supposed to be quite
difficult of comprehension. This is, however, not the case if we
look upon these mirrors as being made up of an infinite number
of small plane mirrors, as shown in figure 3.
1-
H
FIG. 3.
m
H'
F
F
CONCAVE AND PLANE MIRRORS
COMPARED.
Let mn represent a concave mirror; now place a series of
plane mirrors, as a b, b c, c d and d e, so that they will form

18
MICROSCOPY.
tangent planes at the points B, D, E and F. For the purpose of
comparison we can only employ such portions or points of the
plane mirrors as lie in the curvature of m n; that is, the points
B, D, E and F. We will suppose that A B is a ray of light in-
cident on the curved mirror at the point B, corresponding to
the point B in the tangent plane mirror. Erect a perpendicu-
lar B C and make the angle of reflection C B G equal to the
angle of incidence A B C, according to the law of reflection
by plane mirrors. The same will apply to the ray A D inci-
dent upon b c at D, or to any ray incident upon the point of
contact of any tangent plane. It will be observed that all per-
pendiculars to the points of contact of the various tangent
planes meet at the same point, known as the center of curva-
ture. mn is formed by rotating a line equal in length to the
radius about the point C.
It is evident from inspection that since the perpendiculars
(radii) of the various plane mirrors converge, the reflected
rays must also converge in the same proportion.
If we construct a plane mirror b' c' parallel to a b, the in-
cident ray A D would be reflected in the direction D H', ac-
cording to the law of reflection by plane mirrors. It is also
evident that the concave mirror converges the reflected rays.
Before proceeding to the consideration of the formation of
images by concave mirrors it will be necessary to define and
explain some of the terms used. In Fig. 4, m n represents the
diameter or aperture of the mirror, C the center of curvature,
p the vertex or center of the mirror; the infinite line o p which
passes through the center of curvature (C) and the vertex (p)
is known as the principal axis of the mirror. Any other line
passing from C to m n is known as a secondary axis and is a
radius of the sphere of which the mirror is a part.
As has already been stated, all rays of light incident upon a
concave mirror tend to converge. If the incident rays are
parallel they converge or intersect at a point equally distant
from the center of curvature and the mirror, known as the prin-
cipal focus of the mirror. The distance from the principal
focus to the vertex of the mirror is known as the principal
focal distance. From this consideration it is evident that rays
of light emitted from the principal focus will be parallel after
reflection. If the incident rays are convergent they will inter-

MIRRORS AND LENSES.
19
sect at some point lying between the principal focus and the
mirror, while if they are divergent they will intersect at some
point between the principal focus and the center of curvature.
Rays of light emanating from the center of curvature would
again be reflected to the center of curvature. It is also evident
that incident rays diverging from a point converge to a point
after reflection; these points are known as conjugate foci be-
cause light emanating from one point is brought to a focus at
the other.
FIG. 4.
m
n
CONJUGATE FOCI OF CONCAVE MIRRORS.
It is an easy matter to determine the relative distances of the
conjugate foci if the radius of the mirror and the distance of
one focus are known. Mathematically the relationship of the
conjugate foci may be expressed as follows: In Fig. 4 let
Cp, the radius of the mirror, equal r; the distance of one
focus, A p, equal m and the distance of the other focus, a p,
equal n. The angle Aba is bisected by C b; therefore A b
:ab: A Ca C. If the arc bp is quite small, b A and ba
are approximately equal to A p and a p.
Ca
Cp-apr-n
CA Ap-C p=m-r, substituting

20
MICROSCOPY.
We have
(1) nmn-rr-m
(2) nr-nm-nm — mr
(3) nr-2 nm-mr
(4) nr=2 nm-mr
(5) nr=m (2n-r)
(6) m=
n r
211-r
which is the equation for
the distance of the focal point A in terms of the radius of the
mirror and the distance of the conjugate focus a. In a similar
manner we can find the value for m from the proportion
(7) mnr-mn-r. Proceeding as
above we finally get
(8) n =
In r
which is the equation for
2 m-r
the distance of the focal point a in terms of the radius of the
mirror and the distance of the conjugate focus A.
In a concave mirror the size and position of image and ob-
ject varies. If the object is placed between the principal focus
and the mirror, there will be a virtual image which is larger
than the object and erect. If the object is placed at the princi-
pal focus, no image will be formed because the reflected rays
are not brought to a focus.
If the object is placed between the center of curvature and
the principal focus, the image is enlarged and inverted; con-
versely, if the object is placed beyond the center of curvature,
the image is inverted, but smaller than the object.
It is a very simple matter to determine the relative size and
position of image and object in spherical mirrors. All that is
necessary is to remember that focal points of the object must
again be brought to a real or apparent focus after reflection,
indicated by accurate mechanical drawings, having, of course,
due regard to the optical principles involved. For illustration
we shall indicate the formation of an image when the object
is placed between the center of curvature and the principal
focus.

MIRRORS AND LENSES.
21
如
​E
n
FIG. 5.
IMAGE BY A CONCAVE MIRROR.
In Fig. 5, a d represents an object placed between the center
of curvature and the principal focus of mirror m n. If the
image r s were projected upon a screen one could readily find
its distance from p; having in addition the radius C p, we
may readily determine the distance ep by formula (8).
f C may then be calculated. It will be found that the diameter
of the image is to the diameter of the object as the distance
from the image to the center is to the distance from the object
to the center; or, geometrically, from the similar triangles s Cr
and a C d we would get the statement:
sra df Ce C.
The relative brightness of image and object by a concave
mirror is nearly proportional to their surfaces, and to the co-
efficient of reflection; and is inversely as the square of the
focal distances.
2. DIFFUSE LIGHT.
The nature of diffused light has already been explained. It
makes objects visible; the beams of light (sunlight and
electric light) in rooms and elsewhere are only rendered vis-
ible by the irregular reflection from the particles of dust which
float in the air. Tyndall has shown conclusively that if all

22
MICROSCOPY.
floating matter is removed from an enclosed space the beam
of light is invisible. If it were not for the diffused light all
space not illumined by direct of regularly reflected light would
be pitch dark, as, for instance, the shaded sides of buildings,
as well as their interiors. Twilight is produced by the diffusion
of light from particles in the upper layers of atmosphere.
II. REFRACTION OF LIGHT.
As we have just learned, in reflection the ray of light under-
goes a change in direction in the same medium, usually air.
Refraction is a change in direction which the ray of light
undergoes in passing obliquely from one medium to another.
It is necessary to say obliquely because a ray perpendicular to
the refracting surface is not changed in its course, but con-
tinues in a straight line. It need scarcely be mentioned that
refractive media are more or less transparent; that is, the rays
of light can pass through them. Not by any means all of
the light falling upon a refracting medium passes through
it; some of it is regularly and irregularly reflected and
some of it is absorbed or disappears. The amount of re-
fraction varies greatly with different media. Mathematically
it has been shown that the direction of refraction depends
on the relative velocity of light in the two media; the velocity
of light is less in the more highly refracting medium. In non-
crystalline media the ray of light is refracted singly, while in
certain crystalline substances, as Iceland spar, selenite, etc.,
the incident ray is divided and refracted in two directions.
This is known as double refraction. We shall here confine
the discussion to single refraction.
When a ray of light passes from one medium to another of
different refractive power the following laws prevail :
I. For any two media the ratio of the sine of the incident
angle to the size of the angle of refraction is constant.
2. The incident and refracted ray are in a plane which is
perpendicular to the surface separating the two media.
The constant ratio referred to in the first law is called the
index of refraction and is found by dividing the sine of the
angle of incidence by the sine of the angle of refraction. If we
let i equal the index of refraction and R and S the incident

MIRRORS AND LENSES.
23
angle and angle of refraction respectively, then, according to
the statement just made,
sine R
i
sine S
We have thus far considered only the comparative index
of refraction; that is, the index obtained when the ray of light
passes from one medium into another. The absolute or prin-
cipal index is found when the ray passes from a vacuum into
the refracting substance. The index also varies with the
purity or homogeneity of the refracting substances. This ac-
counts for the variance in the tables of refractive indices given
by different authors. The following is a table of absolute re-
fractive indices taken from Ganot's physics:

Diamond
Phosphorus
Flint glass...
Crown glass ...
Plate glass (French)
Rock salt
Alcohol
Oil of Cassia
.2.47 to 2.750
2.616
1.779
1.608
1.587
1.545
1.363
1.600
•
1.336
In
Water
gases the index of refraction is very small. The follow-
ing table gives the absolute indices of a few gases:
Chlorine
Carbonic acid
Air
...
Oxygen
Hydrogen
·
.
1.000772
1.000449
1.000294
1.000272
1.000138
I. MEDIUM WITH PARALLEL SURFACES.
We shall now consider refraction in a medium with par-
allel plane surfaces. Such refracting media are in constant
use by the microscopist, as slides, cover-glasses and the many
mounting media which are placed between covers and slides.

24
MICROSCOPY.

R
FIG. 6.
REFRACTION BY A MEDIUM WITH PARALLEL PLANE SURFACES.
In Fig. 6 let R represent some refracting substance with
plane parallel surfaces, as a piece of plate glass, a medium
more dense than air, hence the incident ray S will be
refracted toward the perpendicular a g.
The emergent
ray S' will be refracted from the perpendicular d f. Substitut-
ing for the formula on page 23, we have:
(1) i=
sine a c S
sine e cg
(2) sine a c Si sine e c g. The angles
de cand e cg are equal, hence their sines are equal.
(3) i=
sine S'e f
Therefore
sine de c
sine a c S
sine S'e
(4)
and
sine e c g
sine de c
(5) sine a c S= sine S' ef, from which it
follows that the incident ray (S) and the emergent ray (S')
must be parallel.
In the case of an object mounted on a slide for exam-
ination under a high-power compound microscope, the light
must pass through several refractive media whose surfaces are
all parallel to each other, as the slide, the mounting medium
(balsam, glycerine-jelly, etc.), and the cover-glass; never-

MIRRORS AND LENSES.
25
theless the incident and emergent rays are parallel to each
other. The case becomes different in the use of immersion ob-
jectives, as will be explained later.
2. PRISMS.
A transparent medium bounded by inclined planes is known
as a prism. The angle included by the planes is called the re-
fracting angle and the planes are deviating planes.
As is generally known, the prisms will separate the beam
of white light into its component colors, as violet, indigo, blue,
green, yellow, orange and red. This property is also in a
measure possessed of lenses, as we shall learn later, and is a
serious hindrance to the making of perfect lenses. For the
present we shall, however, consider only the deviation of the
ray on its passage through the prism without considering the
dispersing effect.
S
d-
C/--
-
FIG. 7.
REFRACTION BY A PRISM.
The total deviation by a prism is equal to the sum of the
angles of incidence and emergence diminished by the refract-
ing angle. In Fig. 7 S is the incident ray.
Erect the perpen-
dicular a d c; the ray on its passage through the prism is
bent toward this perpendicular; upon its emergence it is bent
away from the perpendicular erected at e, upon the side i r.
It is evident that S' f h is equal to the total deviation.

26
MICROSCOPY.
(1) S' fh=fde+fed
(2) Sda-cde=

S'e g
―
ced
(3) S' fh= (Sda-cde) + (S' fh-
ced) = (Sd a+S' e g) - (cde+
ced). Because of the perpendiculars
a and g, ie c and c d r are two similar right-angled tri-
angles; ther fore,
(4) ice-dre.
But
(5) ice-cde+ce d, therefore
(6) c de ce d=dre and
(7) S' fh=Sda+S' eg-dre, which
is what we sought to prove.
3. LENSES.
Lenses are transparent media having one or two curved
surfaces. They are commonly made of crown glass, which is
free from lead, and of flint glass, which contains lead and is
more refractive than the former. Various mineral substances
enter into the glass used in the construction of modern lenses,
as has been stated.
The surfaces of lenses are combinations of spherical sur-
faces or of spherical surfaces with plane surfaces, which gives
rise to six kinds of lenses commonly used. Four of these are
formed by spherical surfaces, two by a plane and a spheri-
cal surface, as shown in Fig. 8.
E
DOD LIC
FIG. 8.
FORMS OF LENSES.
A is a plano-convex lens having one side plane, the other
convex. It is a segment of a sphere, or, compared with B, it
is one-half of a double convex lens.


MIRRORS AND LENSES.
27
B is a double convex lens. There is a common base and
two equally or unequally convex surfaces.
C is a meniscus or a convexo-concave lens. There is one
convex surface and the other concave. The convexity always
exceeding the concavity.
D is a plano-concave lens, having a plane surface and a
concave surface. As compared with E it represents one-half
of a double concave lens.
E is a double concave lens. There is a common base, with
two equally or unequally concave surfaces.
F is a concavo-convex lens. One surface is concave, the
other convex, the concavity always exceeding the convexity.
A, B and C are thicker at the center than at the border
and converge the rays of light. D, E and F are thicker at the
margin and diverge the rays of light. Of these six lenses we
need only illustrate the optical properties of B and E, since the
others are only modifications of these.
The convex lens may be compared to an indefinite number
of prisms placed base to base, as shown in Fig. 9 A, and the
concave lens may be compared to an indefinite number of
prisms with their apices in contact (B).

B₁
FIG. 9.
COMPARISON OF LENSES AND PRISMS.
In order to compare the path of the ray of light in a lens
with that in a prism we proceed similarly as in the case of
curved mirrors; that is, the surface of the lenses are sup-
posed to be formed of an infinite number of small plane sur-
faces, forming tangent planes at the points lying in the curv-
ature of the surface of the lens.

28
MICROSCOPY.

FIG. 1O.
FOCI OF A DOUBLE CONVEX LENS.
The focus of a lens is the point where the refracted rays or
their prolongations meet. As in spherical mirrors, there may
be real and virtual foci.
When rays parallel to the principal axis (o p) fall upon
the lens they are refracted and meet or cross at a point known
as the principal focus (C). The distance from the principal
focus to the surface of the lens is the principal focal
distance. It is constant in the same lens, but varies with the
index of refraction and the radii of curvature. In crown
glass lenses where the radii of curvature are equal the prin-
cipal focus corresponds to the center of curvature (C). If a
point of light should be placed at C the emergent rays would
be parallel. If the point of light is placed beyond C the re-
fracted emerging rays would converge at a point on the other
side of the lens and we would have conjugate foci, as in
curved mirrors.
The double convex lens has a virtual focus when the lumi-
nous object is placed between the lens and the principal focus.
Although we have spoken of the principal focus as a point
this is practically not true, as all parallel rays do not cross at the
same point; it is only approximately true of lenses with small
apertures, that is, the angle formed by joining the edges to the
principal focus. Where the aperture is more than 8° or 10°
the marginal rays focus nearer the lens than the rays which
pass through the lens nearer the axis. This causes the phe-
nomenon known as spherical aberration by refraction.

MIRRORS AND LENSES.
29

d
FIG. II.
FOCI OF A DOUBLE CONCAVE LENS.
In double concave lenses there are only virtual foci, no
matter what the distance of the object; only the prolongations
of the refracted rays intersect, as may be seen from Figure II.
The principal focus of a convex lens may be determined
by exposing it to the sun's rays, which are practically parallel.
Receive the emergent pencil of light upon a ground glass or
paper screen; move the lens backward and forward until the
spot of light projected is smallest and brightest. This is the
focus of heat as well as of light. Retaining the lens at focal
distance soon ignites the screen or paper. On account of this
heat-concentrating effect convex lenses are sometimes called
"burning glasses." Germans speak of the focal distance as
the Brennweite (burning distance).
To determine the focal distance of a double concave lens,
one surface may be covered with some opaque substance, leav-
ing two small apertures equidistant from the principal axis,
but on opposite sides in the same principal section. A pencil
of sunlight is then received on the other surface, and the rays
emerging from the apertures are received on a screen, which
is moved backward and forward until the spots of light on the
screen are twice as far apart as those on the lens; the dis-
tance between screen and lens is then equal to the focal dis-
tance.

30
MICROSCOPY.
The focal distance of a double convex lens may also be de-
termined from the relative diameters of object and image as in
the case of a concave mirror.

FIG. 12.
S
OPTIC CENTER OF A CONVEX LENS.
The incident and emergent ray which cuts the points of con-
tact of two parallel tangents will be parallel according to the
principle of refraction by any medium having parallel plane
surfaces (see page 24). The point where such a ray cuts the
principal axis is called the optic center. The position of the
optic center varies with the form of the lens. It may be de-
termined by the following construction:
In Fig. 12 SS' is the ray cutting the optic center, a and b
are the points of contact of the parallel tangent planes. Ca
and C'b being perpendicular to these tangent planes are
parallel to each other, therefore
(1) Cab-C'ba and
(2) aPC=C'Pb, from which it follows that CaP
and C'bP are similar triangles.
Represent radius C'b by r' and radius Ca by r. Further
let t equal the thickness of the lens measured on the axis and e
the distance of the optic center from the surface of the lens.
From the similarity of triangles

MIRRORS AND LENSES.
31
(3) C'P:CP: :C'b:Ca; that is, the distance of the
optic center from one center of curvature is to the distance
of the optic center from the second center of curvature as one
radius is to the second radius. Substituting we have
(4) r'-er-(t—e): :r':r.
(5) r'r-re-r'r-(r't-r'e)=r'r—r't+r'e.
(6) r'e+re=r't.
(7) e (r'+r)=r't.
r't

(8) e=
r'tr.
This formula will serve to de-
termine the optical center of double concave and concavo-
convex lenses. In lenses with one plane surface this point
lies at the intersection of the axis by the curved surface.
Every straight line passing through the optical center, but
not passing through the centers of curvature, is a secondary
axis. A secondary axis may be represented by a ray of light,
which may be treated as a rectilinear, because the slight devia-
tion which the ray undergoes in passing through a medium
with parallel plane surfaces may be entirely ignored.
As long as the angles formed by secondary axes and primary
axis are small it will be found that rays of light from any
point along a secondary axis will again converge to a point
on the same axis on the other side of the lens, forming either
a real or virtual conjugate focus, this depending upon whether
the incident point is farther away or nearer than the principal
focus.
The relative distances of conjugate foci in double convex
lenses may be determined when the refractive index and radii
are known. Let r equal the index of refraction and assume
that the angles of incidence and refraction are so small that
their ratio is the same as the ratio of their sines.

32
MICROSCOPY.
$
FIG. 13.
CONJUGATE FOCI OF A CONVEX LENS.
In Figure 13 let C and C' represent the conjugate focal
points of ray CabC'. Angles eaC and dac are equal.
(1) dacbac: x1, by division
(2) dac-bacbac a::x-ISI, substituting
(3) dab:bac: :x-1:1, similarly
(4) dba:abc: :x—1:1, (3)+(4)=
(5) dab+dba:bac+abc: :x-1:1, but
(6) dab+dba-gdC'=dCC'+dC'C and
(7) bac+abc=ecr=crr'+cr'r; therefore
(8) dCC'+dC'C :crr'+cr'r: :x—1:1, but
the angles at C, C',r and r' are as the reciprocals of their dis-
tance from S; therefore substituting in (8) we have
I
r's
:—————————— + —————————::x-I:I and
rs
I
I
I
(9)
+
CS C'S
I
I
(10)
I
I
+——=(x—1) (—————-to-
CS C'S
rs rS
If we wish to determine the principal focus we must assume
I
CS
I
I
as equal to —, but =0; letting the principal focus
x
-
x
equal F, formula (10) becomes

MIRRORS AND LENSES.
33
I
(II)
F
I
I
rs r's
=(x—1) (——+
The reciprocal of the principal focal length of a lens is called
its power. From (10) we see that
I
I
I
(12) + =(x—1) (—— +
CS C'S
I
(13) =(x—1) (——+
F
rS
I
and from (II)
r's

I
I
therefore
rs r'S
I
I
I
(14)
+-
from which
F
CS C'S
we get the formula for the power or magnification of a lens.
(CS) (C'S)
(15) F-
CS+C'S
From the above considerations it is easy to deduce the
formula for the power of a combination of lenses equivalent to
a single lens.
1
1
T
B
FIG. 14.
A
FOCI OF A COMBINATION OF LENSES.
In Fig. 14 let P be a ray of light parallel to the axis in-
cident at r and s of the combination of lenses; also, let st be
the emergent ray whose focus is at t. Draw rc parallel to st;
ac will represent the focal length of a single lens having the
same deviation as the above combination; ar is the focal length

34
MEMICROSCOPY.
of a.
Let a equal the distance between the lenses. If we re-
gard t as one of the foci then must be the virtual conjugate
focus of lens b. Let f equal the focal length of lens a and f'
the focal length of lens b. From formula (14) on page - we
get

(1)
I
I
I
the sign changing because
f'.
bt
bx
the focus br is virtual. But
(2) bx-f-a, therefore
(3)
I
I
I
f
bt
f—a
I
I
I
(4)
+
bt
f'
f-a
I
f'+f—a
(5)
bt
f' (f-a)
From the similar triangles acr, bts, xar and xbs we get
(6) bt:ac: :bs :ar: :bx:ax, but ac equals the focal
distance of the combination, therefore
(7) bt:F: :bs:ar: :bx:ax. Substituting
f' (f-a)
(8)
-
f=F (f-a). Recriprocal of F=
ƒ'+f—a
ƒ'+f—a
f-a
I
(9)
-;
; when
f' (f—a)
f
F
lenses are in contact a=o and (9) becomes
(10)
I
I
I
+
F
f
f'
,
that is the power of a com-

MIRRORS AND LENSES.
35
bination of lenses in contact is equal to the sum of their re-
spective powers.
From the above mathematical considerations, aided by ac-
curate mechanical drawings, it is an easy matter to illustrate
the relationship of objects and images by the various lenses.

III. INTENSITY OF LIGHT.
The light-giving power of the sun is about 600,000 times
as great as that of the moon and about 15,000,000,000 times
as great as that of the brightest fixed stars. It has been esti-
mated that 670,000 wax candles placed at a distance of one
foot would be necessary to produce light equal to that of the
sun. Fifty Bunsen cells will produce light one-fourth as
strong as sunlight.
The intensity of illumination is represented by the quantity
of light received upon a unit of surface and is subject to the
following laws:
I. The intensity of illumination varies inversely as the
square of the distance from the source of light.
2. The intensity of illumination received obliquely is di-
rectly proportional to the cosine of the angle which the ray of
light makes with the perpendicular to the illuminated surface.
The first law requires no special discussion. The second
law may be explained as follows: In Fig. 15 let Sm and S'n
bound a beam of light falling upon the oblique surface mn;
opr or snm would be the angle which the illumined surface
makes with the beam of light. If t is the total quantity of
light which falls upon mn and u that which falls upon the
unit of surface then we would have
(1) u=—
t
mn
that is, u= the intensity of light
upon the unit of surface; sn, the section of the pencil of light,
is a projection of mn on a plane perpendicular to the beam
of light. From trigonometry we know that
(2) sn=mn cos snm and

36
MICROSCOPY.
sn
(3) mn=
substituting in (1),
COS smn
t
(4) u=
COS snm. t and sn being constant,
sn

therefore u must be proportional to the cos of the angle of
inclination.
The relative intensities of different sources of light may be
determined by an apparatus known as the photometer. Rum-
ford's photometer consists of a ground glass screen, in front
of which is fixed an upright opaque rod; the lights to be com-
pared are so placed that each one projects on the screen a
shadow of the rod. The lamps are moved back and forth
until the intensities of the shadows are the same. Then care-
fully measuring the distances of the lights from the shadows
the relative intensity of the lights may be estimated from the
first law. Bunsen's photometer consists of a movable paper
screen, with a grease spot in the middle; this is placed be-
tween the lights to be compared and moved back and forth
until the grease spot becomes invisible, which indicates that
the intensity of illumination is the same on both sides of the
screen.
S
0-
m
T-
S
FIG. 15.
INTENSITY OF LIGHT UPON AN OBLIQUE SURFACE.

MICROSCOPES.
37
CHAPTER III.
MICROSCOPES.

I. SIMPLE MICROSCOPES.
Simple microscopes consist of a single lens or a combination
of lenses, generally in close contact, acting as a single lens.
They are usually designated as magnifiers. The lens or lenses
are generally mounted in metal or hard rubber convenient for
holding in the hand, carrying in the pocket, or resting on
some fixed support. Their magnifying power ranges from
two to twenty diameters.
I. GENERAL CONSTRUCTION.
The majority of simple microscopes consist of a single lens
of the convex type. The better and more expensive forms
consist of several lenses, one of which may be double concave;
but the combination has the general effect of a single convex
lens.
As has already been indicated, the spherical lens, considered
theoretically, gives the highest magnification, but in practice
it has been found that spherical and chromatic aberration in-
creases with the increase in magnification, so that the spherical
lens has been long since abandoned for the convex lenses of
considerable length of focus and hence comparatively low
magnification. Even with these the aberration referred to is
sufficient to be annoying and hence combinations of lenses are
used to correct these errors. (See Aberration, Chapter V.)
Images by simple microscopes are virtual, erect and larger
than the object. The magnitude of the image by any one lens
depends upon the distance of the object from the principal
focus. The highest magnification is obtained when the object
is just in front of the principal focus. If the object is placed
at the principal focus no image is formed; if placed just be-

38
MICROSCOPY.
yond the principal focus a real, inverted and enlarged image
is formed. As the object approaches the lens the image be-
comes smaller. It is not advisable to place the object at the
distance of highest magnifications, since it will be found that
spherical and chromatic aberrations increase very rapidly at
the margin. This can be readily noticed upon examining the
cross-hatching of fine cloth in bright light; on moving the
cloth, spread flat, from the lens a position will be reached
where the entire field shows the meshes about equally well,
and where the threads of the cloth seem nearly parallel; mov-
ing the cloth back still more the meshes near the margin be-
come blurred and the threads seem to widen out from the
center, forming curved lines, while prismatic colors begin to
show near the margin of the field of vision.

FIG. 16.
FORMATION OF VIRTUAL IMAGE BY A CONVEX LENS.
In Fig. 16 let ab be an object placed between the principal
focus F and the lens o. Upon emergence rays ac and bd form
diverging angles with the secondary axes, hence it will be
necessary to produce the focal points corresponding to a and b.
They are at A and B respectively, hence the image is virtual
and erect. It is also magnified because its diameter subtends
the angle A o B at a greater distance than that of the object.
Practically all the lenses of simple microscopes now in use
are made of glass. Some have been made of precious stones,
as rubies and diamonds, because these substances have great

MICROSCOPES.
39
refrangibility and comparatively little dispersion. Such lenses
are, however, no longer made, because the aberrations of form
and color can be remedied in a much cheaper way. It is also
stated that scientists have used lenses of water, oil, Canada
balsam and other transparent liquids and semi-liquids.
These were made by placing a drop of the liquid in a small
hole in wood or metal, which would form lenses of the convex
type, approaching mostly the plano-convex. Such lenses are
naturally very unsatisfactory and no longer used excepting as
curiosities in demonstrating optical principles.
The glass from which lenses are to be made must be of pure
material and must be free from air bubbles. It must be as
transparent as possible and free from color. For single lens
microscopes pure homogeneous glass of high refractive in-
dex is used.
It has been found that the form of the lens modified spheri-
cal and chromatic aberration considerably. The radii must
stand in a certain ratio with reference to the refractive index
of the glass to be used. Mathematically this ratio is expressed
as follows:

r
r'
4-2i2+i
2i2+i
in which r and r' are the radii of
curvature of the two surfaces of any convex lens and i the
refractive index of the lens. Lenses constructed according
to this formula are called lenses of best form, and are as free
from aberration as it is possible to make a single lens. With
a glass whose refractive index is 3-2, r'=r6, which means that
one radius must be six times the other. In such a lens one
surface would be much more convex than the other. It must,
however, not be supposed that aberration is entirely corrected
in lenses of best form. In order to correct spherical and
chromatic aberration effectively it is necessary to combine
lenses of different sizes, different form, and of different glass,
as will be explained later.
Aberration is greatest in lenses of the double convex form.
It is considerably less in the plano-convex form, and, as just
explained, least in lenses of best form. It should be remem-
bered that the surface of shorter curvature is turned toward

40
MICROSCOPY.
the object; if used in the reverse position it gives a maxi-
mum of aberration.
2. USE OF THE SIMPLE MICROSCOPE.
Simple microscopes are used in investigations where a
comparatively low magnification is desired. The manner in
which it is used will depend largely upon the nature of the
work upon which one is engaged and upon the form of the
microscope. Strictly speaking, eye-glasses for myopia,
presbyopia, astigmatism and weak eyes are forms of the
simple microscope and used to correct the defects named.
The reading glass is generally used by artists and engravers
for the purpose of examining details in their work. The
watchmaker's glass is used by the watchmaker in making his
repairs, etc. Each specially constructed simple microscope has
its special use, and will be readily understood by those who
have occasion to use it. We shall here content ourselves with
giving some general advice.
Always keep both eyes open. Nothing looks more unsci-
entific and absurd than to see any one distorting his features
in attempting to keep one eye closed. This facial distortion
actually becomes permanent in those who use the micro-
scope considerably and who persist in keeping one eye closed.
Closing one eye has no advantage. It is true at first the ob-
server is considerably annoyed by objects seen with the un-
occupied eye, but only a little practice is necessary to accus-
tom one to totally ignore these images. If there are any who
cannot refrain from closing one eye it would be well to use
binoculars only; that is, simple microscopes having a lens for
each eye.
In high power (hence smaller) lenses it is necessary to bring
the eye very near the upper surface of the lens because this
secures a larger field of view. With the larger and com-
paratively high power lenses experience will indicate the proper
distance they should be placed from the eye. With the read-
ing glass and lenses of like power this distance is about ten
or twelve inches for most persons.
As regards the distance between the
the following advice may be followed:

object and the lens,
Move the lens, with

MICROSCOPES.
41
the
eye in proper position, until the object to be examined is
comparatively near; that is, until it is well within the focal
distance. This gives a low magnification with comparatively
little distortion; now move the lens up or away, when it will
be found that magnification increases, but at the same time
dimness, due to spherical and chromatic aberration, begins to
show itself around the margin; this continues until there is
only a small central area which is comparatively free from
distortion and which shows the highest amplification the lens
is capable of giving. It is therefore advisable to make the
general examination with the object well within the focus,
and to examine any special detail with the desired area just
within the focus.
The objects to be examined should be well illumined, while
the
eye should be well protected. As regards the manner of
securing the proper illumination, of holding the lens, the ob-
ject, etc., no special advice can be given here. It will all de-
pend upon the nature of the work that is to be done. In
any case no special instruction is necessary, provided common
sense prevails.
It is not advisable to purchase expensive simple micro-
scopes, as it will be found that the cheaper instruments will
answer fully as well and generally better. A simple lens of
medium magnifying power, convenient for carrying in the
pocket, will generally satisfy all the demands ordinarily made
of this class of instruments.
The magnifying power of a simple microscope may be
roughly estimated from its focal distance. A single lens of
one-inch focus magnifies about ten diameters; two-inch focus,
five diameters; five-inch focus, two diameters; one-half-inch
focus, twenty diameters; one-fourth-inch focus, forty diame-
ters, etc. As a rule, the larger the lens the lower the magnifica-
tion. The ratio between the diameter of a lens and its focus is
approximately 1:2; for example, a lens two inches in diameter
has a focus of four inches. The magnifying power of any
lens may be very readily and accurately determined by actual
measurement. Focus the lens upon a piece of paper with a
ruler lying upon it; fix the lens in position at the distance at
which the magnification is to be determined. With one eye
look at the spaces on the ruler; with the other eye look at the


42
MICROSCOPY.

point of the pencil; indicate on the paper two lines bounding
a space on the ruler. Now remove the lens and find how
many spaces on the ruler will be contained in the space you
have indicated on the paper; the quotient will indicate the num-
ber of amplifications. Or hold a ruler in contact with the
surface of the lens and focus upon a second ruler or spaces
ruled on paper like those of the ruler, and compare by direct
observation.
3. FORMS OF SIMPLE MICROSCOPES.
The following are some of the more common forms of the
simple microscope. There are a host of different patterns of
each kind placed on the market, each one said to have some
special redeeming qualities, which as a rule do not exist:
1. READING GLASSES.-These are double convex lenses, hav-
ing a diameter of two or more inches and correspondingly low
magnifying power. The lens is mounted in a ring of metal
and provided with a handle. It is used in reading fine print
and in studying details of drawings, photographs, paintings,
engravings, etc. Although it is a very convenient lens to use,
its magnifying power is not sufficient to be of much value in
scientific work. (Fig. 17.)
BAUSCH & LOMB OPT.Co.
2.
FIG. 17.
READING GLASS.
FOLDING MAGNIFIERS.-Under folding magnifiers is
here included a great variety of forms of simple microscopes,
consisting of convex medium-power lenses, mounted in a
frame suitable to be carried in the pocket. The mounting
material is usually vulcanite or hard rubber. Sometimes the
lenses are mounted singly; again, two and three lenses of dif-


MICROSCOPES.
43
ferent magnifying power are combined, swinging on a com-
mon pivot so that one, two or all three may be used,
thus giving a wide range of amplifications. Among these the
bug-hunter and plant-collector will find the most suitable
lenses. The lenses are not corrected for spherical and chro-
matic aberration, a fact which should be kept in mind, espe-
cially in using the higher magnifications.

FIG. 18.
SINGLE LENS
8
FIG. 19.
MAGNIFIER.
TWO LENS MAGNIFIER.
FIG. 20.
THREE LENS
MAGNIFIER.

44
MICROSCOPY.

3.
CODDINGTON LENS.-This is a misnomer, since Sir
David Brewster was the real inventor of this lens. The mis-
take is due to the optician Carey, who constructed one of these
lenses for Mr. Coddington and supposed he was the inventor.
The lens is really the central portion of a sphere with a cir-
cular groove at the middle which is blackened. This cuts off
nearly all of the marginal rays, which distort the image, but
it also reduces the field of view very much. It has a com-
paratively high magnifying power and focusses very near the
object. The lenses are mounted in a folding support of Ger-
man silver or nickeled brass. Fig. 21 shows a Coddington
magnifier and the lens in optical section.
co
FIG. 21.
CODDINGTON LENS.
4. STANHOPE MAGNIFIERS.-The Stanhope magnifier con-
sists of a thick double convex or plano-convex lens so ground
that the plane or the less convex surface is just in focus for
the object. This lens, in combination with another larger
double convex lens of about one-inch focus, is very exten-
sively sold by street venders.
The Stanhope magnifier is very extensively used by the
amateur in examining sections of plant and animal tissues,
drops of stagnant water, vinegar, wings of insects, scales of
butterflies, etc. When liquids are examined it embodies the
principles of an immersion lens. In many respects it is a very
satisfactory lens.
5. LINEN TESTER.-This is a folding microscope, so con-
structed that when opened the base rests on the object to be
examined at the focal distance, usually one inch. The base
has a square opening of one-fourth inch diameter. As the
name would indicate, it is largely used in examining textile

MICROSCOPES.
45
products, for counting the number of threads in the given
standard area. It can, however, also be used in the examin-
ation of other substances. They are made of different sizes
and fold very neatly for carrying in the pocket. Fig. 22 shows-
the form of one-inch focus.

FIG. 22.
LINEN TESTER.
6. WATCHMAKER'S GLASS.-This usually consists of a lens
having a focus ranging from one to two inches, mounted in
one end of a short, hard rubber tube. The other end of the
tube spreads out bell-shaped and is made to fit in the socket
of the eye near the outer rim. As its name indicates, it is
used by watchmakers in repairing watches, etc. Some botan-
ists also use it, especially in herbarium work. The manner of
holding the lens is annoying to some. (Fig. 23.).
FIG. 23.
WATCHMAKER'S GLASSES.

46
MICROSCOPY.
7. ENGRAVER'S GLASS.-Constructed much like the preced-
ing and used by engravers in inspecting their work. (Fig. 24.)
St
8.
FIG. 24.
ENGRAVER'S GLASSES.
APLANATIC LENSES.-These differ from those already
mentioned in the following particulars: They consist of two
or more lenses so constructed and placed with reference to
each other as to secure a maximum of magnification and com-
parative freedom from spherical and chromatic aberration.
Since great care and considerable extra workmanship is re-
quired in their construction, they are quite expensive. They
are recommended in all investigations where accuracy is neces-
sary, if it is permissible to speak of accuracy in connection
with the work that can be done by the aid of low-power
lenses.
In some of these magnifiers the lenses are in close contact
and mounted in a suitable metal frame to be carried in the
pocket. The better forms give a clear field, with considerable
magnification (8 to 20 diameters). In others there are two
lenses or two systems of lenses not in contact.
These are
nearly altogether used with dissecting microscopes. The fol-
lowing are some of the more special forms of aplanatic simple
microscopes:
a. Wollaston's Doublet.-This consists of two plano-con-
vex lenses in contact, with their plane surfaces directed to-

MICROSCOPES.
47
ward the object. This combination gives a clear field and was
at one time highly famed and very extensively used.
b. Steinheil's Aplanatic Triplets. Of these there are sev-
eral forms. The correction is obtained by a double convex
lens of crown glass cemented to two concavo-convex lenses
of flint glass. They give a magnification ranging from ten to
twenty diameters, have a clear field and comparatively long
focus. (Fig. 25.)
о
FIG. 25.
STEIN HEIL'S APLANATIC TRIPLET.
C. Double Achromatic Lenses.-These consist of two cor-
rected systems, which are, however, not in contact. They
show a large, clear field and have a long focus. Magnifica-
tion about five diameters. They are specially adapted for dis-
secting work, although they may be used like the ordinary
pocket lenses. (Fig. 26.)
BAUSCH & LOMB OPT.CO
FIG. 26.
DOUBLE ACHROMATIC LENS.
d. Hastings' Aplanatic Triplet.-This is a modification of
form (b). It has a long focus and wide field.
9. DISSECTING MICROSCOPES.-A dissecting microscope is

48
MICROSCOPY.
usually a simple microscope in which the magnifiers are fixed
to some support so as to leave both hands free for handling
the object and working the focus. The following are some
of the desirable qualities in a dissecting microscope: a. Wide
field, with freedom from spherical and chromatic aberration.
b. Suitable arrangement for vertical and lateral adjustment
of magnifier. c. Medium to comparatively high amplification.
d. Long focal or working distance. e. Contrivance for il-
lumining opaque objects. f. Glass stage with dark and
white background. g. Solid, well-made stand, with or with-
out hand rests. To some the hand rests are only a nuisance,
while those whose hands tremble or incline to tremble will find
them very useful.
There are a large variety of dissecting microscopes on the
market. Most of them are monocular, some binocular. There
are also compound dissecting microscopes for those who desire
amplifications greater than those that can be obtained from the
simple form. In my estimation it is never advisable to invest in
a compound dissecting microscope, as they are quite ex-
pensive. It is certainly very rare that its use would be very
necessary. The low-power compound microscope (1-in. obj.
and 1-in. ocular) can be used as a dissecting microscope.
The following are a few of the more common forms of the
dissecting microscope:
a. Tripod Magnifier.-This consists either of a single con-
vex lens, or two convex lenses separated by a diaphragm,
having a magnifying power ranging from eight to twelve
diameters. The lenses are mounted in a brass or German
silver ring which screws into a second ring, with three legs.
The screw serves for focussing. It is quite extensively used
in botanical work for beginners. (Fig. 27.)



MICROSCOPES.
49

TRIPOD
FIG. 27.
MAGNIFIER.
b. Handy Dissecting Microscope.-This consists of an iron
base having a glass plate top, which serves as the stage upon
which to dissect. A steel upright fastened into the base serves
to hold the magnifiers. These may be removed from the steel
stem and used as hand magnifiers. Fig. 28 will further ex-
plain its construction and use.
FIG. 28.
HANDY DISSECTING MICROSCOPE.
F

50
MICROSCOPY.
C.
Laboratory Dissecting Microscope. This dissecting
microscope is complete in all its parts, excepting the hand
rests. Fig. 29 will explain its construction. The mirror in
the position shown in the figure is for illumination by trans-
mitted light. It may, however, be detached and fastened to
the stage for vertical illumination in the examination of opaque
objects.

LABORATORY
FIG. 29.
DISSECTING
MICROSCOPE.
II. COMPOUND MICROSCOPES.
Optically a compound microscope consists of two lenses or
two series of lenses so arranged that the observer beholds a
magnified virtual image of the object. The object itself is
not seen through the compound microscope, a fact which am-
ateurs are loth to believe. The first lens or series of lenses
forms an enlarged and inverted, but real, image of the object;
by means of the second lens or series of lenses the observer ex-
amines the magnified virtual image of the inverted real image.

MICROSCOPES.
51
The purely mechanical parts of the compound microscopes
are accurately made, and subserve the following purposes:
To hold the optical parts in their proper position.
༨.
2. To secure careful focussing of the lenses upon the
object to be examined.
3.
To provide suitable illumination.

I. FORMATION OF IMAGES.
In its simplest form the compound microscope consists of
two lenses of the convex type; one of comparatively short
focus and turned toward the object is known as the object
lens or objective; the other lens of longer focus is turned to-
ward the eye and is known as eye-lens, eye-piece or ocular.
The first compound microscopes actually had only two lenses,
but those in use at the present time always have more. The
simplest form will, however, serve best to illustrate the forma-
tion of the images.
F
R
FIG. 30.
IMAGES BY THE COMPOUND MICROSCOPE.
In Fig. 30 let R represent the object lens and E the eye-
lens. If an object a b be placed a little beyond the principal

52
MICROSCOPY.
focus F of the lens R, a real, inverted and enlarged image will
be formed at A B on the other side of the lens. The eye-lens
E must be so placed that the image of R will be between the
lens E and its focus F'. The eye will then perceive the vir-
tual image A' B' of the real image A B. Thus the observer
beholds an inverted virtual image of the object. It is evident
that the magnifying power must change with the relative
change of position of the lenses; for example, if lens E were
moved nearer lens R, the image A' B' would be smaller. The
highest amplification of a compound microscope is attained
when the object is placed just without the focus of the ob-
jective and the real image is formed just within the focus of
the eye-lens. The full magnification is found by multiplying
the magnification of the objective by that of the ocular. If
the objective magnifies 40 diameters and the ocular 10 diame-
ters, the total amplification would be 40 x 10 diameters, or 400
diameters. As regards the objective, it is found that the di-
ameter of the object is to the diameter of the image as the
distance of the object from the lens is to the distance of the
image from the lens. The amplification of the oculars can be
determined as for simple microscopes.
If
Theoretically it would seem possible to construct compound
microscopes of an indefinite magnifying power by simply in-
creasing the number of lens systems. In all of the micro-
scopes in use there are only two, as has been described.
we should place the eye-lens so that the real image of the
objective would form outside the focus, a second inverted but
real image would be formed on the other side; we might
even introduce a third and fourth system, finally placing the
ocular in the proper position for examining the image last
formed. If we could realize such a construction, we would
be enabled to make wonderful discoveries. Actual experi-
ence has, however, taught that one objective system and one
eye system is all that can be made use of practically because
more systems disperse and absorb too much light and as a re-
sult the image becomes too indistinct.
2. THE PARTS OF A COMPOUND MICROSCOPE.
The parts of a modern compound microscope may be di-
vided into two sets; the purely mechanical parts, which con-

MICROSCOPES.
53
stitute the stand, and the optical parts, consisting of mirror,
condensers, oculars and objectives. All the first-class instru-
ments of the different manufacturers resemble each other
comparatively closely as to size and the presence of certain
essential parts. There is, however, considerable difference as
to the details of mechanism, a fact which must not be over-
looked in the selection of an instrument. We shall discuss in
detail the important parts of a compound microscope, hoping
that the student will gather therefrom sufficient information
to understand its mechanism and to use it intelligently.

A.
Mechanical Parts, or Stand.
At the present time nearly all of the stands are made after
what is known as the Jackson model. Some years ago another,
the Ross, model was very popular in England. In the Jackson
model the body of the microscope has the rack-work for
coarse adjustment attached to it, and is supported for the
greater part of its length upon the firm arm. In the Ross
model the body is fixed to a transverse arm, which has a
racked stem extending downward. The Jackson is far su-
perior to the Ross stand, principally because in it the coarse
adjustment is much less liable to cause vibrations and un-
steady motion of the body, and is much more durable. It is
still possible to find Ross stands on the market, especially in
France.
Stands are also classified according to the length of the tube.
In the English or long model the tube is from eight to ten
inches in length. This was at first quite generally adopted
by American manufacturers. In the German continental or
short model the tube is about six inches in length. The
short model is to be preferred because it gives greater firm-
ness and compactness; optically it permits better illumination.
American manufacturers are now largely adopting the short
type. The English persist in the long model, while the German
manufacturers are quite unanimous in adopting the short
model. Unfortunately there is no uniformity in designating
the tube length, as will be seen from the table of tube lengths
adopted by different manufacturers. This table was orig-
inally prepared by S. H. Gage and is here adapted from the
work of E. Bausch.

54
MICROSCOPY.
Tube lengths of different manufacturers indicated by the
parts measured:
T
Powell and Lealand
Wales, W....
Spencer Lens Co.
--C
ad Grunow
Reichert
Leitz, E....
Nachet et Fils
..
·
Milli-
meters.
•
.254
.254
.160 or 235
....203
.. 160 to 180
. •
170
.146 or 200
Klonne und Muller.. 160-180 or 254
.160 or 250
Zeiss, C....
b- d
Swift and Son... ..165 to 2281
Bezu, Hausser et Cie..
.220
Seibert, W. and H..... ...190
Bausch & Lomb Opt. Co.160 or 216
a-g
Gundlach Optical Co.... ...254
Winkel, R.....
..220
c-d, Ross and Co...
----dc-e, Beck, R. and J…………….
..254
...254
---e
-f
Green, J......
...254
Hartnack
.. 160-180
---9
c - f
Verick
..160-200
.
Watson and Sons. ...... 160-250
FIG. 31.
VARIATIONS IN TUBE LENGTHS.
The differences of tube-length cause no trouble as long as
the optical parts of the same maker are used with any given
stand. All of the higher objectives, particularly the immer-

MICROSCOPES.
55

(
E LEITZ WETZLAR
FIG. 32.
THE COMPOUND MICROSCOPE.

56
MICROSCOPY.
sion objectives, are, however, very carefully corrected for a
definite tube-length; hence a deviation from this length re-
duces the effectiveness of the objective; therefore it is never
advisable to use a high-power made for a long tube with a
short tube and vice versa. This also applies to low powers,
but in a lesser and scarcely appreciable degree.
We shall now describe the important parts of the stand, be-
ginning with the base. (Fig. 32.)
I. Foot or Base. This constitutes the support of the in-
strument. It should be heavy, with three points of support
so as to insure firmness on any moderately even surface. It
is usually made in the form of a tripod (English foot) or a
horse-shoe (continental foot). It should be of one piece and
sufficiently broad and heavy to permit the body to be placed
horizontally without overbalancing the instrument. Some are
made of japanned iron, others of lacquered brass or bronze,
the latter being preferred.
2. Fixed Pillar.-The fixed portion of the pillar is the
vertical column firmly attached to the base. The upper end
forms part of the joint upon which the movable pillar and the
rest of the instrument is inclined. The pillar is high enough
to make room for the mirror and sub-stage attachments be-
tween the base and stage. Usually it consists of a single piece;
in some of the larger instruments it consists of two parts.
3. Movable Pillar.-This is the part of the pillar above the
joint, and is united to the fixed portion of the pillar by
means of a tightening screw. Just above the joint it is firmly
united with the stage. The upper portion is usually tri-
angular in form and has the arm attached to it and constitutes
part of the fine adjustment. Careful workmanship is neces-
sary to secure an accurately working and durable joint and
fine adjustment.
4. The Stage. This serves to support the objects (slides
or mounts) for examination. It is firmly attached at right
angles to the movable pillar just above the joint. It really
consists of two parts, the transverse arm, which is attached
to the pillar, and the flattened portion or stage proper. The
stage is either circular in form or rectangular, with the upper
surface coated with hard rubber and having a circular opening
in the middle for the purposes of illumination by transmitted

MICROSCOPE..
57
light. In most microscopes for biological work the stage is
immovable, but in the mineralogist's microscope and special
microscopes for chemists the stage rotates upon a central axis
in a horizontal plane. This is for studying polarizing phe-
nomena. Most of the expensive and high-grade biological

E. Leitz
Wetzlar
Bacillus
tuberculosis
(Sputum)
40
20
10
80
FIG. 33.
MECHANICAL STAGE.
microscopes have mechanical super stages. The slide is placed
upon it and the super stage is moved laterally and to and fro by
means of a rack and pinion and screws. By means of this
stage one is enabled to successively examine all parts of an
object or to locate and find any particular area. It will be
found very desirable only in special high-grade work. The
average worker will do very well without it. Furthermore,
complicated mechanical stages soon get out of order.
slide is held in place by means of clips or a clamp.
1
The
5. The Substage. This is a mechanical contrivance at-
tached to the stage and below it, intended for holding in
place various accessories, as Abbé condensers, diaphragm,
tinted glass discs, etc. It is usually attached to the stage
by means of a bar known as the substage bar. The lower
end of this bar has attached to it a ring into which or to
which the accessories are adjusted and which swings upon
the substage bar in a plane parallel to the stage.

58
MICROSCOPY.
6. Diaphragms.-One of the most essential mechanical
parts of a compound microscope is the diaphragm, which is a
contrivance for regulating the amount of light which passes
through the object to be examined. The cheapest and least
useful forms consist of metal discs or caps with holes in the
center through which the light passes. They are either placed
in the stage opening or in the substage ring. It is necessary
to have a number of these discs with different sized openings.
Each time it is desired to change the quantity of light one
disc must be removed and another replaced; this takes time
and is very annoying, particularly if the caps or discs do not
fit well, which is quite generally the case. Furthermore, even
after a change is made, it will generally be found that it is
not quite the illumination desired.
The dome diaphragm and the wheel diaphragm are quite
an improvement upon those just described. Both consist of a
blackened metal disc, near the circumference of which there
are a series of different sized openings which serve to trans-
mit the light. (Fig. 34.) The dome diaphragm differs from
the other form only in that it is arched dome-like so as to bring
the openings as near as possible to the object. (Fig. 35.)

о
O
О
O
FIG. 34.
DIAGRAM OF WHEEL DIAPHRAGM.
These diaphragms are either attached to the stage or to the
substage in such a position that upon rotation the openings
will pass through the central axis of the stage opening.

MICROSCOPES.
59

FIG. 35.

DOME DIAPHRAGM.
By far the best is the iris diaphragm, which consists of a
number of shutters overlapping each other diagonally. By
means of a lever attachment the shutters may be opened or
closed to any desired degree, and that without interrupting
the observation. The opening for transmitted light may thus
& Ope
Comb Opt. Co.
FIG. 36.
IRIS DIAPHRAGM.
be regulated to any size ranging between the two mechan-
ically possible extremes, something which is absolutely neces-
sary for the most efficient work with the higher powers.

60
MICROSCOPY.

0-10-1
FIG. 37.
IRIS DIAPHRAGM AND ABBE' CONDENSER IN POSITION.
The position of the opening of the diaphragm relative to
the object is of some importance. In case the Abbé con-
denser is not used it should be quite close to the object. The
cap and disc diaphragms and the dome diaphragm satisfy
this demand, but the wheel and some iris diaphragms
are usually removed, at least by the thickness of the
stage. An iris diaphragm is quite generally used in
connection with the substage condenser and is placed below
it, regulating the quantity of light which is to pass through
the condenser. Some microscopes are now constructed with
two iris diaphragms, one mounted in the stage and just below
the object slide, the other below the condenser. By this means
very effective illumination may be obtained. The iris
diaphragm in the plane of the stage, which is an innovation by
the Bausch & Lomb Optical Co., is to be used without the
condenser. Its mechanism and position are shown in Fig. 38.
The vertical section in this figure shows the relative position
MICROSCOPES.
61

D
BAUSCH & LOMB OPTICAL CO.
FIG. 38.
TOP VIEW OF STAGE AND STAGE IRIS DIAPHRAGM.
and attachment of the metal and hard rubber portions of the
stage. This position of the iris diaphragm is, however, not
suited for beginners, as they get out of order easily and become
clogged with reagents carelessly used.
7. The Arm.-The arm supports the body of the micro-
scope and consists of three parts. The vertical portion, about
2 inches in length, has a triangular opening into which the
triangular upper portion of the movable pillar fits accurately.
At the upper end is part of the attachment for the fine ad-
justment. A solid transverse portion connects the first part
and another vertical portion which has a wide groove in
which the body rests and the wheels and pinion for the coarse
adjustment near its upper end. All parts of the arm must be
solid, so as to be as free as possible from vibrations.
8. Fine or Slow Adjustment. This is a contrivance for
-
62
MICROSCOPY.
securing slow and accurate focussing. It consists of a fine
screw, with threads perhaps about 1-50 of an inch apart, and
is called a micrometer screw. It is provided with a large
milled head and acts upon the body either directly or indi-
rectly by means of a lever. The upper surface of the head is
often graduated so that the worker may estimate the thickness
of a section, or the depth of focussing, from the number of
revolutions of the head. Fig. 39 will serve to illustrate the
mechanism of a first-class form of fine adjustment. A fine
adjustment, in order to be useful, must work freely up and

a
ИИИИИИИ
19
FIG. 39.
MECHANISM OF FINE ADJUSTMENT.
down without causing any lateral motion of the body, which
may be observed from the fact that there is no lateral dis-
placement of the image of the object under observation.
9. Coarse or Rapid Adjustment. The coarse adjustment
serves to place the tube in a position of approximate focus.
This is done in several ways. In the cheaper grade of instru-
ments such a focus is attained by simply sliding the tube up
and down in an outer sheath and is designated as a sliding
coarse adjustment. It works very satisfactorily after a little


MICROSCOPES.
63
experience and if the tube moves easily. All the higher grade
instruments are or should be supplied with the rack and pinion
coarse adjustment. The pinion, worked by means of a large
milled head at either end of the pinion axle, is attached to the
arm. The rack in which the pinion works is attached to the
body. The cogs or teeth on the pinion and rack should be
diagonal, so as to preclude any possibility of a jerky motion.
The coarse adjustment must work freely and must be free
from irregular vertical and lateral motion. (Figs. 40 and 41.)

FIG. 40.
COARSE ADJUSTMENT, CROSS SECTION.
FIG. 41.
COARSE ADJUSTMENT, SHOWING RACK AND PINION.

64
MICROSCOPY.
IO. Tube or Body.-The tube or body holds the ocular at
the upper end and the objective at the lower end, and rests
upon the arm as already explained. Its length varies, also its
transverse diameter. All are blackened on the inside to pre-
vent the reflection of light. The upper end is provided with
a collar in which the draw-tube slides. The lower end has a
narrowed part screwed into it, called the nose-piece. The
lower end of the nose-piece bears the "society" or "universal
screw." The society screw is so-called because at a meeting
of the Royal Microscopical Society of London it was decided
that a standard screw of 36 threads to the inch and 0.8 inch
external diameter should be adopted for objectives and nose-
pieces. This recommendation by the London Society has been
quite generally adopted by all of the leading manufacturers of
microscopes, so that it is possible to use objectives of any
make with any tube. This great convenience is, however,
more or less annulled by the difference in the tube lengths,
which tends to decrease the working effectiveness of objec-
tions; especially is this the case with the higher power objec-
tives, as already indicated.
II. Pieces.-Besides
Nose Pieces. Besides the single nose-piece just re-
ferred to there are double, triple and quadruple nose-pieces,
which carry two, three and four objectives, respectively.
These multiple nose-pieces are screwed into the tube, as is
the single nose-piece, and revolve so as to bring the various
objectives to the center and in approximate focus. This does
away with the necessity of changing powers by hand, which
consumes too much time for the busy worker, and in addition,
increases the liability to breaking slides and objectives.
Double and triple nose-pieces give entire satisfaction when

LOMBOPT CO.
FIG. 42.
DOUBLE NOSE-PIECE.

MICROSCOPES.
65
accurately and solidly made. Quadruple nose-pieces are gen-
erally too clumsy and may cause some vibratory motion;
furthermore, a quadruple nose-piece is seldom required.

'05 140
BAUSCH
FIG. 43.
TRIPLE NOSE-PIECE.
12.
Draw Tube.-This tube fits accurately into the outer
tube or body at the upper end. The upper end of the draw-
tube receives the eye-piece or ocular and is provided with a
milled collar to be grasped by the thumb and forefinger of the
right hand in moving the tube. On the outside the tube is
highly polished or nickeled to insure an easy motion and is
provided with marks to indicate the distance it is withdrawn.
The inner surface is blackened to prevent reflection of light.
The lower end is often provided with a diaphragm and society
screw.
The draw-tube is used to vary the magnification and to
make correction for variation in thickness of cover-glass. It
can, however, increase magnification satisfactorily with the
lower powers only, as the higher powers, immersion lenses
especially, are carefully corrected for definite tube-lengths, as
already indicated. The tube must work up and down easily,
otherwise a hard push downward may overcome the retaining
power of the coarse adjustment and force the objective
through cover-glass and slide or the compression of the air
within the tubes may lift out the ocular. The tube should be
moved with a rotary motion.
The advantages of a draw-tube are comparatively few with
low and medium powers, and are generally misunderstood and
abused by beginners. A safe rule to follow is never to use the

66
MICROSCOPY.
draw-tube until there is sufficient cause.. For making cor-
rections for variations in thickness of cover-glasses see chapter
on cover-glasses.
b. Optical Parts.
The optical apparatus of the compound microscope includes
the reflecting and refracting parts, as mirrors, condensers, ob-
jectives and oculars. We shall discuss only the important
parts, such as are used with the better and more recently con-
structed instruments. Reflectors known as Lieberkühns, ox-
eye condensers, etc., are now rarely used.
I. Mirrors.-The mirror is attached below the stage and
is almost wholly used for obtaining transmitted illumination
of translucent and transparent objects. Sometimes the mirror
is attached to the transverse arm of the stage upon which it
swings, so that it may be brought above the stage and used in
illumining opaque objects. The compound microscope is, how-
ever, seldom used for examining opaque objects for the reason
that the simple microscope is much more convenient and more
effective for such work.
Mirrors are generally of two kinds, plane and concave, both
being present in the better-class instruments and mounted op-
posite on a common base. The concave mirror is used when
greater intensity of light is desirable. The plane mirror only
is used with the substage condenser.
The mirrors must be capable of being swung in any direc-
tion. It should rotate upon a horizontal axis and again upon
a pivot at right angles to this axis, and finally the mirror bar
should swing through an arc of 100° or more. Usually the
mirror may be moved up and down upon the mirror bar so as
to bring the focus of the concave mirror upon the object.
silver
Mirrors should be made of pure ground glass, not blown
or cast glass. The reflecting surface should be of
instead of amalgam.
pure
2. Condensers.-This is part of the illumining apparatus
of the compound microscope. It differs from the mirror in
that it is a refracting medium instead of a reflecting medium.
It consists of two or more lenses properly mounted and placed
in the substage attachment for the purpose of concentrating

MICROSCOPES.
67
the light for the illumination of transparent objects. The best
are designated as Abbé condensers, so named after Professor
Abbé, who devised them. They are highly essential with
high-power objectives, immersion lenses in particular. It
should be remembered that all of the condensers are constructed
for parallel rays of light, from which it is evident that the
plane mirror only should be used with them. Furthermore,
considerable intelligent judgment and experience is necessary
to use condensers properly. Figures 44 and 45 will serve to

FIG. 44.
ABBE CONDENSER, LOW
APERTURE (1.20).
FIG. 45.
ABBE CONDENSER, HIGHER APERTURE (1.42).
illustrate the mechanism of the Abbé condensers. Fig. 44 shows
one with two lenses and Fig. 45 one with three lenses. A
quarter section is removed in each to show how the lenses are
mounted. The larger the aperture of a condenser the greater
its concentrating power. They should also be carefully cor-
rected for chromatic and spherical aberration. The wider
angle condensers are used as immersion condensers; that is,
it is necessary to place the immersion fluid in contact with the
lower surface of the object slide and the uppermost lens of
the condenser. We shall again recur to the use of condensers
in the chapter on the manipulation of the compound micro-
scope.

68
MICROSCOPY.
It must not be supposed that the Abbé condenser is the only
kind in existence. There are a great variety of others which
were at one time more or less recommended and used, such as
Webster's condenser, Wenham's reflex illuminator, Wenham's
prism, the hemispherical illuminator, the "half-button," Wood-
ward illuminator, Tolles' illuminating lens, spot lens, parabolic
illuminator, and others. The Abbé condensers or slight modi-
fications of the same are, however, the best and are the only
ones in general use at the present time.
3. Oculars or Eye Pieces.-The same ocular and eye-
piece is derived from the fact that this optical part is placed
nearest the eye. It gives a virtual magnified image of the real
and magnified image of the object produced by the objective.
Oculars may be divided into two kinds-those with a field-
lens and those without a field-lens. The former are usually
designated as negative and the latter as positive; because in
the former the image is formed within the ocular, while in
the latter it is formed outside of the ocular. In making this
distinction the term field-lens is used in a restricted sense as
that lens or combination of lenses of the ocular which simply
refracts the image-forming rays of the objective and does not
aid in forming the virtual image of the ocular. Positive eye-
pieces can be used as magnifiers while negative eye-pieces can-
not, or at least not until the field-lens is removed, as other-
wise the objects cannot be brought into focus.
The negative ocular, known as the Huyghenian, is the one
most commonly used and is so named after the Dutch optician,
Huyghens, who first constructed it for use with telescopes.
The following are the essential parts: A metal tube with a
collar at the middle or the upper end; this tube fits into the
upper end of the body or draw-tube. The lower end of the
ocular tube carries the field lens or collecting lens, which is
generally plano-convex and nearly of the diameter of the tube.
The inside of the tube is blackened and at a distance of about
one-third of the way up and at the approximate focus of the
eye-lens is a diaphragm, which is made of hard rubber and
slides within the tube. The diaphragm cuts off the distorted
marginal rays. It also serves to support the eye-piece mi-
crometer. The upper end of the tube carries the eye-lens.
Figure 46 will serve to illustrate the mechanism of the

MICROSCOPES.
69
Huyghenian ocular as well as its optical properties. Usually
two forms of this ocular are found upon the market; the Eng-
lish form, represented in Figs. 46 and 48, in which the tube is
divided into a broader lower portion, which fits into the tube
or draw-tube and a narrower portion, which is usually pro-
vided with a cap. The continental form has a tube which ...
all the way into the body or draw-tube, being held in place by
a collar. (Figs. 47 and 49.)

FIG. 46.
HUYGHENIAN OCULAR (ENGLISH FORM), ILLUSTRATING
OPTICAL
PROPERTIES.
ப
FIG. 47.
HUYGHENIAN OCULAR (CONTINENTAL FORM), OPTICAL
SECTION.

770
MICROSCOPY.
1 IN.
BAUSCH & LOME OPT CO
BAUSCH & LOMB OPT CO.
FIG. 48.
HUYGHENIAN OCULAR,
ENGLISH FORM.
FIG. 49.
HUYGHENIAN OCULAR,
CONTINENTAL FORM.
Of the positive eye-pieces there are several forms more or
less used. The most important is the solid eye-piece made on
the principle of a Stanhope magnifier. Its construction is
clearly shown in Fig. 50. They are made in high powers only;
-inch focus or less. They are the invention of R. B. Tolles
and are called solid because there is only one piece of glass
instead of several distinct lenses. Other positive eye-pieces
consist of two separate lenses or two separate systems of
lenses, more or less corrected for spherical and chromatic
aberration (Fig. 51). The compensating ocular is over-cor-
rected in order to correct any residual error in objectives and
can be used only with such under-corrected objectives.
FIG. 50.
SOLID EYE-PIECE.

MICROSCOPES.
71


FIG. 51.
ORTHOSCOPIC OCULAR.
The magnifying power of oculars varies. High-power
Huyghenian oculars are short, while low powers are long.
That is, there is a ratio between the focal length represented
approximately by the length of the ocular tube and the mag-
nifying power. The designation of the oculars of different
magnification varies with different makers. Some designate
them as A, B, C, D, etc.; others by 1, 2, 3, 4, etc. The most
rational and sensible rating is that adopted by American and
some European manufacturers. They designate the power of
eculars by the equivalent focal length of a single lens having
the magnification of the ocular under consideration. They are
thus indicated as 2 in., 1 in., I in., & in., in., or the equiva-
lent of the metric scale, with the following corresponding
magnifying powers-5, 7, 10, 14, and 20 diameters. Most
manufacturers make the oculars "parfocal;" that is, they are
so constructed that a change of oculars does not disturb the
focus very much.
Eye-pieces should fit evenly into the tube without friction
and yet so closely that if allowed to drop the compressed air
on the inside of the tube will prevent it from falling rapidly.
The lenses must be free from air bubbles, scratches and other
defects.
4. Objectives.-The objective is that part of the compound
microscope which forms the real and inverted image of the
object under examination. It consists of several lenses or sets
of lenses mounted in a metal tube, which is screwed into the

72
MICROSCOPY.
nose-piece. It is by far the most important and with higher
powers the most expensive part of the microscope. The dis-
tinctness and perfection of the image depends largely upon
the accuracy of its construction. Objectives may be divided
into dry and immersion. With the former air is the only
medium between the objective and the cover-glass. With im-
mersion lenses some liquid, as water or oil, is placed between
the objective and the top of the cover-glass. It is very evi-
dent that objectives of comparatively short focus only can be
used as immersion objectives.
Each objective consists of several systems of lenses and as
regards their position are designated as front or anterior, mid-
dle, and back or posterior systems. The front system is the
one nearest the object or farthest from the eye. Each system
may consist of one, two or three lenses. If a single lens, it is
usually of the convex type; if two lenses, one is usually con-
vex and the other concave; if three lenses, there is usually a
middle concave with a front and a back convex lens. The two
and three-lens systems are usually designated as doublets and
triplets. The use of these systems is to correct, as far as pos-
sible, chromatic and spherical aberration, which is largely ac-
complished by using different kinds of glass, as crown and
flint glass, in the construction of the concave and convex lenses.
The construction of lenses for objectives requires special
care. Only the best and purest glass is used, of which the re-
fractive and dispersive power is carefully measured. Since
objectives are expensive and it is desirable to have them dur-
able, that is not affected by temperature, light and chemicals,
glass must be used which is known to withstand these in-
fluences even if it prove inferior optically. The thickness of
the lens and its radius of curvature is carefully determined.
The grinding, especially, requires great care and can be done
only by skilled and intelligent workmen. Only after each lens.
is carefully examined as to the quality of the glass, its thick-
ness, diameter, curvature, etc., is it combined with other lenses
into systems. Each objective is then tested to see if it comes
approximately near the standard set by the maker and the
scientist. It is, however, a fact that no two objectives are
exactly alike; there are slight differences which seem unavoid-

MICROSCOPES.
73
able even with the best mechanical appliances and the most
skillful labor.
Objectives are designated as low power, medium power and
high power, according to their magnification. Low power ob-
jectives have a magnification ranging from 3 to 15 diameters;
medium powers from 15 to 50 diameters; high powers from
50 to an approximate upper limit of 150 diameters.
American manufacturers apply the same mode of rating to
objectives as they do to oculars, a method which is being
largely followed by European manufacturers. The equiva-
lent focus of the low-power objectives is 3 in., 2 in., 1 in., I in.,
2-3 in. and 3-4 in. The medium powers are usually 1-2 in., 2-5
in., 1-4 in. and 1-5 in. High powers are 1-6 in., 1-8 in., 1-10 in.,
1-2 in., 1-16 in., and even higher. The 1-10 in. and above are
used as immersion lenses. Anything above a 1-12-in. objective
is only rarely used; 1-20 in. and 1-25 in. objectives have been
constructed, but they have not been found practical, besides
being very expensive. Figure 52 represents three different ob-
jectives upon which the equivalent focus is given in the metric
system. These figures also show very clearly the different
systems and their relative positions and the construction of the
entire mount of the objective.

24mm
E.LEITZ
WETZLAR
42mm
E. LEITZ
WETZLAR
80 mm
FIG. 52.
OBJECTIVES, SHOWING ARRANGEMENT OF LENSES.
3. DIFFERENT
FORMS OF COMPOUND MICROSCOPES.
There are a large number of different forms of compound
microscopes of special construction and used for special pur-
poses. We shall briefly describe a few of the more import-
ant and mention their special uses.
1. Petrographical or Lithological Microscopes.-These are
I.

74
MICROSCOPY.
especially adapted for studying the structure of rocks. A
petrographical microscope is provided with a graduated re-
volving stage and a centering apparatus for accurately center-
ing the objective upon the stage. It is also provided with
polarizer and analyzer for studying the polarizing phenomena
of rock crystals.
2. Inverted or Chemical Microscope.-This microscope was
devised by J. L. Smith. The stage is placed above the ob-
jective, which is inverted and optically connected with the tube
by means of a prism. It was especially intended for chemical
investigations. Its special advantages are in many instances
questionable. Figure 53 represents a chemical microscope as
constructed by the Bausch & Lomb Optical Company. This
instrument may be converted into an ordinary microscope if
desirable.
HICINBOTHAM
ROCHESTER NY
FIG. 53.
INVERTED OR CHEMICAL MICROSCOPE.
3. Demonstration Microscope.-This microscope consists
of a tube with sliding coarse adjustment and is provided with

MICROSCOPES.
75
low-power objectives and oculars. The stage with clips is at-
tached to the lower end of the tube. Illumination is obtained
by directing the tube toward the source of light. It is used in
showing objects requiring only low magnification (50 to 80
diameters) to an entire class. It can readily be passed from
hand to hand without disturbing the object or focus. Figure
54 shows its construction.

BAUSCH & LOMB OPTICAL Co.
ROCHESTER NY & NEW YORK.
FIG. 54.
DEMONSTRATION
MICROSCOPE.
4. Portable Microscope. This is really a misnomer, since
all microscopes are portable. Those specially designated as
portable are so constructed that the various parts of the stand,
as base, arm, tube, etc., may be readily separated and packed in
a small case. In some the lower end of the pillar screws into
the lid of the case, which serves as the base. Naturally some
of these microscopes are liable to get out of order and are
more subject to vibrations. Some are well made and very
convenient.
5.
Binocular Microscopes.-A binocular microscope differs
from the ordinary monocular microscope in having a bifurcated
tube with two oculars in adjustable draw-tubes. The rays of
light passing through the objective are divided into two parts
by a prism placed above the objective. The prism can be re-
moved and the microscope used as a monocular.
Ever since the discovery of the compound microscope efforts
have been made to produce efficient binoculars, but apparently
with little success, for only since 1853 have any good working
binoculars been made. Shortly after this date they were very
extensively manufactured and very highly recommended, but

76
MICROSCOPY.
at no time were they ever favorably received by scientists in
general. At the present time scarcely any are in use. They
are efficient only with the lower powers (50 to 100 diameters).
Their special advantage lies in giving a stereoscopic view of
objects. (See Fig. 90 illustrating stereoscopic vision.) Figure
55 will serve to illustrate the construction of the body and
optical parts of the binocular compound microscope.
FIG. 55.
BINOCULAR MICROSCOPE.

IMPORTANT ACCESSORIES.
77
CHAPTER IV.
IMPORTANT ACCESSORIES.
Under this heading we shall describe the structure and use of
a few of the more important accessories employed in working
with the compound microscope. There are a large number of
other accessories for special purposes to which no reference
will be made, as that would not be called for in a work in-
tended for simple biological work. The substage condenser,
mechanical stage and several other accessories have already
been mentioned; others will be referred to in Chapter I of
Part II.

I. MICROMETERS.
Micrometers or micrometer scales are used for measuring
objects examined under a microscope. Two kinds are em-
ployed the stage micrometer and the eye-piece micrometer.
The former consists of a circle of glass which has ruled upon
it one millimeter divided into one hundred equal parts. This
thin circle of glass is mounted on a metal plate and is placed
upon the stage of the microscope when it is desired to use it.
The eye-piece micrometer is a circle of glass, sometimes
mounted in a ring of hard rubber, and has ruled upon it five
millimeters, usually divided into one hundred equal parts. The
number of divisions is immaterial and within certain limita-
tions also the distance between the lines, since the value of this
distance must first be determined in terms of the known value
of the spaces on the stage micrometer. The eye-piece mi-
crometer rests upon the diaphragm of the eye-piece. Gener-
ally it will be found necessary to move the diaphragm up or
down (usually down) in order to bring it in exact focus. Re-
move the eye-lens and insert the micrometer; replace the eye-
lens and insert the ocular in the tube; adjust the light and see
if the lines on the scale appear distinctly. If not, the scale is
not in proper position. It will either have to be moved up or
down. The direction may be determined by partially unscrew-

778
MICROSCOPY.
ing the eye-lens; if the lines become more indistinct it indi-
cates that the micrometer is still too near the eye-lens, hence
the diaphragm must be pushed down farther. Repeated trials
are necessary before the scale will be in exact focus. The
ruled lines of the scale should be uppermost. It does not
matter, however, in what direction the scale is placed on the
diaphragm, as it may be turned in any position of the horizontal
plane by simply rotating the ocular in the tube. Any object or
portion of an object can be brought in proper position to be
measured by moving the slide upon the stage.
As already indicated the measuring value of the ruled lines.
on the eye-piece micrometer must first be determined in the
value of the stage micrometer. The unit for microscopical
measurements is the one one-thousandth part of one millimeter,
known as a micro-millimeter, mu or micron, and usually desig-
nated by the Greek letter μ. An object is said to be so many
mus or microns in diameter.
The measuring value of the eye-piece micrometer is deter-
mined as follows: The stage micrometer is placed in focus on
the stage. It is known that the ruling is one millimeter divided
into one hundred equal parts; therefore any two lines are ex-
actly ten apart. If the eye-piece micrometer is placed in
position and it is found that the one hundred lines ruled upon
it cover, for example, eighty of the stage micrometer, then
we know that the distance between two lines of the eye-piece
micrometer is 8μ; that is,
(1) 100 spaces of eye-p. mic.-80 spaces of stage mic., or
800μ; therefore
(2) one space of eye-p. mic.-1-100 of 8ocu, or 8u.
This is the relative measuring value of eye-piece microm-
eter for a low-power combination of objective and ocular. A
higher power combination may give the following:
(1) 100 spaces of eye-p. mic.-13 spaces of stage mic., or
130μ; therefore.
(2) I space of eye-p. mic. 1-100 of 130μ, or 1.3μ.
Other combinations of oculars and objectives would give
other results. It should be remembered that these values must
be determined for each instrument and each separate com-
bination and for varying lengths of the draw-tube. For in-
stance, the objectives and oculars of the same designations,

IMPORTANT ACCESSORIES.
79
but of different instruments, will give slightly varying results
for the micrometer value.
The measuring value for two given lines of the eye-piece.
scale determined in terms of the stage micrometer is a constant
and should be carefully recorded for each instrument, in order
to avoid confusion. All of the actual measurements given in
the number of spaces or lines must be multiplied by this con-
stant. For example, with the higher power the diameter of a
given cell is found to subtend ten spaces of the eye-piece mi-
crometer, therefore the cell measures 10X1.3μ, or 13μ in
diameter.
There are also special micrometer eye-pieces and special eye-
piece micrometers which we shall not describe. They have no
great redeeming qualities and are expensive. The micrometer
described above, used with the ordinary Huyghenian eye-
pieces, will be found perfectly satisfactory for even the most
delicate and accurate work.
The stage micrometer alone may also be used to measure
microscopical objects, but it is inconvenient and lacking in
accuracy. The object is placed upon the stage and after being
carefully focussed the camera lucida is attached and the ob-
ject to be measured is outlined upon a piece of paper, after
which the stage micrometer is placed upon the stage and its
lines made to coincide with the figure upon the paper. Thus,
if two spaces of the micrometer indicate the distance across the
object its diameter would be 20μ, since the distance across
one space equals 10μ. A simpler and equally accurate method
is to determine the value of an ordinary millimeter ruler in
terms of the stage-micrometer and use that in measuring the
object direct. Focus upon the stage micrometer; look at it
with the left eye, hold the millimeter scale parallel to the mi-
crometer scale and on a level with the stage and look at it
with the right eye; observe how many spaces of the millimeter
rule are contained in one space of the micrometer scale. The
result will give the measuring value of the divisions of the
ruler. We will suppose that ten divisions (one centimeter)
of the millimeter rule are contained in one division of the
micrometer, which, as already stated, is equal to 10μ, we
therefore know that the measuring value of one millimeter of
the ruler, for that particular combination of objective and


80
MICROSCOPY.
ocular, is Iμ. Incidentally, it also shows that the magnifying
power of that combination is 1000 diameters. This ruler may
now be used in measuring microscopic objects direct. In
making these determinations it will, of course, be necessary to
use both eyes, one to observe the object, the other to observe
the divisions of the ruler, which must always be held near the
object and on a level with it. If, for example, 20 millimeters
seem to subtend the diameter of a cell we know that it is 20μ
in diameter.
II. CAMERA LUCIDA.
The camera lucida is an optical contrivance attached to the
upper end of the tube of the microscope, which enables the
worker to see the image of the object projected upon a sheet of
paper. It is used principally in making drawings of the ob-
jects under examination. There are a great variety of camera
lucidas upon the market. Some are simple and cheap, while
others are complex and expensive. Some are used with the
body of the instrument in a vertical position, while others are
used with the body inclined or horizontal. All are alike in that
they are optically so constructed as to apparently project an
image of the object upon paper, where it may readily be traced
in outline by means of a pencil.
The simplest form, known as the neutral tint reflector, is
used with the tube or body of the instrument inclined. It con-
sists essentially of a thin film of glass placed in front of the eye-
lens and inclined to it at an angle of 45°. Figure 56 illustrates
this type of reflector, which may be fastened to the rim of the
BAUSCH-LOMB
OPT.CO
FIG. 56.
FIG. 57.
NEUTRAL TINT REFLECTOR.
WOLLASTON'S CAMERA LUCIDA.

IMPORTANT ACCESSORIES.
81
ocular. The emergent rays from the eye-lens are reflected
from the upper surface of the glass film and apparently pro-
jected upon the paper beneath. The name "neutral tint re-
flector" is derived from the fact that the glass film is tinted
to make a better reflector, but it must not be too highly tinted
else the apparent image cannot be seen distinctly upon the
paper.
A student of average mechanical ability may construct a
reflector for himself. Mount an ordinary thin cover-glass
(No. 1) in a wooden, cork, or wire frame suitable to be ad-
justed to the ocular so that the cover-glass will be at an angle
of 45° in front of the eye-lens.
Another and more efficient form is the Wollaston camera
lucida, also used with the tube inclined. It consists of a
prism which refracts the rays emerging from the eye-lens and
projects them into the eye, so that the apparent image may
be traced upon the paper.
Piffard's drawing prism projects the image directly upon the
paper, where it is viewed and traced by the observer. Figure
58 illustrates this prism. The figure to the right presents a

BAUSCH & LOMB OPT CO
FIG. 58.
CAMERA LUCIDA WITH DOUBLE PRISM.

82
MICROSCOPY.
ELLEITZ WETZLAR
FIG. 59.
DRAWING PRISM IN POSITION.
sectional view, illustrating the optical effect of the prisms.
Figure 59 illustrates the use of a prism with tube inclined.
The best camera lucida is the so-called Abbé camera lucida;
at the same time it is also the most expensive and the most
complex in structure. The prism is mounted in a closed
cylindrical box, which is so constructed that it may be swung
in and out of position over the eye-piece. To the right pro-
jects the mirror bar, which carries the mirror for projecting
the image of the pencil and paper. That is, with this camera
lucida the observer beholds an image of the object under ex-
amination as well as the image of pencil and paper. It is not
necessary to give a full description of the mechanical parts
and the optical principles of the Abbé camera. This knowledge
is best acquired from actual use of the instrument. Figure
60 shows this camera lucida attached to the upper end of the

IMPORTANT ACCESSORIES.
83
The part in outline is the prism holder
swung out of position when not in use.
microscope tube.

Bausch & Lomb Optical Co.
ROCHESTER, N.Y.
FIG. 60.
ABBE CAMERA LUCIDA.
There are a considerable variety of other camera lucidas
which it is not necessary to refer to. The ones described are
the more common forms in use. It must also be mentioned that
there are a large number of workers with the microscope who
never use a camera lucida. The best cameras have their ob-
jectionable features, the most prominent of which are indistinct-
ness of outline and spherical distortion, defects which so far
have been very unsatisfactorily met.
The following suggestions will prove useful in working with
the camera lucida:
I. The object must be in exact focus and properly
illumined.
2. The paper upon which the drawings are to be made.
should be in a subdued light. The amount of shading will de-
pend upon the magnification and the intensity with which the
object is illumined.
3. The
The paper should lie at right angles to the axial portion
of the projected image, otherwise the drawing will be dis-
torted.
4. The dimensions of the drawing will vary with the
variation in distance between eye-piece and paper. The stan-

84
MICROSCOPY.
dard or usual distance is ten inches. This distance is not
absolutely required, but some standard must be chosen and
adhered to, otherwise the drawings will vary in size.
5. The size of the field reflected by the prism should be
the same as that seen through the ocular. If this is not the
case the prism is either too near or too far
and must be properly adjusted.
III.
from the eye lens
MICROPHOTOGRAPHIC APPARATUS.
Within recent years microphotography has become quite
popular, although the amount of satisfactory work that is
actually done is comparatively small. Most of the work pub-
lished is very poor, while in a few instances excellent results
have been obtained, particularly in photographing bacteria.
The persistent efforts that are being made will no doubt bring
about great improvements in this special field of work.
At the present time the vertical apparatus is very largely
used. The old-fashioned horizontal camera is inconvenient.
From inspection of Fig. 61 it will be observed that there are
no lenses with the camera, an ordinary microscope serves to
project the image of the object upon the sensitive plate. It
is by far the simplest apparatus on the market; anyone can
secure fairly good microphotographs by following the simple
directions given below, which are largely taken from a small
work by L. Leitz. The apparatus consists of the following
parts: An iron base supports any microscope with good
achromatic objectives and Huyghenian oculars. To the left
the base has attached to it a vertical pillar consisting of two
parts gliding upon each other and fixed in any position by a
clamp screw. The adjustable camera with the usual acces-
sories is attached to the upper part of the pillar. The follow-
ing directions will be sufficient to explain the use of the
apparatus:
I.
Place the object to be photographed upon the stage of
the microscope and focus it carefully; then place the micro-
scope upon the base of the camera stand so that the tube will
be exactly under the neck of the camera. The microscope
tube must be vertical and the draw-tube should not be used.
The camera can be adjusted to suit the varying heights of
different microscopes.

IMPORTANT ACCESSORIES.
85
2.
Draw the pocket attached to the neck of the camera
over the microscope tube so as to shut out lateral light.
3.
Place the ground glass plate in position, secure the

CUC
FIG. 61.
VERTICAL
MICRO-PHOTOGRAPHIC
APPARATUS.
proper illumination by means of the microscope mirror. The
field of view should be in the middle of the plate.
4.
The size of the field of view can be regulated by chang-
ing the distance between the ocular and the wheel diaphragm
of the camera, which is situated in the neck of the camera tube.

86
MICROSCOPY.
5. Focus the object approximately and by moving the slide
bring the object to be photographed in the proper position.
6. Remove the ground glass plate and insert the transpa-
rent plate, the lower surface of which is marked with a cross
at the center. A simple lens is placed above the cross and
carefully focussed upon it. While the eye is beholding the
field of a view through the lens a sharp focus of the object is
obtained by means of the fine adjustment.
7. After this the entire apparatus should be left untouched
for ten or fifteen minutes. If after this period the object is still
in exact focus it is ready to be photographed. Remove the
glass plate and put in its place the plate-holder with the sensi-
tive plate. Place some opaque object before the mirror of
the microscope. When all is ready expose the plate by re-
moving the plate-holder cover and the opaque object in front
of the mirror.
8. The time of exposure depends upon the source of light
and the magnification employed.
Sunlight is the best for this work. A ground glass plate is
placed in front of the mirror so that no direct rays may fall
upon it. Stained objects require the use of orthochromatic
plates instead of the ordinary gelatine dry plates. These plates
require the yellow glass disc, which is placed in the special
substage attachment. The time of exposure for sunlight and
yellow disc when the 1-12 inch oil immersion and No. 5 ocu-
lar is used is one minute. Lower powers require less time.
The same combination with gaslight equal to a Wellsbach bur-
ner requires ten minutes exposure; if an oil lamp with re-
flector is used, twenty minutes or more are required.
As far as the technique of microphotography is concerned.
it need only be stated that it is essentially the same as in ordi-
nary photography. A dark chamber, red light, developers,
etc., etc., are necessary. The Leitz Optical Company of Wetz-
lar, Germany, have for free distribution a beautiful little work
fully explaining the technique, which is very simple. This
booklet is accompanied by four microphotographs, which well
illustrate the range and scope of this kind of work.
In conclusion it may be stated that experience is necessary
before effective work can be done and we would advise the


IMPORTANT ACCESSORIES.
87
beginner not to become discouraged because of frequent
failures.
IV. TEST OBJECTS.
Some convenient means for testing the optical qualities
of objectives is very desirable. Various objects properly
mounted are used for this purpose; of these the most carefully
prepared may be had of dealers in microscopical supplies and
the financial outlay is fully warranted in consideration of the
fact that it will enable one to fully test the working qualities
of any instrument. They should be kept near at hand in order
to be used as an aid in judging what details should be re-
vealed in the material under examination. Any object may,
however, be used as a test object, provided it is carefully
mounted and the student is sufficiently familiar with its his-
tological details so as to know whether or not they are prop-
erly disclosed to the eye by the lenses in use. For example, if
one desires to test the comparative merits of similar com-
binations of objectives and oculars of different makers any
mounted object suited to the magnifying power of the combi-.
nations will prove satisfactory. If, however, it is intended to
test any one combination irrespective of its comparative merit
the beginner is obliged to use test objects of which the his-
tological characters and markings are known and fully
described. For low powers the probocis of the blow-fly is
usually recommended; also the scales of butterflies. These
objects should appear distinct in detail, flat and free from
marginal coloring. For medium powers stained micrococci
and bacteria are generally used. Starch grains are also very
useful. For high powers, especially immersion objectives,
various diatoms are used. The use of these test objects will
be better understood from a description of the construction and
use of the test plate. This plate consists of a series of twenty
diatoms arranged in the order of the relative number and
fineness of the markings or lines on the silicious covering.
The test consists in finding how many lines to the inch the
combination under consideration will reveal. It is not essen-
tial to have the twenty diatoms. The number of lines or
strix to the inch vary from 4,000 to 95,000. Out of the
twenty diatoms Navicula lyra Ehrbg., with 23,000 to 30,000


88
MICROSCOPY.
striæ to the inch, would serve for testing low powers; Pleuro-
sigma augulatum Sm., with 44,000 to 49,000 lines to the inch,
for medium and high power dry objectives, and Amphipleura
pellucida Ky., with about 95,000 striæ to the inch, for im-
mersion objectives.
The Nobert test-plate consists of a plate of glass with lines
ruled upon it ranging from 10,000 to 200,000 to the inch. This
is particularly valuable in testing for spherical aberration. The
ordinary micrometer scales may also be similarly used.
To bring out the full working and resolving capacity of
the compound microscope a full knowledge must be had of the
relationship of the mechanical parts to the optical parts. Only
too often this is not understood and as a result the instrument
is decried as inadequate or lacking in good qualities, which it
really possesses.
In the use of high powers, dry and immersion, special at-
tention must be given to the thickness of cover-glass, as shall
be explained later; further lateral illumination, fine adjust-
ment, use of substage condensers, and other adjuvants to suc-
cessful work must be properly understood and manipulated.


COMPOUND MICROSCOPE,
89
CHAPTER V.
OPTICAL AND WORKING PROPERTIES OF THE
COMPOUND MICROSCOPE.

In a general way the optical properties of the compound
microscope have already been explained. It remains to dis-
cuss more fully the highly important optical and working prop-
erties of the microscope in order that the proper use of the
instrument may be understood.
I. ABERRATION.
The meaning of aberration in microscopy has already been
explained. So far opticians have been unable to construct
lenses or combinations of lenses entirely free from chromatic
and spherical aberration, but the best objectives and oculars
are so constructed that the residual aberration is not suf-
ficient to interfere with effective work.
I. SPHERICAL ARERRATION.
In discussing foci and images formed by spherical lenses it
was assumed that rays of light emitted from a point would
again be brought to a single point after refraction. Actually
this is not the case. Practically it is true of lenses whose
apertures do not exceed 10° or 12°; that is, the distortion of
focal points after refraction is inappreciable when the angular
aperture is small. When the aperture is large, that is, con-
siderably more than 12°, the marginal rays are refracted to
points nearer the lens than rays which pass nearer the axis.
This phenomenon is known as spherical aberration by refrac-
tion and is similar to spherical aberration by reflection pro-
duced by curved mirrors. Fig. 59 will serve to explain the

90
MICROSCOPY.
a b
C

FIG. 62.
REFRACTION OF LIGHT IN AN UNDERCORRECTED LENS.
FIG. 63.
REFRACTION OF LIGHT IN AN OVERCORRECTED LENS.
spherical aberration of a non-corrected or undercorrected com-
bination of lenses. The marginal rays are brought to a focus
at a, while the rays nearer the axis are brought to a focus at b
and c, points more remote. Fig. 63 serves to explain
spherical aberration by an overcorrected combination of lenses.
Inspection of these figures and comparison with Fig. 62 will
serve to make clear the differences.
Spherical aberration is very objectionable in lenses be-
'cause it greatly lessens the sharpness and definition of an
image. The object will appear sharply defined in the center,
but indistinct at and near the margin, or vice versa in over-
corrected lenses. Fig. 64 will serve to explain the optical
effects of spherical aberration. A is a ruled square as seen
by a corrected lens or combination of lenses; the lines all
appear straight. With an undercorrected combination the
same ruled square would appear as in B, while with an over-
corrected it would appear as in C.
As already explained, diaphragms are used to cut off the
defective marginal rays in the cheaper lenses and lower powers.

COMPOUND MICROSCOPE.
91



A
B
FIG. 64.

OPTICAL EFFECTS OF SPHERICAL ABERRATION.
In telescopes the spherical aberration is corrected by grinding
the proper curvatures to the surfaces of the lens; in micro-
scopes by using sets of wider back lenses. In another chap-
ter we shall also refer to the effect that cover-glasses of dif-
ferent thickness have upon aberration.
2. CHROMATIC ABERRATION.
In order to understand chromatic aberration it must be re-
membered that a ray of ordinary white light consists of seven
simple colors, which are capable of being separated by vari-
ous refractive media, as prisms and lenses. These simple
colors are violet, indigo, blue, green, yellow, orange and red.
Chromatic aberration is due to the unequal refrangibility of
these simple colors. A lens not only refracts the ray of light,
but also decomposes it, as does a prism. Chromatic aberra-
tion is more marked in proportion as the lenses are more con-
vex and as the point at which the rays are incident is farther
from the optical axis. From this it is evident that dispersion
increases with the increase in refraction. Newton concluded
that refraction was impossible without dispersion, but it has
long been proven that this is not the case; that is, refrac-
tion does not vary in the same ratio as dispersion, hence re-
fraction is not destroyed at the same time as the dispersion,
which makes it possible to construct lenses which are prac-
tically achromatic. Fig. 65 serves to illustrate chromatic
*In reality there are only three primary colors; red, yellow
and blue. The others are mixtures of these.

92
MICROSCOPY.
5
S
FIG. 65.
CHROMATIC ABERRATION.
aberration by lenses. The ray S incident upon the lens is
refracted, but at the same time it is also separated into its
simple component colors, red being least refrangible, while
violet is most refrangible, the other colors occupying inter-
mediary positions. This ray could therefore not enter into
the formation of a clear image. By combining lenses having
different refracting angles and different dispersive powers,
white light may be refracted without being dispersed. To il-
lustrate, let us suppose that two lenses, one a double convex
که
A B
ACHROMATIC
FIG. 66.
COMBINATION OF LENSES.
lens of crown glass, the other a plano-concave of flint glass,
are so ground that each will separate the red ray 1° from
the violet ray. In order that this may be the case the crown
lens, whose dispersive power is .036, must be so ground that
it will refract light 27°42', because
flint lens, whose dispersive power is .052, must refract the ray
1°
.036
=27°42'. The

COMPOUND MICROSCOPE.
93
I°
.052
19°12', because
19° 12'. If the lenses are placed
as shown in Fig. 66, the crown lens (4) will refract the ray
downward 27°42', and the flint lens (B) will refract it up-
ward 19°12'. Now the dispersive powers of the two lenses,
acting in opposite directions and with the same force, will
just neutralize each other. The colors are therefore reunited
in the emergent ray, and still it is refracted downward 8°30'
(27°42'- 19°12'-8°30'). This, however, corrects only two
prismatic colors, namely red and violet, leaving a residue of
other colors as green and blue, (secondary spectrum); further
correction is therefore necessary to get rid of most of the blue
and purple and part of the green. Opticians make the correc-
tion for those colors which affect the eye-sight most powerfully,
namely the orange, yellow and green comprised between the
Frauenhofer lines D and F (Fig. 67). In the so-called apo-
chromatic lenses, especially recommended for use in micro-
photography, correction is also made for the secondary spec-
trum by the use of a very complicated system. As a rule the

вс D
E
F
G
FIG. 67.
INTENSITY CURVE OF THE RAYS OF THE SPECTRUM.
secondary spectrum does not prove objectionable in micro-
scopic work. It becomes more pronounced when high-power
objectives with high-power oculars are used, especially with
oblique illumination.
II. APERTURE.
By aperture is meant the opening made by the extreme
marginal image-forming rays of the objective meeting at the
focus. It is necessary to say image-forming rays since ob-
jectives may be constructed so as to transmit rays of light
which do not enter into the formation of an image and hence
could serve no purpose and could not enter into the consider-

94
MICROSCOPY.
ation of aperture. Remarkable as it may seem, only a com-
paratively few years ago considerable controversy existed as
to aperture, some maintaining that high-angle objectives were
best, while others favored low angles. Since light is radiated
from an object in all directions, it certainly requires no great
mental effort to comprehend the advantages of wide or high-
angle objectives. The greater the number of rays of light
which enter into the formation of an image the more ac-
curate will be the presentation of details. Different objec-
tives of the same magnifying power but of different angular
apertures will have resolving powers directly proportional to
the apertures.
In speaking of aperture two terms are employed, namely
angular aperture and numerical aperture. The numerical
aperture is found by multiplying the refractive index of the
medium interposed between the lens and the cover-glass by
the sine of half the angular aperture. If we let i equal the
refractive index of the immersion fluid, then
with dry lenses i=1.00,
with water-immersion lenses i
1.33,
and with oil-immersion lenses i = 1.52.
Numerical aperture may be expressed by the following
formula:
Na
= i
sin a
2
This numerical aperture represents the working efficiency
of lenses. The natural and not the logarithmic sine is under-
stood.
It should be kept clearly in mind that there is a close
relationship between the aperture, resolving power, illuminat-
ing power, penetrating power, working distance, focal distance,
magnification and price of an objective. This shall be sum-
marized in a table at the close of this chapter. Yet it must
not be supposed that the optical properties of objectives vary
in exactly the same ratio; some allowance must be made in
favor of one or the other property, that depending some-
what upon differences of workmanship, the intended special
purposes of different objectives and the somewhat varying
opinions of opticians.


COMPOUND MICROSCOPE.
95
The advantages of wide aperture become very apparent
upon comparing the efficiency of the same lens when used
dry and when used as an immersion lens. It need hardly be
stated that it is not practicable to use low-power objectives,
which always have a comparatively long focal distance, as
immersion lenses. By placing some transparent substance be-
tween the cover-glass and the front lens of an objective we
may cause rays of light to become optically effective which
would have been lost or useless otherwise; provided, the im-
mersion fluid has a higher refractive index than air. Fig.
68 will aid in making clear the differences in the illuminating
power of dry lenses and immersion lenses; remembering
that the resolving power or real efficiency of lenses is pri-
marily dependent upon the quantity of light emanating from
the object which the lens is capable of utilizing in the for-
mation of the image. L represents the front lens of an

L
OA
G
FIG. 68.
PROPERTIES OF IMMERSION LENSES.
objective, G the cover-glass, o p the optical axis, A dry objec-
tive, to compare with O, which is to illustrate the immersion
objective. In the dry objective it is plain that the rays of
light between b c and c S will not enter the lens. By inter-
posing the immersion fluid (O), for example cedar oil, which
has the same refractive index as the cover-glass (1.5), the
ray S', instead of emerging parallel to the incident ray, is

96
MICROSCOPY.
continued in a line with the ray in its passage through the
cover-glass, thus causing an angle of light equal to b c S to be
added to the ordinary air angle o a b. This additional quan-
tity of light increases the brightness and distinctness of the
image.
From time to time opticians have made bold statements as
to the wideness of aperture that they have given to lenses,
some maintaining they had made lenses with an air angle "in-
finitely near 180°." While we do not question the statement,
and admitting that some wide-angle lenses have been made,
the fact remains that makers of microscopes very rarely con-
struct objectives with a greater air angle than 140°; in fact,
it is difficult to reach even this angle; 130° is practically the
limit. It is therefore through the use of immersion fluids
that an increase in aperture is readily obtained. For instance,
an objective with an air angle of 130°, having a numerical
aperture of .91, attains an aperture of 1.43 when used as an
oil immersion.
The numerical aperture of lenses is given in catalogues so
that it is not necessary to determine the angular aperture.
In conclusion, we shall, however, give a few methods for de-
termining the angular aperture.
I. Focus upon any object, incline the tube to a hori-
zontal position; place a lamp with a small wick or burner in
front of the objective and a short distance from it. The flame
must, of course, be on the same level as the tube. Now move
the lamp to left and right of the axis, mark the extreme points
where the illumination causes distortion or distinct blurring
of the image; this will mark the opening of the efficient cone
of light. The angle may then be measured by means of a
protractor.
2. Focus upon an object as before, incline the tube hori-
zontally, remove the mirror and put in its place a small toy
candle and proceed as above. Instruments with the mirror
swinging upon an axis in the plane of the stage are very con-
venient for this method, especially if the circular part of the
axis is graduated.
3. There are special instruments (apertometers) upon the
market by means of which the angular aperture is measured.
Methods 1 and 2 are lacking in accuracy, but they give an


COMPOUND MICROSCOPE.
97
approximate idea of the relative wideness of apertures of the
various objectives used.
III. FOCAL DISTANCE AND WORKING DISTANCE.
By working distance is meant the distance between the object
and the front lens of the objective when the object is in focus.
It is in no direct relation to the focal distance; only it may
be stated that the working distance is always less than the
equivalent focal distance. For instance, an objective marked
as having a focal distance of one-fourth inch will have a
working distance less than one-fourth inch; a one-twelfth-
inch objective focusses much nearer than one-twelfth inch.
Working distance decreases with the increase of mag-
nification and of aperture. It was at one time believed that
aperture and working distance varied according to a fixed
law, but that has proven to be wrong.
Considerable controversy has arisen from time to time
as to the importance of working distance. Since working dis-
tance depends upon magnification and aperture, the question
really is, are narrow or wide apertures most desirable in ob-
jectives? Since it has been proven that wide apertures are
more effective, it is but natural to be in favor of compara-
tively short working distance. Working distance becomes an
important factor only with the high powers, since due al-
lowance must be made for the thickness of cover-glass. The
immersion fluid very materially increases the working dis-
tance, thus rendering it possible to use cover-glasses with high
powers.
There seems to be no rational argument in favor of long
working distance after an object is once carefully mounted
and covered with a suitable cover-glass. Of course, in dis-
secting microscopes it is desirable to have considerable space
between lens and stage in order to permit of manipulations
in dissecting. Also in the examination of opaque objects
considerable working distance is desirable to permit of the
proper illumination.
The available working distance is frequently decreased by a
projecting metal tube of the objective for the protection of
the front lens, and by the thickness of the cover-glass.


98
MICROSCOPY.
In short, working distance is a quality of objectives de-
pendent upon their construction and therefore is really not
worthy of special discussion; but owing to the fact that in
rare instances it does become a factor of some significance
it is explained somewhat in detail.
Focal distance as applied to objectives is a meaningless term
unless its significance is understood. For instance, an objec-
tive is marked one-fourth inch, which means that the com-
bination of lenses in it have the same magnifying power as
a single lens of one-four-inch focus. It does not give the
magnification nor, as indicated above, the working distance.
It does point out a way for finding the magnification which
is as convenient as though the actual magnifying power were
given. In order to determine the possible magnifying power
of an objective divide 10, the magnifying power of an ob-
jective (or lens) of one-inch focus, by the number on the
objective. Provided, of course, that this number represents
the optical value as in the American objectives. Thus an
objective marked two inches would have a magnifying power
of 102 5 diameters; a one-sixth inch, 10-1-6-60 di-
ameters; a one-eighth inch, 101-8-80 diameters; a one-
twelfth, 101-12 120 diameters, etc. Eye-pieces that are
rated in the same manner makes it easy to determine the theo-
retical magnification of any combination of objective and ocu-
lar; thus a one-fourth inch objective with a one-inch ocular
could give a magnifying power of 40×10 diameters, or 400
diameters. It should, however, be remembered that tube-length
modifies the magnification of any system of objective and
ocular.

IV. PENETRATING POWER.
By penetration is meant the power of seeing differ-
ent planes of an object at the same time. It depends upon
the aperture and amplification of the objective and the depth
of accommodation of the eye. Penetrating power decreases
as the aperture and magnifying power increases, and is there-
fore a natural condition dependent upon these optical qual-
ities.
Since the accommodating depth of the eye varies with dif-
ferent persons, penetrating power is also variable. It should

COMPOUND MICROSCOPE.
99
furthermore be remembered that while the focussing depth of
the lens or objective varies with the aperture and amplification,
the accommodation of the eye does not vary in the same ratio.
Low powers have therefore great penetration, while medium
and high powers have proportionately low penetration. The
lack of sufficient penetration in high powers is the principal
reason why objects do not appear in perspective. For ex-
ample, a cylindrical cell of Spriogyra appears rectangular in
outline. This lack of penetration also makes it necessary for
the student to continually manipulate the fine adjustment in
order to bring different planes of the object into focus. Since,
as indicated, penetration is an unavoidable condition, depend-
ing upon the optical properties of lenses, it cannot be avoided,
and any further discussion would be useless. The relationship
between aperture and penetration is represented by the fol-
lowing formula: P= ; the penetrating power of an ob-
jective with a numerical aperture of 1.00 being taken as the
standard. Thus penetration of an objective with a numerical
aperture of 1.30 would be

I
Na
I
= .769.
1.30
V.
DEFINITION.
Definition depends upon correction for spherical and chro-
matic aberration. An objective with perfect definition will
cause the object to appear distinct from the center to the
very edge; there must be no distortion or coloration. Since
aberration and correction for aberration have already been
discussed, definition requires no further mention.
VI. RESOLVING POWER.
Resolving power makes it possible to show minute details.
of structure and depends upon magnification, aperture and
correction for spherical and chromatic aberration. The greater
the resolving power the greater the number of lines to the
inch that can be distinguished. The normal eye can distin-
guish about 200 lines to the inch. If, therefore, a microscope
gives an amplification of 400 diameters it should be possible to

100
MICROSCOPY.
distinguish 400 times 200 lines, or 80,000 lines to the inch;
provided, resolving power is perfect.
l
Resolving power is proportional to angular aperture and to
the length of light waves, and is indicated by the formula
= R, in which R= resolving power, l-wave
2(Na)
length and Na-numerical aperture. For example, with
white light the wave length (1) is about .527μ.
(1) is about 527μ. If an objec-
tive has a numerical aperture 1.40, the above formula would
become R=
=.188μ; that is, two fine lines or two
.527μ
2.80
points on an object must be .188 μ apart in order to recognize
them as distinct. In one inch there are 25,400 μ, hence we
could distinguish 25,400.188-135,106 lines to the inch.
Thus if the numerical aperture of an objective is known, its
resolving power can very readily be estimated.
Since resolving power varies with the length of light waves,
red and orange have the longest vibrations and give the least
resolving power; blue and violet the most. Intensity of light
must, however, also be considered; the rays between the Frau-
enhofer lines C and F. have the greatest luminosity and are
most satisfactory in working with the microscope.
In addition to the above there are a host of other factors
which must be considered in estimating resolving power; such
as condition of material to be examined, mounting media,
thickness of cover-glass, exactness of focus, illumination, con-
dition of the eye, position of the eye, etc.; in fact, all factors
concerned in bringing out the effectiveness, mechanical and
optical, of the compound microscope.
Naturally resolving power also varies with the tube-length
and the magnifying power of oculars. A high-power ocular
will resolve better than a low-power, provided it is properly
adapted to the objective under consideration. The range of
optically effective oculars with any given objective is, however,
not very great.
VII. MAGNIFYING POWER.
The prevailing notion among the uninitiated in microscopy
is that the magnifying power of the microscope is the optical
property of greatest importance. That magnification is im-

COMPOUND MICROSCOPE.
101
portant cannot be denied, but magnification without due re-
gard for the other optical properties is of little value. For
instance, a badly corrected one-tenth inch water immersion
objective will prove of much less value than a properly cor-
rected one-fourth inch dry objective. In this connection it
must be stated very frequently high-power objectives are de-
cried as defective or worthless simply because they are not
properly used.
It should be remembered that high powers
are, or should be, carefully corrected for definite tube-lengths
and for definite thicknesses of cover-glass (in dry or water im-
mersion lenses). Also, in the case of adjustable objectives,
the manipulator frequently neglects to make the necessary ad-
justments, and blames the objective for what is really due to
carelessness or perhaps ignorance.
The following conditions accompany magnification:
I. Focal distance decreases with magnification.
2. Working distance and penetration decrease with mag-
nification.
3. Illumination decreases with magnification.

4.
Transverse diameter of front lens decreases with mag-
nification.
5. Aperture increases with magnification.
6. Resolving power increases with magnification.
7.
Increase in tube-length increases magnification.
8. Need of careful correction increases with magnifica-
tion.
9. The need of care and skill in manipulation increases
with magnification.
IO. Price of objectives increases with magnification and
aperture.
The approximate magnification of any combination of ob-
jective and ocular may be determined in several ways. If
the equivalent focal distance of objectives and oculars is given,
this becomes an easy matter, as already explained in a previous
chapter. Since, however, the ratings in focal distance are
not always given, and since tube-length and other conditions
vary, it is best to determine the amplifying power for each
instrument and each combination. This may be done as fol-
lows: Place the stage micrometer on the stage and focus the
ruled lines carefully. Observe the lines on the micrometer

102
MICROSCOPY.
scale with one eye; with the other eye observe the divisions
on a millimeter rule held on a level with the stage. If it is
found, for instance, that one division of the stage micrometer
(1-100 mm.) is just equal to one mm. of the ruler, then we
know that the combination magnifies 100 diameters. If we
find that one division of the micrometer scale is equal to 3.5
mm. of the ruler, then we know that the combination mag-
nifies 350 diameters, etc. From this it becomes evident that
the ruler may also be used as a substitute for the eye-piece
micrometer after the measuring value of the millimeter di-
visions have been once determined in terms of the stage
micrometer. For instance, in the above combinations a milli-
meter division of the ruler would have a measuring value of
IOμ and 2.85μ respectively. These values are readily
determined, knowing that each division of the stage microm-
eter is equal to 10μ.
Magnifying power may also be determined by means of the
stage micrometer and the camera lucida.
By means of the
camera lucida project an image of the micrometer divisions
upon a sheet of paper placed at a right angle to the optical axis
of the eye and on a level with the stage. Mark the divisions
of the scale and determine the magnification by comparing
with a ruler as above.
The following table is a resumé of the optical properties
of objectives as it applies to the more important powers of
leading manufacturers of microscopes in America, England
and Continental Europe. It is very difficult to present an
average of properties in a tabulated form, especially as it
applies to aperture. It must be remembered that the cheaper
objectives have much narrower apertures than the more ex-
pensive or professional series. Again, the apertures cited in
catalogues are usually somewhat excessive. The table gives
the water and oil apertures for all the objectives, when in re-
ality the powers of less magnification than the one-eighth
inch are used as dry objectives. I have intended to show
the advantage of immersion lenses in increasing resolving
power. Quotation of prices is as variable as aperture; in fact,
the cost of objectives varies almost directly with the aper-
tures. It might also be stated that the three-inch and two-
inch objectives at one end and the one-sixteenth inch and


COMPOUND MICROSCOPE.
103
one-eighteenth inch at the other extreme are rarely used.
The former do not give sufficient amplification, while the
latter are very difficult of construction, very expensive and
are not more effective than a well-constructed one-twelfth inch
oil-immersion objective.
RESUME OF PROPERTIES OF OBJECTIVES.

Equivalent Focus.
Angular Aperture.
f
Numerical
Penetrating
Power.
Aperture.
Na i sin (2)
Dry Wtr Oil
(P. p.)=(Na.)
Resolving Power
in lines to the inch.
25,400μ
(R.p.)=
(2 Na.)
Magnifying Power.
Cost.-Dollars.
10
In. MM
i= i=
1.00 1.33 1.52
Dry Water. Oil.
Dry. Water.
Oil.
214
f
3 75.
5° .04 .05 .06 25.
20. 17.
3,848 4,794 5,545 3.3
6
2 50.
10° .09 .12 .14 11.
8.3
7.1
8,759 11,599 13,368 5.
6
1½ 37.
15° .13 .17 .20 7.7
5.8
5.0
12,512 16,387 19,318 6.6
6
14 30.
20° .18 .24 .26 5.5
4.1 3.8
1 25. 30° .26 .35 .40 3.8
2.8
2.5
3-4 18. 40 .34 .46 .52 2.9
1-2 12.5 50° .42 .56 .64 2.3
2.2
1.9
1.8 1.5
1.5
1-3 9.2 609.50 .66 .76 2.0
1-4 6.3 70° .57 .76 .87 1.8
1.5
1.3 1.1
1.3
1-5 5. 80 .64 .85 .98 1.6
1.2
1-6 4.3 90° .71 .94 1.07
1.4
1.1
1-7 3.4 100° .77 1.02 1.16
1.3
1-8 3.2110° .82 1.09 1.24
1-9 2.7 120
1-10 2.5 130° .91 1.20 1.38 1.1
1.2
.97
.91
.87 1.15 1.32
1.1
.87
.83
1-12 2.1 140° .94 1.25 1.43 1.1
.80
1-16 1.6 150° .97 1.29 1.47 1.0
8
25,400 35,775 38,485 10. 10
32,564 44,561 49,804 13.3 15
40,316 54,043 61,951 20. 15
47,925 63,500 72,571 30. 18
56,070 73,410 83,883 40. 20
1.0 61,695 81,935 94,873 50. 30
.90 68,537 90,714 103,211 60. 40
.86 71,345 99,214 111,894 70. 60
.81 79,127 105,088 119,529 80. 70
.76 83,883 110,917 127,254 90. 75
.72 87,585 115,981 133,684 100. 80
.69 90,714 120,493 138,043 120. 100
.77 .68 93,151 124,289 142,569 160. 125
16,933 23,090 25,400 8.
1-18 1.2 160 .98 1.30 1.48 1.0
.77
P7
.67 94,873 120,434 142,696 180. 150

104
MICROSCOPY.
CHAPTER VI.
THE MANIPULATION AND CARE OF THE COM-
POUND MICROSCOPE.
Equal in importance to being thoroughly familiar with the
mechanism and optics of the compound microscope is a cor-
rect knowledge of its care and manipulation. Ignorance or
carelessness in this direction will prove both expensive and
annoying.
A safe rule to follow is never to touch or handle any
instrument or any of its parts until its mechanism is well
understood. Every manipulation should be for a definite and
justifiable purpose. A well-constructed modern compound
microscope is a durable instrument if properly handled, but if
improperly handled no instrument is more readily injured or
rendered useless. These statements should suffice to place
the beginner on his guard and to impress the importance of
familiarizing himself with its mechanism and proper manipu-
lation.
I. MANIPULATION OF THE INSTRUMENT.
I. POSITION OF INSTRUMENT AND WORKER.
The microscope should be placed on a solid table or desk,
which is free from vibrations and large enough for the work in
hand. The back of the instrument represented by the pillar
should be directed toward the worker and the body should
be vertical whenever necessary. A vertical tube gives a hori-
zontal stage, which prevents the object slide from gliding off
and also prevents the movements down the incline of particles
in the liquid mounting media. Usually it will be found con-
venient to incline the tube at an angle; as when the table is
too high, the chair too low or the person short. A slight de-
gree of inclination is generally desirable in the examination
of permanent mounts, as it frequently permits of better il-
lumination and reduces the necessary forward inclination of
the head.

CARE OF COMPOUND MICROSCOPE.
105
The chair
The worker should sit behind the instrument.
should have a narrow back and no arm rests. The narrow
back and the absence of arm rests permits free movement
of the arms. The back is essential to resting the upper part
of the body from time to time. A long continued forward
inclination of the head and body produces fatigue, and should
be counteracted as much as possible. The seat of the chair
should be low enough to permit the feet to rest flat upon the
floor. The table should be high enough so that when the
tube is slightly inclined the eye may be placed to the ocular
without any considerable stooping or sagging of the body.
The body should incline forward at the required angle from
the hips up and should be supported by resting the elbows
upon the table on either side of the microscope.
Stooping forward or sagging of the body is pernicious, as
it retards the heart's action as well as free respiration. It also
tends to produce dyspepsia, and if long continued will produce
stooping shoulders and curvature of the spine. A long-con-
tinued forward inclination of the head tends to produce con-
gestion of the conjunctiva and interferes with free cranial
circulation. High or tight standing collars should not be
worn, as they prevent free movement of the head and tend
to cause congestion of head and face. The worker will find
it highly beneficial to rise occasionally and to go through a
few lung and muscle gymnastics. This will invigorate the
body, retard fatigue and make vision more acute, besides
making the brain more active. On account of some necessary
forward inclination of the body and some slight unavoidable
sagging of the internal viscera, it is never advisable to work
immediately after a heavy meal, as it will retard digestion and
very soon bring on dyspeptic troubles. It is rarely advisable
to prolong a single session of work with the compound micro-
scope beyond two hours. Each session should be followed
by some physical exercise.

2. ILLUMINATION.
The best light for microscopical work is the reflected sun-
light. Direct sunlight is never to be used. Experience has

106
MICROSCOPY.
determined that the light reflected from a white cloud and
coming through a window with northern exposure is most
suitable. The microscope should be on a desk or table a few
feet from the window and so placed as to get the illumina-
tion from the side or front. If the light from a window facing
south or any other direction having an exposure to direct sun-
light must be employed, translucent linen shades must be used
to convert the direct rays into reflected light.
Indirect sunlight is always preferable to artificial light, even
on a very cloudy day. Very frequently beginners have dif-
ficulty in recognizing the difference between brightness of
field and clearness of image, and will persist in using the light
from a lamp instead of reflected sunlight, simply because it
gives a bright, glaring field. In fact, the average worker will
rarely have any occasion to use artificial light.
Of artificial lights, oil and gas light are most objectionable
because of the annoying yellow tint. This may, however,
be sifted out or neutralized by using properly tinted glass
shades or by glass discs placed in a ring of the substage. If
a flat flame is used, the edge should be turned toward the
mirror, as this gives the greater intensity and the rays are
more nearly parallel.
Electric light, acetylene, Wellsbach and other lights of the
incandescent order are less objectionable because they give
greater intensity and have less of the objectionable yellow
tint. These lights must, however, be properly shaded so as to
protect the eyes. An open gas light is objectionable because
of the unsteadiness of the flame.
There are a large variety of microscope lamps upon the
market which we cannot take the time to describe. The stu-
dent who is obliged to use artificial light occasionally will find
the ordinary oil lamps or gas light sufficient.
Having selected or decided upon the best available source
of illumination, the next step in microscopical work is to se-
cure the brightest illumination of the field of view. With the
microscope in proper position and the objective in approximate
focus, place the eye to the ocular and adjust the mirror. The
mirror bar should be vertical for all ordinary purposes, and
the mirror should be swung upon its two crossed horizontal
axes until the field appears brightest. Be careful to keep

CARE OF COMPOUND MICROSCOPE. 107
hands, books and other objects out of the way of the light and
mirror.
Lateral or oblique illumination is obtained by swinging the
mirror to right or left. In high-power work lateral illumina-
tion will reveal details of structure not brought out by cen-
tral illumination. The amount of obliquity that may be em-
ployed will depend upon the required resolving power and
upon the aperture of the objective. Naturally, transmitted
illumination by means of the mirror would be impossible if
it were swung beyond the angular aperture.
Oblique illumination has its objectionable features in in-
creasing any existing chromatic aberration. In general it
may be stated that the use of oblique illumination is very
limited and that experience will readily suggest when to em-
ploy it, and experimenting with each individual object will
soon limit the working efficiency or advantages to be derived
therefrom. Oblique illumination is never permissible with a
substage condenser.

3. FOCUSSING.
The essential feature in focussing consists in so manipu-
lating the coarse and fine adjustments as to produce a clear
and distinct image of the object under examination. A prevail-
ing notion is that focussing is an insignificant mechanical detail
in microscopical work. This is far from the truth. Skillful
focussing requires experience guided by intelligence, and is
practically commensurate with the working ability of the
microscopist.
In order to focus, an object must be placed in its proper
position upon the stage. Any of the test objects mentioned
will serve very well. If they are not available, one can readily
prepare a mount of potato-starch. By means of a pocket
knife scrape a small bit from the cut surface of a potato,
place it on the center of the glass slide and cover with cover-
glass, making sure that the space between cover and slide is
occupied by water. Place this temporary mount upon the
stage and secure illumination as already directed.
It is always advisable to begin focussing with a low power
(1 in. or in. objective), as the higher powers require greater
care and skill. The proper way to focus is downward, al-

108
MICROSCOPY.
though many teachers advocate focussing upward. To focus
downward the objective must always be farther removed
than its working distance. With the eye in position turn the
body of the instrument downward by means of the coarse
adjustment. The rate with which the adjustment may be
operated depends in a great measure upon the skill of the
manipulator. The beginner must work slowly, keeping the
eye fixed upon the field. As the objective approaches the
focus dust particles on the cover-glass (on upper and lower
surfaces), particles suspended in the mounting medium, ir-
regularities in transparency of glass and medium, the object
itself, will appear in the field of view. At first the object will
appear as an indistinct shadow. This is the signal to work the
adjustment more slowly. As soon as the object reveals some
details of structure the fine adjustment should be employed
to focus accurately. It need scarcely be stated that care and
caution in focussing increases directly with the amplification
and aperture of objectives.
After once knowing the focal distance of the various com-
binations of objectives and ocular, little difficulty will be en-
countered in properly focussing downward. Focussing down-
ward has many advantages. It teaches one to always keep the
objective above the focal distance when not in use, thus
making room for the proper manipulation of the object-slide
in removing it or placing it on the stage. It does away with
the necessity of that careful sidelong squint to see whether
or not the objective is within the focal distance or close to the
slide.
In working with medium and high powers the thumb and
first finger of one hand should grasp the milled head of the
fine adjustment and move it back and forth in order to bring
the different horizontal planes of the object into focus. Upon
this constant but careful manipulation of the fine adjustment
depends much of the accurate work of the biologist. This
constant manipulation may very aptly be compared to the
manipulation of the rudder of a ship in order to give a steady
and direct course.
In focussing immersion lenses, watch the objective from
the side and bring the front lens of the objective in contact
with the fluid on the cover-glass and then turn the objective


CARE OF COMPOUND MICROSCOPE.
109.
back until it is certain that it is above the focus; then with
the eye in position, focus downward, as already explained.
II. HOW TO USE THE EYES.
There is a right way and a wrong way of using the eyes
in working with a compound microscope. The student may
know how to manipulate the instrument correctly, but if he
does not know how to use the eyes he can never accomplish
good results.
In the first place, only a comparatively few persons know
how to look; that is, to look with a view to seeing details of
form and structure. Of most persons it may justly be said
that they have eyes but see not. This is essentially due to a
lack of proper training. An all-pervailing opinion exists that
all that is necessary is to direct the eyes toward the object in
order to perceive it in its entirety. This is by no means the
An intelligent, well-trained mind must be back of the
eye to direct it and to properly interpret the impressions trans-
mitted to the brain center by way of the optic apparatus. The
eye thus directed will give attention to form, color, to slight
variations in refraction, to slight variations in light and shade;
in fact, to a hundred and one details which the uneducated
eye will overlook entirely or perceive only indistinctly.

case.
Working with the microscope has the same effect upon the
eyes as any other work. It is not any more injurious than
reading good print. This is a fact not generally believed by
the laity, but it is nevertheless true. Beginners in working
with the microscope are apt to deny the statement, but nearly
every case of apparent contradiction is found to be due to a
lack of knowing how to manipulate the instrument properly.
The difficulty may be due to dust or finger-marks on the
lenses, there may be some of the mounting fluid on the front
lens of the objective or on the top of the cover-glass; the nose-
piece may not be swung in its proper position, the mirror may
not be properly adjusted; the object may be too large or too
thick. These and many other evidences of a lack of knowing
how to properly manipulate the instrument have come to my
notice from complaining students.
In regard to normal and abnormal vision, the student is

110
MICROSCOPY.
referred to the special chapter on that subject. We shall here
only state that if glasses are worn in reading, they should also
be worn in working with the microscope, and that the eyes
should not be overexerted by long sessions behind the mi-
croscope.
With the monocular microscope one eye only can be used at a
time, but this does not imply that the eye not in use should be
closed. In all forms of microscopical work both eyes should
be kept open. Most biologists use the right eye nearly alto-
gether, but it is a good plan to use the eyes alternately, so as not
to fatigue one eye through excessive use; however, this would
apply only in case that the power of vision is equal or nor-
mal in both eyes.

In
As regards the position of the eye, it should be near the
eye-lens of the ocular. With high powers it must be nearer
than with low powers in order that the largest number of
rays of light may enter the eye (eye-point). The entire field
of light should be seen and the margin clearly defined.
working with high powers the eyelashes may even be brought
so near as to rest upon the lens and moisture evaporating
from the surface of the eye-ball may condense upon the
cooler lens, producing more or less disturbance of vision.
Frequently it becomes necessary to use both eyes; one to
observe the image, the other to observe a ruler in measuring
or a pencil and paper in drawing, etc. It may be stated that
at first there will be some difficulty in keeping both eyes open,
as the eye not looking down the tube will perceive extraneous
objects, which cause considerable annoyance, but only a few
days or a week of patient persistence will enable the student
to learn to ignore these extraneous objects entirely. It may
be necessary to shade the eye not in use until it has learned
not to focus upon objects and the mind has learned not to
give attention to the impressions received from this eye.
III. CARE OF THE INSTRUMENT.
We shall here confine ourselves to a few brief statements,
hoping that they may serve more as hints rather than full
information.
I. The instrument when not in use should be kept in its

CARE OF COMPOUND MICROSCOPE.
111
case in a dry, shaded or dark place, away from all chemicals.
Before placing it in the case all external parts should be
carefully cleaned and wiped dry; the metal parts with a clean
linen or cotton cloth or a piece of chamois skin; the lenses
with Japanese (dentist's) paper. No finger-marks should

show anywhere.
2. To remove and adjust objectives grasp the collar be-
tween thumb and first finger of the left hand and fasten or
unfasten it with the thumb and first finger of the right hand;
at no time should the hold upon the objective be lost, other-
wise the objective may fall upon the stage, the floor, or the
table and jolt it sufficiently to discenter the lenses, if not to
break them.
3. Lenses of objectives and oculars may require cleaning;
if so, remove dust by means of a small camel's hair brush and
a soft cloth, or the above-mentioned paper. Never unscrew
lenses of ocular or separate the objective systems unless it is
absolutely necessary. Always clean immersion lenses immedi-
ately after being used; remove oil by means of a soft cloth
and wipe perfectly dry by means of the Japanese paper.
4. All of the gearings, sliding adjustments, screws and
hinges of the stand require cleaning from time to time. For
this purpose wash them with Japanese paper dipped in xylol
or benzol and afterwards rub on a little simple vaseline as a
lubricant. Never use oil, as it will gum or become sticky.
5. Never allow mounting fluids or chemical reagents to
come in contact with objectives. Many objectives are ruined
by not giving attention to this precaution. The reagent may
corrode the metal and loosen the lenses, or it may work its
way between the lenses of the front system, producing opacities
which will render the objective useless until cleaned and reset
by a skilled workman in optics.
6. Never touch or manipulate a single part of the instru-
ment unless it is fully understood what the touch or manipula-
tion signifies. This advice is especially intended for beginners,
who persist in all sorts of experimentation to find out "how it
will work." It is true, much may be learned from such experi-
mentation, but it will in all probability be experience which
must be dearly paid for in cold cash.
7. Objectives should not be subjected to extremes of tem-


112
MICROSCOPY.
perature, as the unequal expansion of glass and metal may
loosen or crack the lenses. Lenses should also be kept out of
direct sunlight, as they may become discolored and ruined.
8. When it becomes necessary to have the stand or lenses
repaired, always take them to the manufacturers of the instru-
ment, as they know best how to make the repairs.
9. Loose pinions or hinge-joints should be properly tight-
ened at once.
IO. Be careful not to let alcohol and other corrosive chem-
icals come in contact with the stand, as they remove the
lacquer. The lacquer serves to prevent the metal from oxi-
dizing.
II. In carrying the instrument, grasp it by the pillar and
arm, holding it in front of you, being careful not to knock
against anything. Never grasp it by the tube nor carry it
swinging at the side. In inclining the body always do so by
grasping the arm and not the tube or body.
IV. HINTS ON THE PURCHASE OF A MICROSCOPE.
Since compound microscopes are expensive, it is quite
important that a prospective purchase be carefully considered
with a view to securing the instrument best adapted to the
required needs. The following suggestions will no doubt
prove useful:
First of all, the would-be purchaser must know what he
wants. He must have some definite opinion as to the nature
of the work to be done. If possible, consult some biologist,
stating the nature of the work, and let him select the most
suitable instrument. If such a specialist cannot be consulted,
the following advice should be heeded:
1. Deal only with reliable dealers. Be sure to select an
instrument bearing the stamp or name of a reputable firm.
2.
Never purchase a second-hand instrument unless you are
competent to pass judgment upon its merits.
3. The stand should be of the recent "Continental" type.
It should be possible to incline the body horizontally without
upsetting the instrument. The body should remain in position
at any angle. The stage should be large, solid and lined above
with hard rubber.

CARE OF COMPOUND MICROSCOPE. 113
4.
There should be a double or triple nose-piece.
5. There should be a rack and pinion coarse adjustment,
with diagonal cogs. All gearings must work smoothly.
6. Special attention should be given to the fine adjustment.
It should work smoothly and must be guaranteed as to dura-
bility.
7. A graduated draw-tube should be present.
8. There should be a plane mirror and a concave mirror.
9. An iris diaphragm and a substage condenser should be
present. The iris diaphragm may be mounted in the stage or
below the stage, preferably the latter. The condenser can be
dispensed with more readily than the diaphragm.
IO. The objectives and oculars should be carefully ex-
amined and tested before purchasing. Any reliable dealer
will do this willingly or will at least permit it to be done. For
work in vegetable and animal histology the following objectives
and oculars will be most suitable: 3-4 inch objective and 1-6
inch objective, combined with a 1 in. ocular and a 1-2 inch
ocular. For special high-grade work in histology and bac-
teriology a good 1-12 inch oil immersion lens should be added
II. As to special accessories and appliances do not pur
chase them until needed.
12. Refer to the chapter on the "Parts of the Compound
Microscope," where the mechanical parts are more fully
described.


PART II.

MICRO-TECHNIQUE.
CHAPTER I.
MATERIAL.
We can consider only the more important material to be used
in doing micro-biological work. Special work will necessitate
special material, which may perhaps be readily prepared or
can be purchased of dealers in microscopical supplies. A little
forethought, coupled with some mechanical ingenuity, will
enable one to make many of the mechanical conveniences and
thus save a considerable financial outlay. The advice already
given in connection with the purchase of accessories for the
microscope may well be repeated here; that is, purchase only
necessary material.
I. MICROSCOPE DESKS, TABLES AND CHAIRS.
For the active worker with the microscope nothing is
superior to a well-made solid desk. It should be made of
heavy, well-seasoned oak; it should be of the proper height
and with a flat, roomy, rectangular top. There should be
vertical drawers with locks on either side. The top should
be smooth, but not polished, varnished, painted or covered
with a cloth or anything else. It may be paraffined from
time to time and should always be clean. Some of the more
expensive tables are made of quartered oak, glass tops and
beautifully finished. Figure 69 shows such a desk, which also
has a very convenient top reagent case. The drawers serve to
115

116
MICRO-TECHNIQUE.
hold notes, manuscript, drawings, glass utensils, etc., etc. One
of the larger lower compartments will hold the microscope.
Any carpenter will be able to construct a much cheaper desk,
provided he has the seasoned oak and is definitely instructed as

FIG. 69.
MICROSCOPE DESK.
to the dimensions, number of drawers, etc. The top of the
desk should not be too high, and should be suited to the
height of the individual; 29 inches to 32 inches will be found
convenient heights for different individuals when the chair or
stool is 20 inches high. The size of the top should be about 50
inches by 32 inches. Glass tops are objectionable, as they be-
come worn and chipped with active work, besides being liable
to crack.
Microscope tables usually have revolving tops and are a
special convenience when a number of persons wish to exam-
ine objects already mounted. They should be solid and well-
made. They will scarcely serve the purpose of a desk and are
more of a luxury and convenience than a necessity.

MATERIAL.
117

B&L OPT.CO
FIG. 70.
MICROSCOPE TABLE WITH REVOLVING TOP.
The chair for the microscopist has already been mentioned.
Like the desk and table it should be solid. Stools are also
recommended by some, simply differing from the chair by the
absence of a back; they are specially desirable in laboratories
where the backs would be in the way. The height of the chair
or stool must be adapted to the height of the desk or table and
to the height of the worker, as already indicated.
II. MICROTOMES AND SECTION KNIVES.
Microtomes are a special apparatus for cutting sections.
They may be divided in two classes; those which regulate the
thickness of the section automatically and those in which the
thickness of the sections is regulated by the cutter.
I. SIMPLE MICROTOMES.
With the simple microtomes the thickness of the sections to
be cut is regulated by hand. There are many different kinds.
upon the market; some are made to be clamped to the desk,
118
MICRO-TECHNIQUE
some have a broad base so as to rest upon the desk or table,
some are to be held in the hand while cutting the sections.
A
very convenient form is shown in Fig. 71. To the lower

BAUSCH & LOMB OPT. CO.
FIG. 71.
SIMPLE CLAMP MICROTOME.
left-hand side is a clamp screw for fastening the microtome
to the desk or table; to the lower right side is a very
BAUSCH & LOMB OPT.CO
O
3
FIG. 72.
BASTIN'S
HAND
MICROTOME.

MATERIAL.
119
accurate micrometer screw, by means of which the thickness
of the section is regulated. Near the top is a clamp screw for
clamping the material to be sectioned. The razor or section
knife glides over the glass top in cutting the sections. Figure
72 shows a hand microtome as designed by the late Professor
Bastin. Sometimes it is desirable to harden the material to
be cut by freezing. To do this it is necessary to attach a freez-
ing apparatus to the microtome.

2. AUTOMATIC MICROTOMES.
With the automatic microtomes the thickness of the sections
is mechanically regulated by a special attachment, which is
operated by one hand or by the to and fro movement of the
section knife carrier. The thickness of the section is usually
indicated by a certain number of clicks; each click represent-
ing about 2 u. To explain the mechanism of the automatic
microtomes would be a waste of time and space. The best
plan is to study the apparatus itself and learn to manipulate
it under the direction and guidance of a competent teacher.
There are a great variety of automatic microtomes upon the
market; all are quite expensive. Some are more specially
FIG. 73.
AUTOMATIC MICROTOME.

120
MICRO-TECHNIQUE.
adapted for sectioning paraffine material, some for celloiding
or collodion material, others again for both. Naturally the
student will have no occasion to use this instrument until he
is fully familiar with the compound microscope and has
studied many sections made by hand or with the simple
microtome and will therefore readily learn how to use the
instrument and to take proper care of it. Figure 73 shows an
automatic microtome with knife in position.
III. GLASS SLIDES, OBJECT SLIDES.
Objects for examination under the compound microscope
are mounted upon rectangular slides or slips of thin plate
glass three inches long by one inch wide (Fig. 74). The thick-
ness is somewhat less than ordinary window-glass. They
should be made of a good quality of white glass free from air
bubbles and opacities. The edges should be ground. Rough
edges scratch the stage of the microscope and, furthermore,
indicate a poorer quality of glass. The thickness of the differ-
ent slides should be uniform, which is generally not the case.
In a case of necessity ordinary window-glass may be cut into
slips of the regulation or required size and used. A good
quality of mica may readily be cut with a pair of scissors and
FIG. 74.
GLASS SLIP.
similarly used. However, these substitutes are not to be used
if the glass slips can be obtained.
There are a great variety of modifications of the ordinary
glass slips for special biological work. The glass slip with
FIG. 75.
GLASS SLIP WITH
CONCAVE CENTER, PROFILE VIEW.

MATERIAL.
121
concave center is much used in studying living bacteria, dia-
toms, some protozoa and other minute organisms. Figure 75
shows a cross section of such a slide with cover-glass in posi-
tion. A drop of the bacterial culture, etc., is suspended from
the middle of the lower surface of the cover, which is fastened
to the slide by means of plain vaseline to prevent evaporation
of moisture. Larger algae and protozoa are studied in life
boxes which resemble the above, only the space occupied by
the organisms is much larger. Life slides, current slides,
syphon slides, observation slides, stage aquaria, etc., are all
special contrivances for studying special biological subjects.
Some are important, others are merely conveniences and per-
haps none are absolutely necessary or even desired by the
average student. Furthermore, the ingenious student will be
able to construct most of the special slides for himself as occa-
sion demands it.
IV. COVER GLASS.

Cover-glasses are very thin pieces of glass, circular or rec-
tangular in form, which are placed upon the object to be ex-
amined. They serve to protect and flatten the object and keep
the object and mounting media in place. They should be made
of a good quality of glass, entirely free from air bubbles,
opacities, irregularities in surfaces or irregularities in refrac-
tion. Recently covers have come to notice in which numerous
minute, more highly refractive specks were noticeable, appar-
ently due to incompletely dissolved crystals of silica. These
irregularities in refraction proved annoying because they could
be mistaken for micrococci.
There are various sizes of covers made, given in diametrical
measurement, as in., § in., & in., 7 in., and I in. One inch
is the largest, as that is the width of the slide. For temporary
mounts in general the larger covers should be used, especially
when using corrosive micro-reagents, as they lessen the liabil-
ity of the media coming in contact with the objectives.
As to whether the square or circular covers should be used,
it may be stated that the square covers are especially desir-
able in mounting serial sections, otherwise the circular forms
are preferable. For instance, if the zoologist wishes to mount
122
MICRO-TECHNIQUE.
serial sections of the earth-worm, embryo of chick, etc., he
finds the square covers most suitable because the sections can
be arranged in rows of equal length.

FIG. 76.
COVER GLASSES.
The thickness of covers also varies. Manufacturers usually
make about four grades as to thickness. The thickness is of
great importance, as it is directly concerned with the working
capacity of objectives. That is, opticians correct spherical
and chromatic aberration of objectives according to a standard
thickness of cover-glass and a standard of tube-length, as
already mentioned.
The significance of the cover may be
set forth as follows: If an optician should correct an objective
completely for a given standard of tube-length without refer-
ence to cover-glass, he would find it very effective upon exam-
ining uncovered objects, but as soon as a cover-glass is placed
upon the object more or less distortion of form and color would
appear, because the cover-glass modified the refraction, a thin
cover differently from a thicker cover, hence the necessity
of giving special attention to thickness of cover-glass when
correcting aberration of form and color. Unfortunately differ-
ent opticians do not use the same standard of thickness. Fur-
thermore, the different covers of a given lot designated as hav-
ing a certain thickness show considerable variation, hence the
'necessity of measuring the thickness before using them with
the higher powers. Within certain limits correction for thick-
ness of covers can be made by means of the draw-tube. If
the cover is too thin the tube should be drawn out; if too
thick the tube should be shortened. Adjustable objectives are
for the purpose of ready adaptation to varying thickness of
covers.
The following table shows the different standards of thick-

MATERIAL.
123
ness of covers adopted by the different manufacturers of micro-
scopes:
IO-100 mm.
Swift & Son.
10-100-15-100
10-100-15-100
66
Bezu, Hausser et Cie.
66
15-100
66
15-100
66
15-100
16-100
66
Nachet et Fils.
Gundlach Optical Co.
W. & H. Seibert.
R. & J. Beck.
Bausch & Lomb Optical Co.
66
17-100
E. Leitz.
66
17-100
R. Winkel.

18-100
66
C. Reichert.
18-100
66.
Klonne und Müller.
66
15-100-20-100
C. Zeiss.
664
20-100
Watson & Sons..
16-100-25-100
66
Ross & Co.
66
25-100
W. Wales.
66
25-100
Powell & Lealand.
66
25-100
66
25-100
66
25-100
Spencer Lens Co.
J. Grunow.
J. Green.
These different standards must be kept in mind in using.
the instruments of the different makers. Catalogues are usu-
ally very explicit about stating the thickness of covers to be
used with dry objectives. With oil immersion lenses the
thickness of the cover makes no difference only in so far as it
must be thin enough to permit proper focussing. The im-
mersion fluid has the same refractive index as the cover, hence
there can be no disturbance of refraction with varying thick-
nesses of cover. With immersion fluids that are not homo-
geneous, as water, correction must be made as with dry objec-
tives. The thickness of covers is determined by a special
apparatus known as a cover-glass gauge (Fig. 77). The
instrument is not expensive and is really a necessity for the
careful worker with the higher power objectives.
In a case of necessity pieces of good mica may be used as
covers. Instances have come to notice where pieces of broken
slides were used as covers, but such covers can be used only
with very low powers.

124
MICRO-TECHNIQUE.
V. TURN-TABLE. SLIDE CENTERER.
Turn-tables are used principally to hold mounts in place
while placing a ring of shellac about the edge of the cover-
glass or for placing rings of shellac upon the slide. They are
FIG. 77.
3
2400/M
TICAL CO.
YORK CITY
61
1510
THOUSAND7
OF INCH
NON ADJUSTABLE
OBJECTIVE
CORREGTED FOR
2
//
5
8/1
0.16 Mm COVER GLASS 2
TUBE LENGTH 82 Inches/6
81
COVER GLASS GAUGE.
also used in mounting to center the mount carefully. It is
almost impossible to make neat permanent mounts without a
turn-table. Its construction and manipulation is simple and
may readily be understood from an inspection of Fig. 78.
Some turn-tables are self-centering, while with others the
slide must be centered by hand.
Those who do not have a turn-table can readily prepare a
slide centerer as shown in Fig. 79. Rule a rectangle the size
of a slide upon heavy cardboard; draw diagonal lines to lo-
cate center, and then draw squares and circles about this cen-
ter equal to the diameter of the cover-glasses used. This will

MATERIAL.
125


FIG. 78.
TURN TABLE.
be found quite as satisfactory as the turn-table for mounting
purposes.
VI. ACCESSORY MATERIAL.
Under this head we shall mention a few articles which will
be found quite essential in micro-technique. Many others
FIG. 79.
SLIDE CENTERER.
could be mentioned, but the average student of biology would
not find them absolutely necessary. Furthermore, students
are earnestly advised to work with a minimum of cumbersome
apparatus and appliances. Again in many instances the stu-
dent of average mechanical ability will be able to construct
desirable material and appliances as occasion demands.
I. WATCH GLASSES.
The Syracuse watch glasses will be found very useful in
dehydrating, staining and clearing sections for mounting.

126
MICRO-TECHNIQUE.
These glasses contain the reagents and are stacked, one above
the other, to prevent evaporation of reagents. Six to one dozen.
will be sufficient for ordinary purposes. Figure 80 shows a
stack of six Syracuse watch glasses and will suggest how they
are to be used without further explanations. Some skill is
necessary to handle them properly.
TR.SEC. TONGUE-CAT
HEMATO XYLIN
DIST. WATER
ALCOHOL
ABS ALCOHOL
OIL OF CLOVES
FIG. 80.
SYRACUSE WATCH CRYSTALS.
2. SECTION LIFTERS.
Section lifters are required to lift sections from one medium
to another. The blade should be thin, quite broad, flexible
and well nickeled. The broadly angular ebony handles are
FIG. 81.
SECTION LIFTERS.

MATERIAL.
127

superior to the cylindrical smooth metal handles, as they per-
mit of greater security in handling. They are made in all
sizes and curvatures. Figure 81 shows two lifters which will
prove very effective for ordinary work.
3. DISSECTING NEEDLES.
Dissecting needles are required in making temporary mounts,
particularly in animal biology. There is a great variety of
dissecting needles upon the market. They can readily be
made by the student. Simply fasten needles, eyes down, into
wooden handles (pen-holders will answer very well) of suit-
able size and length. The needles must be highly polished
and sharp. The needles may be bent in any desirable curva-
ture by first heating to redness. Never attempt to work with
rusty needles.
4.
SLIDE BOXES.
These are wooden boxes specially made to hold slides with
mounts upon them, each box holding twenty-five slides (Fig.
BAUSCH & LOMB OPTCO.
FIG. 82.
SLIDE BOX.
82). The boxes should be well made of well-seasoned wood,
otherwise they will come to pieces soon.
There are also
special boxes for mailing slides, slide trays, slide cabinets, etc.
I.
5. OTHER ACCESSORY MATERIAL.
Glass-stoppered reagent bottles are desirable, each with
a glass rod with which to transfer a drop of the reagent from
bottle to the slide or section. Reagent bottles should be wide-
mouthed and not too large. They should be closed when not

128
MICRO-TECHNIQUE.
in use and kept away from dust. Some reagents must also
be kept from sunlight.
2. Pincers, forceps and clamps for handling cover-glasses,
sections, and for clamping the cover until the mounting
medium has had time to harden somewhat, will be found use-
ful.
3. Camel's hair brushes will be found useful for removing
dust from objectives and also for removing sections from the
section knife and for other purposes.
4. Watch crystals and evaporating dishes will be found
useful. The former for holding sections, material, etc.; the
latter for heating material, melting paraffine, etc.
5. Japanese tissue paper used by dentists is excellent for
wiping and cleaning lenses, oculars and objectives.
6. Glass rods for reagent bottles, pipettes, medicine drop-
pers, scissors, etc., will be required on occasion.
7. Labels for slides and reagent bottles will be desirable.

REAGENTS.
129
CHAPTER II.
REAGENTS.
The reagents described are those most commonly used in
ordinary microscopical work. Some will be required in the
preparation of temporary mounts, some for permanent mount-
ing and still others for both. Efficiency in the results obtained
from the use of reagents will depend upon a correct knowledge
of their physical and chemical properties and experience in
using them. For convenience of reference the reagents are
described in alphabetical order. Percentage solutions are made
either by weight or by volume.
I.
I. REAGENTS IN ALPHABETICAL ORDER.
1. Acetic Acid. This is essentially a clearing agent. A
one or two per cent aqueous solution is employed for fixing
the nucleus and showing its structure more clearly. It is
frequently employed in combination with methyl green or
gentian violet for staining purposes.
A strong solution, fifty per cent or more, is an excellent
medium for clearing up tissues and revealing the structure of
the cell-walls. It is also an excellent aid to the study of
crystals of calcium oxalate, as they are not soluble in it.
2. Alcanna Tincture.-This is a coloring agent used in
studying resins, which become a deep red in a short time. The
tincture may be prepared by placing some chips of alcanna
(alkanet) root in alcohol. It should never be prepared in large
quantities, as it deteriorates very rapidly. The reaction may
also be obtained by placing a small thin chip of the root with
the preparation mounted in fifty or seventy-five per cent alcohol.
3. Alcohol. This is doubtless the most important reagent
in micro-technique. It is found useful in all degrees of
strength. Forty to seventy-five per cent alcohol is a very use-
ful preservative for most organic substances. The strength

130
MICRO-TECHNIQUE.
required will depend upon the size and nature of the sub-
stance. Animal substances require a somewhat greater
strength than vegetable substances. For hardening and dehy-
drating purposes all strengths of alcohol are required, as will
be explained under "Methods." Alcohol is one of the best sol-
vents and will be required for cleaning slides, covers and occa-
sionally also lenses. It is used with various other substances
in temporary mounting and to give micro-chemical reactions.
Absolute alcohol is quite expensive. It should be kept in a
hermetically sealed can or bottle, otherwise it will take up
moisture. In most cases 95 to 97 per cent alcohol will serve
all the purposes of absolute alcohol.
4. Ammonia.-A strong aqueous solution is a good clear-
ing agent for vegetable sections. It will also be found useful
for softening dry herbarium specimens preparatory to macro-
scopical study, as well as microscopical examination. With
nitric acid it gives the so-called xanthoproteid reaction with
protoplasm, characterized by a yellow coloration.
5. Aniline Blue.-This stain is also known as Hoffmann's
blue and is used for staining protoplasm and the callus of
sieve-tubes. Make a saturated solution in 50 per cent alcohol
or in water and filter. Of this stock solution add five or six
drops to 15 gm. of distilled water. This dye is excellent for
staining bacteria. It also stains cellulose membranes, but it is
readily washed out. After staining wash the vegetable sec-
tions in water and mount in glycerine.
6. Bichloride of Mercury.-This reagent will be found very
useful. A saturated aqueous solution is a good fixing and
hardening agent for animal and vegetable tissues. Weaker
solutions may also be similarly used. In the strength of one
part to 1,000 or 2,000 of water it is an excellent antiseptic
wash.
It should be remembered that bichloride of mercury, also
known as corrosive sublimate, is a very powerful poison. As
an antidote give an emetic, white of egg, flour and milk.
7. Canada Balsam.-This is used as a mounting medium
dissolved to the consistency of thin syrup in xylol, benzol, tur-
pentine or oil of cloves.
8. Carbolic Acid.-A nearly saturated alcoholic or aqueous
solution is an excellent clearing and dehydrating fluid. A 21

REAGENTS.
131
to 5 per cent solution is an excellent antiseptic wash. A 5 per
cent solution is employed as a mordant in bacterial staining.
9. Cedar, Oil of.-Used as a clearing agent and as an im-
mersion fluid with oil immersion lenses.
IO.
Celloidin.-Dissolved in equal parts of ether and abso-
lute alcohol it forms a much-used infiltrating medium. A 21
per cent and a 5 per cent solution are generally used. Celloidin
deteriorates with age. The 2 per cent solution will also be
found exceedingly useful to form a protecting coating over
slight cuts, abrasions, burns, etc. Collodion may be similarly
prepared and used.
II. Chloral Hydrate.-A saturated aqueous solution is an
excellent clearing fluid, especially useful in the study of pollen-
grains.
12. Chloroform.-An excellent solvent of fats and oils.
Also used in the place of xylol in paraffine infiltration. It will
be found useful for killing or anæsthetizing insects and other
animals.
13. Chloride of Sodium.-A per cent aqueous solution is
much used as a macerating and teasing solution. Salt is also
a temporary preservative of animal tissues.
14. Chlor-iodide of Zinc.-This is also known as chlorzinc.
iodine, iodized chloride of zinc and Schultze's solution. Sat-
urate pure hydrochloric acid with zinc and allow the solution
to evaporate to the consistency of thin syrup or strong sulphuric
acid. Keep metallic zinc in the solution during this process.
Now saturate the solution with iodide of potassium and metallic
iodine. Keep in a dark place.
This reagent is much used in vegetable histology. It gives
color reactions with starch, cellulose and lignified tissues.
15. Chromic Acid.-This is a fixing agent and is used in a
per cent to I per cent aqueous solution in the study of the
cell-walls of alge, starch granules and the plasmodia of
Myxomycetes. Strong solutions slowly dissolve all cell-walls
excepting those of suberized cells.
The stopper of the chromic acid bottle should be coated with
vaseline, otherwise the crystals which form in the neck will
cause it to stick.
16. Cloves, Oil of.-A very popular clearing fluid pre-
paratory to mounting in Canada balsam. It should, however,

132
MICRO-TECHNIQUE.
be remembered that it slowly decolorizes stained sections. It
dissolves celloidin and collodion.
17. Dammar Gum.-Dissolve in warm turpentine, xylol or
benzol to about the thickness of syrup. An excellent mount-
ing medium.
18. Eosin. This is a red stain derived from phthalic acid.
Dilute aqueous or alcoholic solutions are employed. It stains
protoplasm and nuclei. It is much used in staining bacteria.
19. Formalin.-This is an excellent preservative for both
animal and vegetable substances.
substances. It is superior to alcohol
because it does not change the natural colors very much and
does not make tissues so brittle. A 4 per cent to 6 per cent
aqueous solution is employed. It is an irritating poison.
20. Fuchsin.-This is one of the aniline dyes and much
used in general staining. Dilute alcoholic or aqueous solu-
tions are made. To 100 grams of a 5 per cent aqueous carbolic
acid solution add one gram of Fuchsin and ten grams of alco-
hol; filter. This solution is much used for staining tubercle
bacilli. The stain is very permanent.
21. Gentian Violet.-This is undoubtedly one of the best
general stains. It stains quickly, evenly and the color is quite
permanent. Dissolve in water or dilute alcohol to which a lit-
tle carbolic acid, acetic acid or formic acid has been added.
Use only dilute filtered solutions. Concentrated solutions
stain too quickly and heavily. Gentian violet in aniline water
or 5 per cent carbolic acid is used for staining bacteria.
22.
Glycerine.-Pure glycerine is used as a mounting
medium. Equal parts of glycerine and water forms an excel-
lent softening agent for vegetable tissues. One part of glycerine
and two of water is an excellent clearing fluid for temporary
mounts. Equal parts of glycerine, alcohol and water also forms
an excellent clearing fluid.
23. Glycerine Jelly.-Prepared as follows: One part, by
weight, of the best French gelatine soaked for two hours in
six parts of distilled water; to this add seven parts of pure
glycerine. By way of a preservative add 1 gm. of carbolic acid
to 100 gms. of the mixture. Heat the entire mass for ten or
fifteen minutes, stirring continually, until the fluid is clear and
all flocculence has disappeared. Filter through glass wool. It

REAGENTS.
133
is advised to purchase it ready prepared if possible. Glycerine
jelly is in many respects a very superior mounting medium.
24. Gold Size.-A finishing agent for microscope mounts.
Apply with a camel's hair pencil.
25. Gum Arabic.-An excellent mounting medium. About
10 per cent aqueous solution will be found very useful for
mounting living protozoa and other rapidly moving small
organisms, in order to check their movement sufficiently to
make their study possible.
26. Hydrochloric Acid.-The pure acid is used with a 2
per cent alcoholic phloroglucin solution to give the red lignin
reaction. To the section add one drop of HC1 followed by one
drop of phloroglucin. A per cent to per cent alcoholic
solution of HCl acid is used to decolorize overstained prepa-
rations. It is also used as a clearing agent.
27. Iodine. An alcoholic solution (tinct. of iodine) is
used in testing for starch.
The official tincture may be
employed, but it should be diluted. Five parts of chloral with
two parts water and a little iodine added is used to decolorize
chlorophyll bodies and demonstrate the presence of starch con-
tained therein.
28. Oils.-Oil of lavender, oil of lemon and oil of marjo-
ram are used as clearing agents like oil of cloves and cedar oil.
Linseed oil is used for diluting gold size.
29. Mercury.-See bichloride of mercury.
30. Methyl Violet.-A good stain prepared and used much
like gentian violet.
31. Nitric Acid.-Used as a clearing fluid and solvent.
32. Osmic Acid.-A fixing agent. It is a very corrosive
poison. Preferably it is kept in crystalline form in hermetically
sealed tubes and dissolved in water when desired for use. A
I per cent solution is useful for killing and fixing protoplasm,
nuclei, plastids, and other plasmic substances. It is especially
useful in the study of nuclear division and the structure of
lower forms of animal and vegetable life. Osmic acid is re-
duced by fats and the tissue is blackened in time if not thor-
oughly removed by washing.
-
33. Paraffine. This is used for infiltrating and imbedding.
Two grades, one comparatively hard, the other comparatively
soft, are used. These may be mixed in various proportions.

134
MICRO-TECHNIQUE.
34. Phenol.-Used like carbolic acid.
35. Picric Acid.-A 2 per cent aqueous solution is used
as a fixing agent. Combining with sulphuric acid improves it,
as it is more readily washed out of the tissues. To a satu-
rated aqueous solution of picric acid add too parts of sulphuric
acid. The material fixed in picric acid must be thoroughly
and repeatedly washed in water to remove all traces of the
acid.
36. Potash.-A 5 per cent solution will be found an excel-
lent clearing agent. Concentrated solutions are also used for
various purposes.
Potash solutions should be kept in glass-
stoppered bottles; the stoppers must be coated with paraffine
or vaseline to prevent their sticking fast.
37. Safranin.-An excellent plasmic and nuclear stain.
Make a saturated alcoholic solution and dilute with an equal
amount of distilled water. It is a slow stain.
38. Shellac.-Use only perfectly clear material and dis-
solve in alcohol to the consistency of syrup. It forms an ex-
cellent mounting medium.
39. Turpentine.-A solvent of Canada balsam and a clear-
ing fluid. Turpentine Canada balsam does not become as brittle
as xylol balsam.
40. Xylol.-A most excellent clearing fluid and invaluable.
in paraffine infiltration. It is also very useful in cleaning
gearings, etc., of microscopes and other scientific instruments.
II. REAGENTS ARRANGED ACCORDING TO PROPERTIES.
It will be observed that the following classification of
reagent is somewhat confusing, as one and the same reagent is
mentioned under several different groups. Experience, directed
by judgment, will teach the proper use of the different reagents
included under the same head. The numbers preceding the
reagents named refer to the numbers of the preceding alpha-
betical list.
I. Fixing Agents.-These reagents have the property of
instantaneously killing plasmic substances without contracting
them and of coagulating and hardening albuminoid substances.
They are therefore also hardening agents. The fixing fluid
must be added in large quantities and should act as rapidly as

REAGENTS.
135
possible. The material must therefore be in small pieces. The
fixing agent must be thoroughly washed out again afterwards.
6. Bichloride of mercury.
15.
Chromic acid.
32. Osmic acid.
35. Picric acid.
II. Hardening Agents.-Hardening agents act by coagu-
lating albuminoid matter or by abstracting water, or both.
3. Alcohol.
6. Bichloride of mercury.
15. Chromic acid.
32. Osmic acid.
35. Picric acid.
—
Tannin, for gelatinous substances and for injecting
blood vessels.
III. Softening Agents.-These have the opposite effect
from the above. Macerating mixtures are also softening
agents. There are a great variety of chemicals which might be
included under this head. Most of the alkalies and many
acids belong here.
1. Acetic acid.
22. Glycerine.
31. Nitric acid.
36. Potash or caustic soda.
IV. Dehydrating Agents.-These agents act by displacing
the water in tissues. Some of them are also hardening or
softening agents, while others have no special effect either way.
3. Alcohol.
8. Carbolic acid.
22.
Glycerine.
31. Nitric acid.
V. Clearing Agents.-These act in several ways. They
may partially dissolve the opaque substance or substances or
cause them to swell, or the transparent substance fills the inter-
cellular and intermolecular spaces and allows the light to pass
through more readily.

136
MICRO-TECHNIQUE.
I. Acetic acid.
II. Chloral hydrate.
22. Glycerine.
28. Oils-
Cedar.
Cloves.
Marjoram.
39. Turpentine.
40. Xylol and Benzol.
VI. Mounting Media.-A great variety of substances are
used for temporary mounts. Many substances not mentioned
may be used on occasion for permanent mounting, as glues
and cements. I have found "Vanstan's stratena" an excellent
medium. The following media are used for permanent mounts:
7. Canada balsam.
17. Dammar.
22. Glycerine.
23. Glycerine jelly.
25. Gum arabic.
38. Shellac.
VII. Stains.-Stains are coloring agents which impart
their color directly to substance without any chemical reaction.
As a rule the best results are obtained by using dilute solu-
tions with long treatment. The following are only a few of
the more important stains:
5. Aniline blue.
18.
Eosin.
20.
Fuchsin.
21.
Gentian violet.
30. Methyl violet.
37. Safranin.
VIII. Micro-chemical Reagents.-These are used to pro-
duce certain reactions with tissues, the most marked of which
are colorations in or upon cells. Only a few are given.
14. Chlor-iodide of zinc.
27. Iodine.
26. Phloroglucin (see Hydrochloric acid).

REAGENTS.
137
IX. Preserving Fluids.-Any substance, whether in solu-
tion or powder, which will prevent destructive or putrefactive
changes in tissues is a preservative. We shall mention only a
few fluids used for that purpose.

3. Alcohol.
8. Carbolic acid.
19. Formalin.
22. Glycerine.
34. Phenol.

138
MICRO-TECHNIQUE.
CHAPTER III.
METHODS.
There are a vast number of methods for preparing and
mounting objects for examination under the compound micro-
scope. It would be impracticable and wholly unnecessary to
refer to all of these in detail. We shall content ourselves with
referring in a general way to some of the more common
methods, such as have proven to be most satisfactory and
desirable by the average biologist, botanist in particular. It
is only through experience that the student will learn to adopt
or modify the method to suit any particular case.
I. TEMPORARY MOUNTS.
Temporary mounts of substances placed upon a special glass
slide for examination under the compound microscope are
such as are not intended to be kept for repeated examination
and as a rule are not kept longer than a few days or several
weeks. The active botanist will spend most of his time upon
the examination of such mounts; the material for which can
usually be readily obtained. It must be left to the individual
worker's judgment when to prepare permanent mounts. Most
temporary mounts can be converted into permanent mounts
by the proper manipulation, as will be explained later.
I. MOUNTS OF ENTIRE OBJECTS.
The narrow field and limited depth of focus of the com-
pound microscope and the lack of transparency of objects
quite accurately determines the limit of the dimensions of
objects which may be mounted entire. It is true, thick objects
may be flattened somewhat by pressure and the opacity par-
tially overcome by clearing fluids, but even these devices soon
reach their limit of usefulness.
There are a host of substances which may be mounted and
examined entire, such as small insects, hairs, scales of butter-

METHODS.
139
flies, wings of insects, eggs of insects and other small animals,
leaves of moss, pollen-grains, spores, hair-cells, small fungi,
bacteria, diatoms and other algæ, milk, vinegar, crystals and
many other substances. Most of these substances may be
mounted in a drop of water placed on the slide and covered
with a cover-glass. Substances soluble in water, as many salt
crystals, may either be mounted dry or placed in liquid in
which they are not soluble, or they can be examined in a super-
saturated solution. Bacteria may be mounted in water by
mixing them with a small drop, but owing to the fact that these
organisms are very small and colorless it is usually best to
resort to a permanent method of mounting, to be mentioned
later. It is, however, necessary to mount them in water or
some other harmless fluid to determine the presence or absence
of automobility. Beginners in microscopy are at first more or
less interested and misled by a vibratory motion observable
in small objects suspended in liquids, as, for instance, bac-
teria, particles of organic and mineral substances, etc. This
is known as Brownian movement and is supposed to be due to
local variations in temperature. It is a wholly passive move-
ment on the part of the particles. A little experience will
soon make clear the difference between the two kinds of mo-
tion. The beginner is at first also perturbed by the move-
ment of particles due to gravitation. This is of course only
noticeable when the stage is inclined and is the more sur-
prising since the objects seem to move up the incline. This
up-hill movement is, however, only apparent since the motion
as well as the image of objects looked at is reversed.
There must not be too much material placed on the slide, as
that interferes with the transmission of light and makes it
impossible to distinguish the individuals. The beginner is
very liable to place enough material on the slide for fifty or
more mounts, an error which he soon learns to correct by
experience. Many substances in the form of liquids or semi-
liquids must be diluted; as milk, blood, bacterial growths, etc.
All that is necessary is to add sufficient water and mix thor-
oughly.
With many substances it is desirable to increase their trans-
parency by the use of clearing fluids. This is particularly the
case with insects, wings of insects, and other more or less


140
MICRO-TECHNIQUE.
opaque objects. In fact, most substances are more clearly
defined by the use of some clearing fluid. Glycerine diluted with
equal parts of water and alcohol is a splendid general clearing
fluid and is at the same time a good preservative.
It need scarcely be mentioned that in order to examine liv-
ing small fresh-water algae, protozoa, small crustaceans, hydra,
small worms and other minute plants and animals, they must
be mounted in some inert liquid, as water; preferably a drop
of the liquid in which the organisms live and grow. Their mo-
tion may be reduced by mounting in a solution (10 per cent) of
gum arabic.
2. CRUSHED AND POWDERED SUBSTANCES.
(ADULTERATIONS.)
A great variety of substances occur in a crushed condition or
in the form of powder, as commercial starch, dextrin, flour,
almond meal, tooth powder, snuff, ground spices, ground
mustard, ground coffee, powdered drugs, insect powder, con-
dition powders, etc. It is very frequently desirable to examine
these for their natural constituents or to determine whether
they are adulterated. Many of these substances are of suf-
ficient fineness to be mounted and examined at once. All that
is necessary is to place a minute quantity of the powder upon a
slide, mixing well with a drop of clearing fluid and cover with
a cover-glass. Ground coffee, chicory, coarsely powdered
drugs, tea-dust and similar substances will usually require
further reduction. This may be done by hand with a mortar
and pestle. Such substances as macaroni, spaghetti, bread
crusts, crackers, chalk, baked clay, etc., may be crushed in a
mortar or they may be scraped by means of a knife. As
regards the fineness required it may be stated that it varies
somewhat. In a general way it will be found that particles
which will pass through a sieve of from 80 to 100 meshes to
the inch will be sufficiently fine. Thus powdered drugs desig-
nated as No. 80 and No. 100 are well adapted for microscopic
examination. In many instances Nos. 60 to 80 (60 to 80
meshes to the inch) are sufficiently fine, that depending upon
the nature of the histological elements of which the drug is

METHODS.
141
composed and upon the distinguishing characters one wishes
to determine.
Owing to the fact that nearly all of the powdered substances
placed on the market are very frequently more or less adulter-
ated it becomes an important item to be able to detect such
adulterations. In some instances this can be done quite read-
ily. For instance, if wheat flour is mixed with cornmeal it
may be readily detected by the characteristic differences in
the forms of the wheat and corn starch granules. If chicory is
added to ground coffee it will be detected by the presence of the
histological elements peculiar to the chicory root, which are
altogether different from those of the coffee bean. In many
instances careful expert work is required to detect the nature
of the adulteration.
It may be stated here that in order to be able to detect adul-
terants one must be perfectly familiar with the normal struc-
ture of the substance. If one has studied the constituents of
the pure article there is little difficulty in detecting the pres-
ence of a foreign substance. The accurate determination of
the quality and quantity of the adulterant is usually quite
difficult. Rough estimates can, of course, be made quite
readily.
The histological elements of powdered vegetable and animal
substances are more or less fragmentary and comparatively
difficult of recognition, because they occur variously inter-
mingled and in various positions, so that it is advised never to
undertake the study of the powders until a more exact knowl-
edge of the histology has been obtained from the study of
carefully made sections. This, of course, only applies to sub-
stance that can readily be sectioned, such as the different parts
of all higher plants and animals.
Great care and cleanliness are necessary in all microscopical
work, but particularly in the study of powdered substances.
Dust and other foreign substances must be carefully removed
from slides and covers. Great caution must be observed so as
not to get different powders mixed. Never use the same slide
and cover for different powders unless special care has been
observed in cleaning them. If several slides are being pre-
pared for examination be sure to label them, otherwise con-
fusion is sure to follow.


142
MICRO-TECHNIQUE.
3. NEEDLE PREPARATIONS.
(TEXTILE FABRICS.)
Mounts prepared with the aid of dissecting needles are called
needle preparations and may be either temporary or perma-
nent. The great majority of such mounts will, however, be
temporary, as they are usually of substances which can be
obtained readily. Sometimes it is desirable to make needle
preparations of substances which are generally sectioned, such
as fibrous vegetable tissues, fungi, lichens, muscle, fascia,
fibrous connective tissue, etc. This is the case when it is
desirable to examine the isolated histological elements. Sepa-
ration of the tissue elements is much aided bv maceration in
some softening agent, as water, or glycerin and water, salt
solution, potassium hydrate solution, acetic acid, etc.
To prepare the material, place a small bit of it upon the slide
and keep it moistened with the softening fluid, per cent salt
solution or water. By means of the dissecting needles, which
must be free from rust, separate the histological elements. A
dissecting microscope or good pocket lens will be found useful,
although it is not by any means absolutely necessary. Wipe
away all that is not desired and mount the remaining tissue
elements in water or some clearing fluid. Textile fabrics, as
the various kinds of paper, paste-board, blotting paper, filter
paper, dryers, paper currency, cloth of all kinds, cotton, yarn,
etc., may be separated by means of the needles or they may be
scraped by means of a knife and mounted as above.
Any temporary mount, whether it be of an entire object,
needle preparation or sectioned material, may be converted into
a permanent mount by the following general process:
1. Removing Temporary Mounting Fluid.-Carefully lift
off the cover-glass and remove most of the mounting fluid by
means of blotting paper. Be careful not to leave bits of the
blotting paper on the slide or to drag away the material on the
slide by means of the blotting paper.
2. Dehydrating.-Place a drop or two of approximately
absolute alcohol on the specimen and allow it to remain for a
few minutes. The alcohol displaces the water in the specimen
and prepares it to receive the clearing fluid. It must be

METHODS.
143
remembered that alcohol will shrink and distort the tissue ele-
ments more or less.
3. Clearing. After the alcohol has evaporated in part,
place a drop of clearing fluid upon the specimen; xylol or oil of
cloves are excellent. Allow it to remain for a few minutes and
remove the excess by means of blotting paper.
4. Mounting.-See that the tissue is properly arranged
upon the center of the slide (use needles and turn table or slide
centerer). Place a small drop of xylol balsam or oil of cloves
balsam upon the specimen and cover with cover-glass. Or
place the cover-glass on first and add a small drop of balsam
near the margin of the cover and allow it to be carried under
by capillarity.
If it is desired to stain the specimen, it may be done before or
after step 2, that depending upon whether the stain is in the
form of an aqueous or alcoholic solution. A drop of the
stain is added and allowed to remain a few minutes, after
which all excess of stain must be washed away by means of
water and blotting paper. Of course the specimen must again
be dehydrated before clearing and mounting. It must also
be borne in mind that alcohol tends to decolorize stained speci-

mens.
Dehydrating should be done gradually in order to prevent
distortion as much as possible. Gradual dehydration will be
explained later.
4. HAND SECTIONED SPECIMENS.
Most vegetable tissues and some animal tissues may be sec-
tioned without any special preparation. Some substances, as
rock, bone, chitinous coverings of insects, many seeds, are too
hard to be cut by a knife or razor. Muscle, connective tissue,
glandular tissue and similar substances are too soft and yield-
ing for the knife. Most leaves, stems, some seeds, barks,
roots, cartilage, and many other substances, can be cut by
hand, provided a sharp razor or section-knife is used. It is
simply a waste of time and energy to attempt to cut any
section with a dull knife, a fact which is not sufficiently rea-
lized by most students.
Considerable practice is necessary before good hand sec-

144
MICRO-TECHNIQUE.
tions can be made. The following suggestions may prove
valuable:
1. Section-knife or Razor.-A good razor will be found
adequate for all ordinary purposes. Do not attempt to use a
worn-out razor full of nicks or a razor made of a poor quality
of steel. Use a good razor only, one which can be sharpened
to a keen edge and which will retain the sharp edge for some
time. The blade must not be too broad nor too thin and should
be flat on its lower side when in the act of cutting sections.
A very thin blade will bend upon cutting tissues of some
firmness. A thick blade cannot be sharpened readily. The
razor must be kept free from rust.
2. Sharpening the Razor.-Good razors appear on the
market sufficiently sharp to be ready for use. The keen
edge is, however, slightly dulled by the action of atmospheric
oxygen and carbon dioxide, hence it is advisable to "touch it
up" a little on a strop or barber's strap. Students are advised
to use a strop with handle rather than the strap. The razor is
sharpened by laying the blade flat upon the strop and dragging
it evenly and diagonally from the handle end to the opposite
end, then reversing the blade by turning it with the back rest-
ing upon the strop and dragging it in the opposite direction.
Never turn the razor over on its edge. The razor should be
sharpened on the strop very frequently; on an average every
time eight to twelve sections have been cut. Occasionally it
will become necessary to sharpen the razor upon a fine oil
stone so as to remove small nicks and to even up the edge.
Examine the edge occasionally with a pocket lens which will
reveal nicks not noticeable to the naked eye. To test the
sharpness of the razor select a hair from the head an inch or
more in length, hold it between thumb and finger of the left
hand and let the free portion rest upon the upturned edge of
the razor, draw the razor forward and upward. If the hair
is cut in two and not split the razor is sufficiently sharp for
cutting sections.
3. Handling the Razor.-As soon as the sections are cut
close the razor and lay it out of the way. Never move about
with an open razor in your hand, as you may injure yourself
as well as others who may be near you in the laboratory.
When through using, the razor should be wiped perfectly dry

METHODS.
145
and placed in its case.
Do not allow corrosive reagents to
come in contact with the razor.
4. Cutting Sections.-Sections may be cut directly or after
the substance has been subjected to some preliminary prepara-
tion. If the material, as leaf, small stem, root, rhizome, etc.,
is to be cut directly, grasp it between thumb and first finger of
the left hand so that the thumb is a little below the end from
which the section is to be cut, and the tip of the first finger
projecting slightly above. Let the distal end of the razor
blade rest upon the tip of the index finger and push the razor
diagonally forward and to the left through the material. If
the end of the material is ragged or rough, cut away a portion
with a pocket-knife. Use a sharp pocket-knife whenever
rough cutting is to be done and never use the razor for such
purposes, as it is sure to break out nicks. The thickness of
the section can be regulated somewhat by the pressure of the
blade upon the tip of the index finger. Never try to cut sec-
tions by pushing the razor straight forward through the sub-
stance. Moving the razor diagonally forward and to the right
is more apt to cause tearing and separation of tissues, especially
if the razor is not very sharp. It is advisable to keep razor and
material moist. As soon as cut the section should be mounted
or placed in some liquid to prevent its drying and to prevent
the entrance of air.
With some tissues it is difficult or impossible to make sec-
tions without first imbedding or placing them between some
supporting substance, as pith or cork. Many leaves, for
example, are too delicate to be cut as suggested above. I,
however, usually succeed in cutting good sections of such
leaves by rolling them as one rolls up carpet. Some prefer
to place the leaf-blade between two pieces of pith or cork.
Leaves, delicate stems, etc., may also be imbedded in melted
paraffine; after cooling, sections can be cut without any diffi-
culty. Before mounting such sections the paraffine is removed.
5. Kinds of Sections.-Most tissues are cut at right angles
to the long axis, which is usually the long axis of growth of the
tissue. Such sections are therefore spoken of as transverse
sections (a, Fig. 83). When sections are cut parallel to the
long axis they are designated as longitudinal sections. In the
case of cylindrical organs, as roots, stems, branches, etc., two


146
MICRO-TECHNIQUE.
kinds of longitudinal sections may be cut, radial and tangen-
tial. In the former the cut is always made in a radial plane
(c), that is, the knife always moves from the periphery to-
ward the center, or vice versa, from the center toward the
periphery. A tangential section is made by cutting along a
longitudinal plane at right angles to a radius or radial plane
(b). The tangential section should be made as near the
10
d
FIG. 83.
KINDS OF SECTIONS.
periphery as possible. The three kinds of sections will show
the true form and structure of the cells and tissues of organs
of longitudinal growth. It is never advisable to make diag-
onal sections (d, Fig. 83).
83). Great care must be observed in
order to secure true transverse and longitudinal sections.
Beginners and careless workers are apt to cut most sections
more or less diagonally. Such sections are worse than useless
as they convey an erroneous idea of the form of cells.
In order to show epidermal cells in vertical view it is neces-
sary to make sections parallel to the epidermal surface (sur-

METHODS.
147
face sections). Such sections are usually made without any
difficulty. The sections must be made very thin in order not
to include the cells of underlying tissues. Surface sections
should be mounted with the outer surface directed upward.
6. Mounting Sections.-Sections cut by hand may be
mounted in water, alcohol, glycerine, clearing fluid, or any of
the many reagents used in the work. (See reagents.) Place
a drop of the mounting fluid upon the center of the slide, in
it place the section or sections and cover with cover-glass.
Let one edge of the cover rest upon the slide to the left of
the mounting medium and let it come down slowly by means of
a dissecting needle or pincer so as to prevent displacement of
sections and to exclude as much of the air as possible. The
sections may be stained if desired or converted into perma-
nent sections as already explained.
Caution should be observed not to use too much or too little
of the mounting medium. If too much is used it will cause
the cover-glass to float about and the liquid will project above
the margin of the cover or even run over the upper surface,
where it is likely to come in contact with the higher power
objectives. Mounting media, dust, finger-marks, etc., on the
upper surface of the cover-glass, will cause distortion and
indistinctness of the image. If too little of the medium is
used, the object or objects will not be entirely immersed and
cause blurring and indistinctness of image. There should
be just enough of the mounting medium to fill all of the space
between the slide and lower surface of the cover-glass and no
more. Remove any excess at once and make sure that there
is none of the fluid on top of the cover-glass. Under no cir-
cumstances should mounting media of any kind come in con-
tact with objectives. If this should accidentally happen,
remove the liquid at once by means of a dry clean piece of
cloth or Japanese paper.
7. Removing Air Bubbles.-Air bubbles are the bugbear
of the microscopist. They attract the attention of the begin-
ner, who will for some time fail to recognize their identity.
When numerous they interfere very much with the examina-
tion of sections, hence it is desirable to get rid of them. This
may be done in several ways. In the first place care should
be observed to prevent the entrance of air into the section.


148
MICRO-TECHNIQUE.
As soon as cut it should be immersed in some liquid or
mounted at once.. Mounting the sections in recently boiled
water will remove some air bubbles by absorption. Mounting
in alcohol will drive out air bubbles. The air may also be
removed by placing the sections in a vessel of water or mount-
ing fluid under the receiver of an air-pump. The quickest
and most effective and most readily available method is to
heat the mounted section over a flame (Bunsen burner, lamp,
etc.). Hold the slide, section uppermost, over the flame
accompanied by a circular motion so as to heat the slide uni-
formly, otherwise the slide and cover-glass may crack; watch
the section; as soon as a lively escape of air bubbles is noticeable
take the slide away from the source of heat for a second or
two, then hold it over the flame again for a second. Repeat
this from two to four times, when it will be found that nearly
all of the air is driven out. Do not hold the slide over the
flame until no more bubbles are driven off. It must, how-
ever, be borne in mind that the heating process will dissolve
or destroy certain cell-contents, as starch, inulin, resin, wax,
mucilage, etc. The cell-walls will become somewhat swollen,
but not materially altered in form; in fact, heating will restore
shrivelled cell-walls to their normal form. Very active boiling
may crack the cover-glass and break up the section. The
evaporated mounting medium should be replaced at once by
adding more to the edge of the cover-glass and allowing it to
be drawn under by capillarity.
II. PERMANENT
MOUNTS.
Permanent mounts differ from temporary mounts in that the
substances to be mounted are subjected to special processes
which makes it possible to keep them for a long time for
repeated examination. There are many methods for preparing
permanent mounts of animal and vegetable substances. In a
general way the methods applied to animal tissues also apply
to vegetable tissues. It would be impossible in a work of this
kind to explain the many special methods in detail.
I. DIRECT METHOD.
This might also be designated as the method without infil-
tration. That is the substance or substances of which mounts

METHODS.
149
are to be made are not passed through solutions of the infil-
trating media, as celloidin, collodion, or paraffine, preparatory
to sectioning. Any temporary mount may be converted into
a permanent mount, as already explained in a general way.
The following suggestions apply more especially to free hand
sections which it is desirable to mount permanently.
necessary reagents and apparatus must be at hand.
suggestions are general and must be modified to suit special
cases and requirements:
The
The

1. Firing. Place the section in a two per cent solution of
picric acid for a few minutes, after being thoroughly washed in
distilled water. This applies to sections of vegetable tissue
cut from the green or living plant only. Dead tissues require
no fixing, in which case proceed at once with dehydrating.
2. Washing.-Wash thoroughly in distilled water to re-
move all traces of the picric acid, otherwise annoying precipi-
tates will be formed in and upon the cells.
3. Dehydrating.-Alcohol is used for this purpose and it
should be done gradually in order to prevent distortion of
cell-walls. Pass from water into 15 per cent alcohol, from
that into 25 per cent, and so on up to absolute alcohol, each
time passing into a solution 10 to 15 per cent stronger than
the preceding. The section should be left in each solution for
a few minutes. Use section lifters.
4. Clearing. From absolute alcohol the section should be
placed in some clearing fluid, as xylol, benzol, oil of cloves,
that depending upon the mounting medium to be used.
5. Mounting.-Remove superfluous clearing fluid by
means of blotting paper and dry cloth and mount in Canada
balsam diluted with xylol, oil of cloves, turpentine, or some
other suitable diluent. Glycerine gelatine is in many respects
the most suitable mounting medium. There are many other
mounting media which may be employed.
6. Staining.-If it is desirable to stain the section it must.
be done after dehydrating. If the staining is to be done in an
aqueous solution, the section is placed in the stain after fixing
and washing, and then passed up through the alcohols, cleared,
and then mounted. If it is to be stained in an alcoholic solu-
tion, for example, a 50 per cent solution, pass the section up
through the alcohol to the 50 per cent, stain, and continue to

150
MICRO-TECHNIQUE.
dehydrate, clear and mount as before. If the section is over-
stained, decolorize by washing in alcohol or in acidulated
alcohol.
2. CELLOIDIN METHOD.
In this method a solution of celloidin is used as the infiltrat-
ing material. Collodion may be used in its place, as it is in all
respects similar.
For this process the substances to be sectioned should never
be large, nor is it possible to cut very hard substances, as the
celloidin is not sufficiently firm and resisting. Leaves, stems,
roots, should be cut into fragments not greatly exceeding one-
fourth of an inch in any diameter. It is best to do this cutting
at the beginning or after the fixing process, using a sharp
knife or a pair of scissors.
The celloidin or collodion is dissolved in equal parts of
absolute alcohol and ether. Make two solutions, one a 2
per cent (by weight) and another about 5 per cent, of celloi-
din. The substances dissolve rather slowly; several days are
usually required before it is thoroughly dissolved. The
solutions must be kept in well stoppered bottles so as to prevent
the evaporation of alcohol and ether. Do not use these sub-
stances near an open fire, as they are very inflammable.
1. Fixing.-As already described.
2. Dehydrating.-As already described.
3. Infiltrating.-From the absolute alcohol place the tissue
in a 2 per cent solution of celloidin or collodion. Allow it to
remain for at least 24 hours. Now place it in a 5 per cent
solution of celloidin for about 24 hours.
These periods are not absolute. A longer period may be
required in many instances.
4. Imbedding or Fixing Upon Blocks.-The infiltrated
substance may be imbedded upon pieces of cork or small blocks
of wood; the latter are preferable because they give a firmer
support and make it possible to clamp more firmly in the
microtome. The cork or block should be moistened in 75
per cent alcohol. The surface upon which the substance is to
be fastened should be roughened. By means of stick or glass
rod place a drop of the 5 per cent celloidin upon the block or
cork, in it set one of the pieces to be sectioned, holding it in

METHODS.
151
the proper position until the celloidin has begun to harden
a little; now let more celloidin drop over it until it is entirely
enclosed by it. Hold it quietly for a short time (half a.minute
to a minute), and then place it celloidin surface down into a
dish of about 75 per cent alcohol. Never allow it to remain
exposed to the air, as the celloidin would become hard and
brittle in a very short time through the evaporation of the
ether and alcohol. In the course of half an hour the block
is ready for the microtome. Or it may be kept in alcohol for
several days if desirable or necessary.
5. Sectioning.-Fasten the block in the clamp of the
microtome. If it is cork, do not clamp too firmly, as the com-
pression of the cork may loosen the celloidin. Should this
happen it may be refastened by adding a little of the celloidin
solution.
While cutting, the block, as well as surface of section-
knife, should be kept moist with 75 per cent alcohol. Place
it on by means of a camel's hair brush or set a dropping bottle
or similar apparatus above the microtome. As soon as cut
the sections are placed in a dish with 75 per cent alcohol
until ready for staining and mounting.
6. Staining.-Staining is usually done before removing the
celloidin. In staining proceed as already explained.
7. Removing Celloidin and Clearing.-After the section
is stained pass up through the different strengths of alcohol and
finally pass from absolute alcohol into oil of cloves, which
dissolves the celloidin very actively and at the same time clears
the section.
8. Mounting.-Mount in oil of cloves, Canada balsam or
turpentine balsam. It should be remembered that oil of cloves
fades out the stain in time.
The fixing agent employed will depend upon the nature of
the substance to be infiltrated. (See Reagents.)

3. PARAFFINE METHOD.
The essential difference between this method, and the pre-
ceding one is that paraffine is used as the infiltrating material
instead of celloidin or collodion. It has its advantages as
well as its disadvantages. It is preferable because it is pos-

152
MICRO-TECHNIQUE.
sible to make thinner sections, but tissues are apt to shrink
more than in the preceding methods, because of the fact that
heat is employed in the infiltrating process. The following
are the different steps of procedure:
1. Fixing.-As in previous methods.
2. Dehydrating.-As in previous methods.
—
As
3. Clearing. From absolute or approximately absolute
alcohol place in xylol until completely permeated by it.
soon as this has taken place the tissue becomes translucent
throughout. The time required will vary with the size and
consistency of the substance, on an average perhaps twelve
hours.
4. Infiltrating.-From pure xylol the tissue is placed in a
mixture of equal parts of xylol and paraffine of medium hard-
ness. A medium paraffine can readily be made by melting
together equal parts of hard and soft paraffine.
Place the vessel with xylol and paraffine in an incubator
and keep it at a temperature of the melting point of the par-
affine. The temperature of the incubator should be carefully
regulated by means of a Reichert thermoregulator, or some
other good regulator. If the temperature becomes considerably
higher than the melting point the tissues will shrink and
the cell-walls become much distorted.
Allow the xylol to evaporate and add more paraffine from
time to time until finally (in the course of 24 hours, more or
less), the substance is in a highly concentrated solution of
paraffine. It is not necessary to get rid of all traces of the
xylol. Lastly, place the substance in pure melted paraffine
and allow it to remain for several hours, after which it is ready
for imbedding.
4. Imbedding.-Imbed in medium or hard paraffine. Make
paper boxes large enough so that the substance. may be
enclosed on all sides by melted paraffine. Dealers in micro-
scopical supplies have for sale adjustable boxes which may be
adapted to the size of the substance to be imbedded.
Pour a little melted paraffine into the box, now place in it
the substance and pour melted paraffine over it until it is well
covered. Now orientate the substance by means of a dissect-
ing needle so as to have it in the desired position and allow it
to remain quietly until the paraffine begins to harden. The

METHODS.
153
cooling process may be hastened by immersing the box, block
and all, in cold water, or by holding it under the faucet of a
hydrant and allowing the cool water to run over the par-
affine.
After the paraffine is thoroughly set the material is ready for
sectioning.
5. Sectioning.-The simple or automatic microtome is
used. Material infiltrated by paraffine is cut dry. The block
of paraffine is suitably fastened and the sections are removed
from the knife by means of a camel's hair brush and placed in
a small box or directly into the fluid for removing the paraffine.
If the paraffine is very hard and brittle, as it is apt to be in cold
weather, it may be softened to the desired degree by placing
the microtome with block near a radiator or other warmth
yielding appliance. Before beginning to cut sections, trim
away as much of the paraffine as possible, as it must be dis-
solved out afterwards.
6. Removing Paraffine.-Any substance which dissolves
paraffine may be used, as xylol, benzine, benzol, chloroform,
ether, etc. Chloroform and ether are objectionable because
they evaporate too rapidly and cause cells to shrink. Xylol
and benzol are perhaps best. Place the sections in a small
vessel of xylol and allow them to remain for a few minutes;
stirring will hasten the solution of the paraffine; or place the
section on a slide somewhat inclined and allow a few drops of
the solvent to run down the incline over the section, carrying
the dissolved paraffine with it. All the paraffine should be dis-
solved.

-
7. Mounting. After the paraffine is removed the sections
are ready to be mounted in xylol balsam or glycerine jelly.
8. Staining. If it is desired to stain the sections they must
be passed down the alcohols, stained, again passed up the
alcohols, cleared and mounted as already explained. Stain-
ing tissues in toto previous to infiltrating is usually very
unsatisfactory, as the outer layers are overstained while the
inner layers are not sufficiently stained.
Instead of using xylol in the infiltrating process, benzol,
turpentine or chloroform may be used. Benzol acts about the
same as xylol. Turpentine is much used with animal tissues.
Chloroform is objectionable because it makes tissues brittle,

154
MICRO-TECHNIQUE.
besides it forms objectionable bubbles which are difficult to
remove.
III. SPECIAL METHODS.
Under this head might be mentioned a great many methods.
of micro-technique not referred to or fully explained in the
foregoing chapters. We shall briefly describe a few of the
more important ones, such as are simple and have a more or
less close relationship to the methods already explained.
Much might also be said of special methods of staining, as
staining the spores and flagellae of bacteria, double staining,
multiple or differential staining, etc. In a work which is
purely elementary these methods cannot be explained. The
interested student will be obliged to consult a competent in-
structor or special works on the subject.
I. EXAMINATION OF BACTERIA.
Although bacteria belong to the smallest of organisms, they
are easily manipulated by the biologist. They may be tem-
porarily mounted in water or other inert liquid or semi-liquid
to examine them as to motion, manner of growth and other
physiological phenomena. Making permanent mounts is quite
simple, provided one does not desire to demonstrate the pres-
ence of spores or flagellæ. The process is as follows:
I.
1. Placing the Bacteria on Cover Glass.-Carefully clean
and dry a cover-glass. Dip a clean sterilized platinum needle
into the bacterial growth (in liquid, upon culture medium, in
broth, upon potato, etc.), and smear it over the upper surface
of the cover-glass mixed with a small droplet of pure water.
The beginner will generally place too many bacteria on the
cover. As many as will adhere to the very tip of the platinum
needle is usually sufficient. Mix thoroughly with the droplet
of water and spread evenly.
2. Air-drying the Smear Preparation.-This step is pre-
paratory to the next. Allow the bacteria to dry in air. As
soon as the smear changes from transparent to a hazy light
gray it is dry. The time required will depend upon the
amount of water placed on the cover. A few minutes will
usually be sufficient. A moderate heat will hasten the dry-

METHODS.
155
ing, but do not heat too much, as the bacteria would become
boiled in the liquid.
3. Passing Through Flame.-Pass the cover, with bac-
terial side up, four times through the flame of a Bunsen burner.
Four times is not a mystic number nor is it absolutely required,
but it will be found that if passed through oftener the bac-
teria become shrivelled and charred, and if passed through
only once or twice the albuminous matter is not sufficiently
coagulated to cause the smear to adhere firmly. Never hold
the cover in or directly over the flame as the bacteria would
become roasted almost instantaneously. The heating is for
the purpose of coagulating the albumen about the bacteria and
fastening them firmly to the cover-glass.
4. Staining. Place a drop of the stain on the coagulated
smear preparation and allow it to remain until the bacteria are
stained. The time required will depend upon the stain used.
Comparatively rapid stains, as methyl blue and gentian violet,
will require about two minutes to stain the cell-wall and the
cytoplasm. Slow stains, or weak solutions of all stains, will
require from ten minutes to several hours. Spores stain with
great difficulty. Special staining must be resorted to to dem-
onstrate the presence of flagellae.
5. Washing.-Wash off all excess of stain by means of
clean water. Hold the cover under the hydrant or rinse in a
dish of water. If overstained the preparation may be decolor-
ized to the desired degree by washing in alcohol or alcohol
acidulated with hydrochloric acid or acetic acid. For example,
in spore-staining the bacteria must be left in the stain
(Fuchsin) for several hours, with moderate heat applied.
Decolorize in acidulated alcohol until all of the stain is re-
moved excepting from the spores. Although spores stain
with great difficulty, they retain the stain very tenaciously
when once permeated by it.
6. Mounting Temporarily.-Before mounting the prepara-
tion permanently it is advisable to examine it in order to
determine whether it is suitable. Simply turn the cover-glass
bacterial side down, on a slide. The water adhering to it will
serve as a temporary mounting fluid. If the slide proves sat-
isfactory mount permanently.
7. Mounting Permanently.-Wipe the free surface of the


156
MICRO-TECHNIQUE.
cover dry and allow the water on the bacterial surface to
evaporate; then mount in xylol balsam, oil of cloves balsam, or
some other suitable mounting medium.
In steps I to 5 inclusive the cover-glass is held bacterial
side up by means of a pair of pincers. An aqueous or alcoholic
solution of the stain may be employed. It is not advisable to
use a saturated or very strong solution; weak solutions with
long exposures give the best results. It should also be borne
in mind that the heating and staining will cause the bacteria
to become reduced in size though the form remains practically
unaltered.
2. EXAMINATION OF MEAT FOR TRICHINAE.
Within recent years meat inspection has become an impor-
tant commercial factor. In some countries the meat of every
slaughtered hog must be examined microscopically with a
view to determining the absence or presence of encysted trich-
inæ. The method of examination is very simple and is here
given in a condensed form, so that every one in possession of
a compound microscope or a high power dissecting microscope
or pocket lens may make an examination of meats intended for
consumption. It must, however, be stated that thorough cook-
ing of all meats before eating is the best safeguard against
infection. Never eat raw meat sausages, or raw meat or par-
tially cooked meat of any kind, especially of pork. Very gen-
erally trichinosis in man is due to eating raw (smoked) or
partially cooked pork. If the meat happens to be infected with
trichinæ the cysts are dissolved in the stomach, the liberated
trichinæ become sexually mature (intestinal trichina) in the
intestines. The young are produced from eggs, and after a
time burrow their way through the coats of the intestines and
into the voluntary muscles, where they again become encysted
in an immature state. Just how they gain access to the muscles
has not been accurately determined; some investigators main-
tain that they enter via the lymphatics and blood vessels.
To examine meat for trichinæ secure samples from the mus-
cles of the loins, the ribs, eye and tongue, as they usually con-
tain the most organisms. By means of a pair of scissors or a
sharp knife cut strips in the long axis of the muscular fibres,

METHODS.
157
making them about 1 cm. long by .5 cm. wide and 2 to 3 mm.
thick, and place them in a per cent salt solution. Place
several of these pieces of muscle upon a slide and separate
the fibres as much as possible by means of the dissecting
needles. Cover with cover-glass, applying some pressure, and
examine with low power (in. obj. and I in. ocular). If any
trichinæ are present they will be readily noticed by the pres-
ence of oval granular cysts, in which the coiled worms are
situated (Fig. 84 A). There may also be free motile forms
present. B and C of Fig. 84 represent the sexually mature
trichinæ.
It should also be borne in mind that there are several other
organisms which resemble trichinæ. Of these the most com-
mon are the so-called bodies of Rainey or Miescher, which are
animal parasites belonging to the group Sporozoa. They are
elongated granular bodies occurring within the muscular fibres
while trichinæ lie between the fibres. They are very common in

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158
MICRO-TECHNIQUE.
the domestic hog. After one has become familiar with the
appearance of trichinæ they will not readily be mistaken for
any other parasite.
3. MOUNTING SECTIONS OF BONE.
Bone, on account of its hardness, requires special preparation
for mounting. The method of procedure is something as
follows:
I. Secure a well-dried bone, from which fat and other
organic matter has been washed out thoroughly. It is best to
select a long bone. By means of a bone-saw saw off some
transverse and longitudinal sections, making them as thin as
possible. Make the sections from the compact portion of the
bone.
2. Thin down the sections as much as possible by means
of a sharp file.
3. Place the section between two sand stones or Washita
stones and grind until the section is thin enough to bend of its
own weight when one end is raised. The grinding surfaces
of the stones and the section must be kept wet. Grind care-
fully, examining the section frequently.
4. Wash the sections in water and allow them to remain
in water until ready for mounting.
5. The sections are mounted in Canada balsam, but thin
balsam fills up the lacunæ; it is therefore necessary to boil the
balsam until it is quite thick, or about the consistency of thick
syrup while still warm. Allow it to cool until it is about the
consistency of putty or butter. Place a bit of this balsam upon
the cleaned cover-glass and in it imbed the air-dried section of
bone. Now place cover with section downward upon center
of slide. Apply some pressure to force out superfluous balsam;
apply a little heat if necessary, being careful not to liquefy
that portion of the balsam in the immediate vicinity of the
section.
In a well prepared section the Haversian canals, the lacunæ
and the canaliculi should show clearly under a magnification
of two hundred or three hundred diameters.

METHODS.
159
IV. GENERAL DIRECTIONS.
I. Before beginning to work with the microscope see that
the lenses are clean, the mirror free from dust. The gearings
should work smoothly.
2. In using medium and high powers never remove a slide
from the stage or place one on the stage without first turning
up the body of the instrument, so as to avoid the liability of
touching or injuring the objectives.
3. Be careful with corrosive reagents. All acids and iodine
will act upon metal. Potassium and other strong alkalies will
corrode glass. Alcohol will remove the lacquer from stands.
4. Always close reagent bottles and put them back in place.
Handie them carefully.
5. Never touch lenses or mirror, as the cleanest fingers will
leave a trace of oil which must be carefully wiped away.
6. Razors, dissecting needles, pincers and other steel imple-
ments should be kept clean and free from rust. Keep them in
a dry place, away from chemicals.
7. Cleanliness should be observed in every detail. Have
on hand a sufficient quantity of clean rags, Japanese filter
paper, chamois skin, etc., to keep instrument, slides, covers,
objectives, oculars, razors, dissecting needles, etc., perfectly
clean and dry.
8. Keep everything in its proper place. This is very impor-
tant.
9. Before returning an instrument to its case or locker, wipe
it perfectly clean. No finger-marks should show anywhere.
10. Acquire the habit of labeling everything properly so
as to avoid confusion and mistakes. This applies to reagents
used, temporary and permanent slides, material used for study,
etc. Slides may be fully labeled or simply numbered and a
full description of the mount given in a note-book. The label
or description should give the name of the substance upon
the slide; the kind of section or preparation; what method of
infiltration and imbedding; what stain used; mounting
medium and date. Labels should be securely pasted.
II. Poor, useless or undesirable mounts should be dis-
carded and the slides and covers cleaned by means of suitable
solvents and returned to their proper boxes or places.

160
MICRO-TECHNIQUE.
12. All necessary reagents, stains, etc., should be purchased
of reliable dealers. Purchase only the most important mate-
rial; use home material as much as possible. Never purchase
material in bulk, which is rarely used, or used only in small
quantities.
Other special directions are given elsewhere.


THE NORMAL AND ABNORMAL EYE.
This special chapter is not introduced to serve as a "home
physician for the treatment of diseases of the eye." It is hoped
that the suggestions given may serve as a guide to the better
care and use of the eyes and to impress upon the reader the
necessity of calling to aid prompt medical attendance in all
noticeable disorders of the eye and defects of vision. It is also
hoped that this chapter may serve to dispel a prevailing notion
that work with microscopes is specially injurious to eyesight.
The proper use of microscopes is not in the least injurious; it
is the improper use of these instruments, due to ignorance and
carelessness, which often does harm, as has already been
explained.
I. THE OPTICAL PROPERTIES OF THE EYE.
Optically, the eye may be compared to a camera obscura
(dark chamber), which consists of a box (B, Fig. 85), black-
ened on the inside, with an opening (o) on one side, which
may or may not be occupied by a convex lens. The lens was
introduced by the Neapolitan physician Porta, who found that
it gave a much brighter and better defined image. Focal points
of objects (mn) are refracted by the lens and an inverted
image (rs) is formed on the blackened wall opposite the
opening.
m
B
FIG. 85.
DIAGRAM OF CAMERA OBSCURA.
161

162
THE EYE.
DIAGRAM OF CAMERA OBSCURA.
The photographer's camera is simply a modification of the
above. It is supplied with an adjustable lens, so as to project
the image of exterior objects exactly upon the sensitized plate.
In the eye there are several refractive media, as cornea, vit-
reous humor, crystalline lens and aqueous humor, which cause
the inverted and reduced image of outward objects to be pro-
jected upon the retina. In order that the refractive media may
project a clearly defined and perfect image of objects upon the
retina there must be a maximum of transparency of the refrac-
tive media and suitable accommodating power. These we shall
consider somewhat in detail.
I. REFRACTION.
Upon examining an antero-posterior vertical section of an
eye it will be observed that the globe of the eye is spheroidal
and consists of segments of two spheres; a portion of a sphere
of smaller radius (8mm.) forming its anterior transparent
part and is set to the front of the posterior segment of a larger
sphere. The lens takes a position near the front segment.
When the eye is at rest its antero-posterior diameter is about
22.5 mm. Rays of light parallel to the principal axis are
refracted, so as to form the principal focus on the retina, as
shown in Fig. 86. The refractive indices of the aqueous humor
S
bc
d
FIG. 86.
FOCUS OF PARALLEL RAYS OF LIGHT.
(b) and vitreous humor (d) are about the same, 1.33. The
refractive index of the lens is about 1.4. The refractive parts

REFRACTION.
163
of the eye must not be compared to a single convex lens, as may
be observed from the inspection of Fig. 86. The aqueous
humor bounded by the cornea (a) in front and the lens (c)
behind is in reality a convexo-concave meniscus. The lens
itself is a double convex lens of approximately "best form"
adapted to the other refracting media, thus producing a mini-
mum of aberration. The radius of its anterior surface is 10
mm., while that of its posterior surface is 6 mm., the less con-
vex surface being directed toward the objects to be seen. The
convexity of the lens varies, however, with the act of accommo-
dation due to its elasticity and the action of the ciliary muscles.
From what has already been said about lenses and mirrors
the formation of images by the eye will be readily understood.
Rays of light from objects outside of the eye but within the
range of distinct vision are brought to a focus upon the retina,
as shown in Fig. 87. The image formed upon the retina is real,
reduced in size and inverted. The inverted image has been
the source of much discussion by physicists and physiologists.

0
B
FIG. 87.
p
NORMAL FORMATION OF IMAGE UPON THE RETINA.
The principal cause of the controversy has been due to the
fact that the brain was considered as something behind the
eye having the power to perceive the inverted image, which is
not at all the case. The brain is simply conscious of certain
stimuli emanating from the retina due to the images formed,
and does not behold the image itself. The same statement also
applies to the reduced image of the objects perceived. It is
essentially experience that teaches us to form approximately
correct estimates of the position, size and form of objects.

164
THE EYE.
2.
ACCOMMODATION.
The changes taking place in the eye to adapt it for seeing
objects distinctly at different distances constitute accommoda-
tion. It varies greatly in different persons and even at differ-
ent periods in the same person. Usually also the accommo-
dating power of the two eyes differs somewhat. The range
of accommodation in a given person is the distance between
the nearest and most remote points at which an object may be
seen distinctly without artificial aid. Naturally the range of
FIG. 88.
IMAGE OF NEAR OBJECTS.
accommodation varies with the size, form and brightness of
objects. As has already been indicated, the principal changes
to bring about accommodation take place in the crystalline lens.
It was at one time supposed that the external muscles of the
eye were also concerned in this action by changing the antero-
posterior diameter of the eye and the curvature of the cornea.
Actual test has shown that the corneal surface as well as the
general contour of the eye remain unchanged in the process
of accommodation. The crystalline lens acted upon by the
ciliary muscles becomes more and less convex, especially the
anterior surface, thus projecting the real inverted image upon
the retina, no matter where the object may be, provided it is
within the range of accommodation. If an object is brought
too near the eye it can not be perceived, because the accommo-
dating power of the eye is insufficient to bring the image upon
the retina (Fig. 88). It has also been found that in operations

ACCOMMODATION.
165
where the entire lens is removed the eye loses the power of
accommodation entirely, which is conclusive proof that the lens
alone undergoes the changes in form to bring about accommo-
dation. When the eye is at rest, that is when the ciliary mus-
cles are entirely relaxed, as in sleep or when the eyes are closed,
the lens is focussed for objects at an indefinitely great distance.
Rays of light from such objects are practically parallel.
The distance of most distinct vision varies somewhat with
different persons. It is usually 10 to 12 inches for the normal
eye. That is, this is the distance at which the normal eye can
best make out details of structure, no matter what the size and
form of the object may be. If we wish to examine a house,
-
a.
FIG. 89.
BINOCULAR VISION AND RANGE OF VISION.
a tree or any large object minutely, we can only do so by bring-
ing small circumscribed areas of the object within the range
of most distinct vision. Very small objects are usually
brought somewhat nearer to the eye than 10 or 12 inches.

166
THE EYE.
Although we have two eyes, each one receiving an image
of objects looked at, we are conscious of a single image only;
that is because both eyes are directed to the same point. The
muscles of the eyeball are so arranged and controlled by nerve
centers that the impressions upon the retinas of both eyes are
perceived as a single impression, no matter what the position of
the object may be in the range of vision (Fig. 89). But since
the two eyes are directed upon the objects from different posi-
tions, binocular vision differs from monocular vision. Espe-
cially is this noticeable in looking at comparatively small solid
bodies; the right eye perceives a certain area (m o) which the
left eye does not perceive, while the left eye perceives a certain
area (ns) not seen by the right eye; these several independent
visions are combined with the vision common to both eyes
(men) and apparently united upon the retina. We thus get
a view of objects in relief. It is upon this principle that stereo-
scopes are constructed. Two pictures are taken of the same
object from different positions. The slightly dissimilar pictures
are placed in a frame and the images combined upon the retina
by means of lenses or prisms. Fig. 90, A, B and C, will serve
to illustrate the difference between monocular and binocular
vision. A represents a pyramid as seen with two eyes; B
repre-
A
B
FIG. 90.
C
STEREOSCOPIC VISION.
sents the same cone as seen with the left eye, and C as seen
with the right eye. Now place a card vertically between B
and C and look at B and C with left and right eye simulta-
neously, whereupon the two images will be combined and give
the appearance of A. It may be necessary to gaze at the fig-

STEREOSCOPIC VISION.
167
ures a few seconds before they become properly focussed upon
the retina. From this it becomes evident that binocular vision
gives us a much better perspective of objects; it enables us
to obtain a better and more accurate perception of the form and
position of objects. Persons with considerable width between
the eyes obtain a better perspective than those who have the
eyes nearer together. Good judges of form, such as artists,
anatomists, etc., must have normal vision, with the eyes con-
siderably separated. However, the advantage of binocular vis-
ion to see objects in relief decreases directly as the distance
increases.

3. ABERRATION.
The eye is by no means a perfect optical instrument. As is
naturally to be expected, the main defects are chromatic and
spherical aberration. In the normal eye these aberrations are
usually not noticed, but they exist, as has been proven experi-
mentally. Chromatic aberration may be observed by looking
at a small opening in a shutter through which sunlight is enter-
ing; the opening will seem to be encircled by a halo of pris-
matic colors. It may also be observed about dark objects placed
between the eye and any white surface with the eyes focussed
upon the latter. That is, chromatic as well as spherical aberra-
tion is noticeable when objects are more or less out of focus.
Spherical aberration is manifest by blurring and distortion of
images. In the normal eye it is largely corrected by the iris
which cuts off most of the distorted marginal rays.
In connection with aberration we must also mention certain
slight irregularities in curvature of the cornea and lens present
in nearly all eyes, and which produce slight distortion of
images in various planes, causing the phenomena of astigma-
tism, which shall be referred to more in detail later.
4. ENTOPTIC PHENOMENA.
These phenomena are due to imperfections in the refracting
media of the eye; they are not uniformly transparent. The
rays of light, in passing through these media, undergo local
absorption and refraction, causing shadows to be thrown upon
the retina which are thus rendered noticeable.
The most common phenomenon of this kind is the appear-

168
THE EYE.
ance of floating bodies in the vitreous humor, the so-called
muscae volitantes (flying flies) or floating specks. They
assume the form of single beads, rows or groups of beads,
granules or small irregular patches. They change their posi-
tion continually. When an attempt is made to fix vision upon
them they float away. Sometimes they become a source of
considerable annoyance to the microscopist. Quite generally
an upright position of body and head, with the eyes directed
horizontally or upward, causes these specks to disappear almost
entirely, as such a position causes the opaque bodies suspended
in the vitreous humor to gravitate away from the lens and
out of the range of vision.
There are various other entoptic phenomena due to abnor-
malities in the lymphatic and blood-supplying system of the
eye which we can not explain here. The phenomena above
referred to are more or less present in all so-called normal eyes.
Some persons never become conscious of them, while in most
others they never become troublesome. In the case of really
annoying or distinctly noticeable entoptic phenomena it is
advisable to consult a competent oculist.
5. SPECIAL OPTICAL PHENOMENA.
There are a host of optical phenomena with which the stu-
dent should be familiar, and which are usually explained in
the larger works on physiology and physics. They are not
specially concerned in microscopical work, and for that reason
are not more fully explained here. The following are the more
important:
I. Persistence of Impressions on the Retina.-That is the
impression of the image on the retina remains longer than the
stimulus causing it. For instance, the lightning flash which
exists for perhaps twenty millionths of a second is perceived
for nearly half a second. The construction of various toys
depends upon this, as the Zoetrope or wheel of life, the kaleido-
scope, etc.
2. Irradiation.-This causes objects to appear larger or
smaller than they really are. For instance, white objects upon
a dark ground appear larger than they really are. This differs
greatly in different persons; it also differs in the same person.

OPTICAL PHENOMENA.
169
in different periods and with varying physical conditions. It
is particularly noticeable on looking at stars, which appear
much larger than they really are; and also on looking at the
new moon, whose crescent seems to extend beyond the shaded
portions of the disc. The existence of irradiation is often
made use of in dressing to produce an apparent diminution or
increase in size; for instance, dark or black gloves make the
hands appear smaller than light gloves.
the
3. Formation of After Images.-After the primary impres-
sion of the image has passed away there appear secondary
images of the complementary color of the object. For instance,
if one gazes at a window for a few seconds and then directs
eye upon the white wall the window panes will appear dark
and the frame-work light. Or, if a colored object is looked at
the complementary color will appear in the after-image. After-
images are very variable and numerous. They manifest them-
selves in such a multitude of forms that it would be impossible
to enumerate them.
II. DEFECTIVE SIGHT.
The eye is a very delicately constructed organ and requires
special care. We shall here refer to some of the more com-
mon defects met with, not that the reader may resort to home
treatment, but that he may recognize the defects more readily
and realize the importance of prompt attention by competent
oculists and opticians.
I. DIPLOPIA OR DOUBLE VISION.
Diplopia is an affection of the eye which causes objects to
appear double; that is, there are two image-impressions instead
of one.
As has already been indicated, each eye receives an
impression of the image of the object, but the two images are
exactly superimposed in normal vision, thus producing single
vision.
Diplopia may occur in one eye or it may be binocular dip-
lopia; the latter is the more common form. Binocular diplopia
may be due to excessive contraction or to paralysis of the
muscles of the eye, and, as will be explained later, is a phenome-

170
THE EYE.
non accompanying strabismus. It may also be due to improp-
erly constructed and badly centered eye-glasses.
Monocular diplopia is of rarer occurrence. It may accom-
pany some forms of astigmatism and is primarily due to defects.
in the refractive media, causing a bifurcation of the image-
forming rays.
Diplopia, whether monocular or binocular, is, therefore,
usually a phenomenon depending upon some marked defect of
the eye, or eyes, and should receive prompt medical attention.
2. ACHROMATOPSY OR COLOR BLINDNESS.
Color blindness is a condition of the optic apparatus which
renders it impossible to distinguish certain colors. The emi-
nent physiologist Dalton possessed this defect very perfectly
developed and gave it a careful study, on which account color
blindness is often designated as Daltonism.
The commonest form is blindness for red, which color
appears like black and the brighter shades bluish-green or pink.
Yellow appears like green, but brighter. Blindness for green
also occurs, which color appears black, while the lighter shades
appear as red, and yellow as red. Violet blindness is very rare,
and is not well understood. It may be developed artificially
by taking medicinal doses of santonin.
Color blindness is usually congenital and is far more com-
mon among males than females. It is merely an abnormality
and not a disease which is amenable to treatment. There is
no difficulty in recognizing form and outline and in discrimi-
nating between light and shade. It does not cause any incon-
venience or annoyance. Color blindness becomes of great sig-
nificance in the observations of colored signal lights, as used
in the marine and railway service. Railway officials must be
carefully examined for color blindness.
Even persons with perfectly normal vision have difficulty in
recognizing many tints and shades. The difficulty is increased
by the fact that we have no standard for the estimation of
colors and by the confusion caused by the hosts of tints and
shades improperly and unsystematically named. The best
color test for ordinary purposes consists in giving the person
a skein of wool of some color, as red, green, orange, violet,


COLOR BLINDNESS.
171
etc., and requiring him to match it with other skeins which
appear to have the same color in it. In order to guard against
simulation the experiment should be repeated at intervals of
a day or a week.

3. WEAK EYES (AMBLYOPIA).
Weakness of eyes or dimness of vision, technically known
as amblyopia, is of very common occurrence. Persons thus
affected see objects only dimly and are obliged to bring them
near in order to see them distinctly or at all. Usually they are
looked upon as nearsighted (myopic), which is not the case at
all, since the image is properly focussed upon the retina. It
differs only from the image upon the retina of the normal eye
in that it is less distinct.
Amblyopia may be present in one eye or in both eyes, and
may be due to a variety of causes. It may be congenital or may
be acquired. If it is present in one eye only, as is usually the
case in the congenital form, there is more or less squinting
(strabismus) of the affected eye, because the person neglects
to adjust it properly, and as a result loses the muscular control
of it through disuse.
Opacities in the humors of the eye, or the lens and structural
defects of the retina, are the usual causes of acquired amblyo-
pia. Dimness of vision may increase to total blindness (amau-
rosis). It may also be caused by diseases of central origin,
such as diabetes, hysteria, inflammations of the brain and spinal
cord. If it is due to an opacity of the lens, as in cataract, it can
be remedied by removal of the lens. Glasses with convex
lenses are used to magnify the objects, thus making up in size
what is lost in clearness.
If marked amblyopia or total blindness comes on suddenly
competent physicians and oculists should at once be consulted.
4. SQUINTING (STRABISMUS).
Squinting or looking cross-eyed, technically known as stra-
bismus, is both congenital and acquired. It is the result of the
inability to adjust both eyes upon the same object. If one
eye is directed upon the object the other is drawn either too
far inward or outward, and as a result there is squinting inward

172
THE EYE.
(convergent strabismus), or squinting outward (divergent
strabismus). This is shown in Figs. 91 and 92. In Fig. 91
the left eye (A) is directed toward the object; the right eye,
instead of being directed to the same point, is turned in equal
to the distance a b, which measures the amount of squinting,
B
A
a--b
FIG. 91.
CONVERGENT STRABISMUS.
B
a---b
A
FIG. 92.
DIVERGENT STRABISMUS.

STRABISMUS.
173
and is to be considered in correcting the defect. Fig. 92 illus-
trates divergent strabismus. Since the squinting eye does not
perceive the same object seen by the normal eye there must be
double vision, which is really the case. This double vision is,
however, not troublesome, as the person soon learns to ignore
the image of the squinting eye; furthermore, this eye does not
focus as a rule, so that no distinct image is formed upon its
retina. One eye may squint or both eyes may squint alter-
nately. One eye may be employed in looking at near objects
and the other in looking at remote objects. One does not squint
with both eyes at the same time.
Strabismus is the result of some disturbance of the muscular
equilibrium of the eye-ball. If the internal rectus muscles of
one or both eyes should become shortened there would be
single or double convergent strabismus. If the external rectus
muscles are shortened or contracted there would be divergent
strabismus. A similar condition would result if the muscles
should become paralyzed as the result of lead poisoning or some
other cause; but in this case the resulting phenomenon is quite
different. In strabismus the movements of both eyes always
take place, while in the case of muscular paralysis the affected
eye is partially or wholly motionless, presenting a very charac-
teristic contrast.
The important fact to be kept in mind is that strabismus is
always associated with some disturbance of vision; in fact the
disturbance of vision is often the cause of the squinting. Con-
vergent strabismus is usually associated with hypermetropia,
while divergent strabismus is usually associated with myopia.
This indicates the necessity of consulting an oculist in case of
squinting, especially the incipient squinting of children.
I.
The following are the inciting causes of strabismus:
Defects in the refractive power of one eye; especially loss of
transparency of the cornea and lens. 2. Amblyopia or total
blindness in one eye. 3. Hypermetropia and myopia. 4. Dis-
eases of central origin.
As regards treatment it may be stated that latent or incipient
strabismus may be remedied by correcting the defects of vision
by means of glasses and medicines. Long standing and fully
developed strabismus can rarely be corrected without operative
measures; this is particularly true of divergent strabismus.

174
THE EYE.
The operative measures consist in tenotomy; that is, in cut-
ting the shortened muscles. After the operative measure the
defects of vision must be corrected, as far as possible, by means
of the proper glasses, otherwise strabismus will develop again.

5. NEAR-SIGHT (MYOPIA).
Near-sightedness or myopia is by far the most common
defect of the eyes. In this condition rays of light which should
focus upon the retina focus in front of it, as shown in Fig. 88.
The essential difference between the myopic and emmetropic
or perfect eye is that in the former all objects must be brought
very near the eye in order to bring them within the range of
most distinct vision. It must not be confounded with amblyo-
pia, although the two are quite frequently associated. The
myopic eye may have a perfectly normal vision when brought
sufficiently near, but all distant objects are indistinctly seen, a
defect which the patient seeks to remedy by partially closing
the lids, wrinkling the forehead and blinking. Near objects
are seen distinctly without any effort at accommodation, since
myopia is a chronic focussing for near objects.
Myopia is a defect common among the young and is on the
increase in spite of the efforts of efficient oculists. It is a
defect which may be inherited. It would, however, no doubt
be more correct to state that the tendency to myopia is inher-
ited and not myopia itself. The following are the more impor-
tant causes of myopia: 1. First and foremost, over-exertion
of the young, developing eye; long-continued looking, as in
reading, studying, sewing or work of any kind where the child
is obliged to keep the eyes focussed for an hour or more at a
time. This is very evident from the fact that students and
studious children are largely myopic. 2. Amblyopia or weak-
ness of vision which makes it necessary to bring objects near
the eye. After this is persisted in for a long time accommoda-
tion for near objects becomes permanently established.
Insufficiency of the internal rectus muscles. 4. Over-exertion
of the eyes, as by tailors, students, typesetters, lithographers.
5. Poor general health and insufficient nutrition.
It
3.
Myopia, after it is once fully established, can not be cured.
may be corrected more or less completely by wearing con-

MYOPIA.
175
cave glasses. It is in the adjustment of the correct glasses
that great care and skill is required. Once and for all it may
be stated here that no one should allow himself to be duped
into purchasing glasses from mere dealers in glasses and from
traveling charlatans who advertise in the country papers as
specialists.
We should not be doing our duty should we fail to caution
against the continued reading of fine and poor print and the
long-continued work by artificial light, as these are undoubtedly
frequently the inciting causes of myopia.
Those affected with myopia become presbyopic later than
those with normal vision; in fact, they may not become pres-
byopic at all. Myopes and amblyopes are said to be better
natured than those with normal vision because they cannot
notice defects and faults so readily.

6. FARSIGHT (HYPERMETROPIA).
of
Farsightedness or hypermetropia is the reverse
myopia. The parallel rays are brought to a focus behind
the retina, or, more accurately speaking, are not brought to
a focus at all. (See Fig. 89, C, and compare with B and A.)
Objects must be moved away from the eye in order that the
rays may focus, if only partially, upon the retina. It is less
common than myopia, which is fortunate since it is by far
the worse of the two evils. If the hypermetropia is at all
well developed, the objects are held at a distance, which in
itself reduces the clearness, and in addition the powers of
accommodation are called into play very strongly, even for
comparatively remote objects.
All infants are hypermetropic because the antero-posterior
diameter of the eye is too short for the refractive media; as
the child grows this diameter increases until the eye becomes
emmetropic (normal), or it may even elongate excessively,
producing myopia.
Hypermetropia, like myopia, cannot be cured. It can be
readily corrected by means of the proper convex glasses. As
to cause, it is often the result of a diminution in the refractive
power of the media, as cornea and lens. Sometimes it is caused
by luxations of the lens; that is, the lens sinks down so that

176
THE EYE.
rays
of light entering the pupil no longer pass through it. In
such cases accommodation is, of course, lost. Tumors and
other pathological growths may cause hypermetropia. It is
also caused by a shortening of the antero-posterior diameter of
the eye, due to changes in the eye-ball itself.
A
A, EMMETROPIA.
B
B, MYOPIA.
C
C, PRESBYOPIA.
FIG. 93.

HYPERMETROPIA.
177
7. SENILE FARSIGHT.-PRESBYOPIA.
This is farsightedness of the aged, and resembles hyper-
metropia in that rays of light tend to focus behind the retina.
The following are the essential differences: Presbyopia has
to do with the accommodation; that is, the eye is no longer
capable of focussing for near objects; while in hypermetropia
the accommodating power is not affected. Presbyopia occurs
in the aged, while hypermetropia occurs in the young. Both
are corrected by means of convex lenses.
Presbyopia is the result of a flattening of the lens and a loss
of accommodation. This loss of accommodation is particularly
noticeable in attempting to look at near objects. Some persons
become presbyopic quite early in life, while others have normal
vision until a ripe old age.
8. ASTIGMATISM.
Astigmatism is due to irregularities in the refractive media
so that parallel rays are caused to focus at different points on
the retina. That is, rays passing through the media in differ-
ent planes are not all brought to a sharp focus on the retina,
thus producing blurring and distortion of the image in those
planes. The irregularities in refraction may be due to irregu-
larities in the curvature of the various media, as cornea and
lens. If these curvatures in the various planes are uniform,
the resulting astigmatism may be corrected by means of cylin-
drical lenses. But if the curvature in the same plane is irreg-
ular, it is practically impossible to correct it. This latter
form is usually known as "irregular astigmatism."
A small amount of astigmatism is perhaps present in all
eyes. For instance, upon looking at Fig. 94 it will be found
that the parallel radiating lines will appear blurred or less
distinct in certain planes; this is due to astigmatism. If the
defect is not marked, it need cause no anxiety; but if it is
very distinct, so as to cause great distortion in certain planes,
it is necessary to have the defect corrected by means of
cylindrical lenses.
Regular astigmatism is usually congenital and equally devel-
oped in both eyes.

178
THE EYE.
111
14
13
15
12
11
10
9
2
FIG. 94.
TEST FOR ASTIGMATISM.
5
9.
INFLAMMATORY CONDITIONS OF THE EYES.
Inflammatory conditions of the eyes are very numerous and
due to a great variety of causes. We shall call attention to
them only in a general way.
The most frequent seats of inflammatory conditions are the
cornea and the sclera or sclerotic coat. Of the protective
tissues the conjunctiva and lids are frequent seats of inflam-
mation. The inflammation may be due to an injury, to some
specific infection, or it may be the result of some systemic
disease, as diphtheria and diabetes. It may also be the result
of some defect in vision, and finally to uncleanliness. No
matter what the cause of the inflammation or irritation may be,
it should receive prompt attention, as it may otherwise result
in permanent injury to vision.
I.
III. HINTS ON THE CARE OF THE EYES.
Illumination.-Do not use the eyes when facing strong
light. Place yourself so as to have the light at your back or

ASTIGMATISM.
179
at your side. Objects to be inspected should be well but not
excessively illumined. Never read or similarly use the eyes
in direct sunlight or in strongly reflected sunlight. Never
gaze at a very bright light, as the sun in midday
or a strong electric light. Even a short exposure may
do permanent harm. Never use the eyes more than is neces-
sary in artificial light and never in a flickering or unsteady
light. The arc light is especially objectionable because of its
unsteadiness. The ordinary gas light is also unsteady. Do
not use the eyes for a long time in poorly lighted rooms or
where the light is so deficient as to necessitate straining of the
eyes. If one is obliged to be in strongly reflected sunlight, as
in a sand desert, sand beach, snow-covered ground or on the
water, it is well to protect the eyes by means of smoked
glasses. In Russia and other snow-bound countries blindness.
is far more prevalent than in other countries, due to the glare
of light reflected from the snow. Sudden and extreme
changes in the intensity of light are very injurious.

It
2. Use of Shades.-Shades for the eyes should be used
only when it is necessary; that is, when the eyes are weak or
when there is inflammation or other condition requiring
such a protection as prescribed by a competent physician or
oculist. The continued use of eye-shades when they are not
necessary weakens the eyes instead of benefiting them.
may be compared to an athlete who would seek to improve his
muscles by going to the gymnasium with his arms in slings and
sitting down instead of exercising. The normal eye should be
properly exercised without protection, provided the illumina-
tion is approximately suitable. All bright artificial light, as
electric light, Wellsbach light, strong gas light, and others of
equal or greater luminosity should be partially or totally cov-
ered by translucent shades. Opaque shades for light are
generally objectionable because the contrast between the
shaded area and the non-shaded is too great. Most artificial
lights should be sufficiently powerful to be placed at a distance
of five to ten feet from the head. By so doing one is less
affected by flickering and variations in intensity due to move-
ments of the head and body.
3. Wearing of Glasses.-Eye-glasses and spectacles are
essentially of two kinds, those worn to protect the eye against

180
THE EYE.
excessive or colored light and those worn to correct some
defect in vision. The former consist usually of glass with
parallel surfaces, colored various shades and tints of blue and
gray. As stated, the surfaces are always parallel, but as a
rule not plane but curved, with the convexity outward, as
this form also protects somewhat against lateral illumination.
As regards the glasses worn to correct some optical defect of
the eyes, we have already given the more important sugges-
tions under defective sight. According to the defect to be
corrected such glasses are convex, concave or cylindrical.
Prisms are also used in special cases. All the lenses have a
long focal distance. The unit is the convex lens with a prin-
cipal focal distance of one meter; it is known as a meter lens,

000000
FIG. 95.
LENSES, SPHERICAL AND CYLINDRICAL.
and its refractive power is one diopter (D). A lens of double
this refractive power (2 D) has a principal focus of 50 cm.
The oculist usually has a box of lenses ranging from .25 D
to 20 D which he uses in testing eyes for glasses. Concave
glasses are spoken of as negative and rated as above, but pre-
ceded by the sign.
—
Cylindrical lenses are segments of cylinders of various
diameters and may be convex or concave. (See Fig. 91.)
They are used in correcting astigmatism. Prisms are some-
times used alone and sometimes combined with lenses.
Lenses for eye-glasses and spectacles should be of clear
glass, free from air bubbles and other defects: They should

EYE-GLASSES.
181
be well mounted in a good frame, which should be as light
as possible. If the lenses are heavy, the bridge piece should
be broad and specially protected so that it will not work itself
down into the tissues and bones of the bridge of the nose.
The center of the lenses should be exactly in front of the
pupils. Both lenses should be in the same plane. As soon
as glasses cause distortion of vision or disturbance of accom-
modation or annoyance of any kind they should at once be
taken to an oculist (not optician or dealer in eye-glasses) for
inspection and correction. Never wear glasses when they are
not needed. The monocle and lorgnette are largely an expres-
sion of affectation. The monocle is especially objectionable
because it invariably causes temporary or permanent unilateral
facial distortion and disturbance of vision. Never be without
glasses when they should be worn. Always wear the glasses
intended for the special work that is being done, such as long
and short distance glasses.
In conclusion we will mention the stenopæic eye-shade. This
consists of a blackened plate of metal in which there is a nick
or slit. It is used for correcting vision in localized opacities
of the cornea where lenses would be of no avail. The disc is
held before the eye so that the slit is in front of the trans-
parent area, the opaque areas being shut off by the disc. This
gives, of course, a very small field of vision; furthermore, the
eye must not be moved; but it enables the patient to read
some or to do other work which would be impossible without
this aid.
4. Exercise of the Eyes.-The eyes should be used in vari-
ous kinds of work so as to develop them to their highest capa-
bilities. Non-use of the eyes is injurious, sooner or later
causing weakness and dimness of vision, due principally to a
lack of tone in the nerve apparatus and the powers of accom-
modation. Proper exercise can do wonders in developing the
powers of quick and detailed perception of objects, the correct
perception of size, color, form, and position. Mere opening of
the eyes is not sufficient. It is, in fact, the eye which indicates
the nature and character of the individual. Perception of the
stupid and ignorant is always deficient. They either fail to
see certain details entirely or only after special instruction and
the use of artificial aids to vision.


182
THE EYE.
We cannot enter into details as to how the eyes should be
exercised in each particular case. The eyes should not be
used until they become fatigued, as is evidenced by blurring,
flickering, trembling of lids or painful sensations in the eye
itself or in the head. Frontal headaches are frequently due to
overexertion of the eyes or to some defect of vision, as already
indicated.
In using the eyes the position of the body should be consid-
ered. Trunk and head should be erect so as to allow free
circulation of the blood and free, unrestricted respiration.
Tight collars or anything which tends to interfere with the free
circulation of the blood to the head is very injurious. Both
eyes should be directed upon the object at about the same angle.
For instance, the position of the average school-boy in writing
is highly injurious and is a common cause of irregularities in
binocular vision.
The eyes should not be used for a long time while the body
is at rest, as in reading, sewing, and microscopical work.
Reading in particular is fatiguing. One should never read for
more than an hour while sitting perfectly still, especially in hot
weather or in a warm room. Fine print is very objection-
able because it strains accommodation. According to recent
experiments, print from II-point Roman type, with leading,
is most legible. The print of this book is of this kind.
Working with dirty lenses, no matter whether they belong
to eye-glasses or microscopes, is very injurious; likewise work-
ing with poor lenses not properly corrected for aberration.
Those wearing glasses should be sure that they are properly
fitted and adjusted. It should be borne in mind that the
lenses for eye-glasses must be changed from time to time, as
the defects of vision progress or become otherwise modified.
Dim objects, indistinct images, or images not in focus cause
straining of the eyes in attempting to focus upon them.
5. Care of the Eyes.-The proper care of the eyes in a
large measure also applies to the proper care of the entire
body. The eyes should be frequently bathed in warm water,
using a very mild soap for the lids (and face) and wiping them
with a soft towel. Uncleanliness is very frequently the cause
of inflammatory conditions of lids and conjunctiva, and is cer-
tainly liable to bring about infection with various bacteria,

CARE OF EYES.
183
causing serious damage to cornea and conjunctiva. If there
is a slight inflammatory condition, producing dryness of the
eyes, burning sensations, and causing the lids to glue together
upon rising in the morning, it is well to wash the eyes care-
fully before retiring and rubbing a little plain vaseline on
the lids. The trouble may be due to some incipient defect in
accommodation or refraction, or it may even indicate the
presence of some disease, hence the advisability of consulting
a physician or an oculist.
If some foreign substance, as a particle of sand, dust, cinder,
etc., gets into the eye it should be removed at once, otherwise
it may work its way into the cornea or sclera, necessitating its
removal through operative measures. To remove the foreign
substance let the person sit in a chair with the head thrown
back; a second person raises and turns back the lids by means
of the thumb and fore-finger of the left hand; let the patient
rotate the eye as far as possible so as to enable the examiner
to see the substance and wipe it away by means of a soft,
clean cloth dipped in warm water. If it does not come away
by gently brushing the rolled or folded cloth over it, bandage
the eye and consult an oculist. Small particles that are not
lodged in the tissues will soon work their way to the inner
corner of the eye, from whence they are readily wiped away.
Persons with large eyes, or widely separated lids, not well pro-
tected by long lashes, are very much troubled with foreign
particles entering the eyes. Occupation is very largely con-
cerned in the liability to injury to the eyes. The plasterer in
slacking lime is exposed to the particles of lime; the machinist
to particles of steel filings; the chemist to acids and other
injurious substances, etc. Injuries entailing destruction of
tissue result in opacities, which may produce partial or total
blindness.
Those who use alcoholic drinks to excess quite generally
have poor eyesight. The excessive use of tobacco, in partic-
ular, is very injurious, producing the so-called "tobacco
amblyopia." Irregular habits, especially late hours, are very
often the cause of inflammations of the eye, and resulting
dimness of vision. Regular, temperate habits are necessary to
maintain good eyesight, as well as good general health.


GENERAL INDEX.

A
Abbé Condenser, 67.
Aberration, 39, 89.
chromatic, 91.
of eye, 167.
spherical, 89.
Accessory material, 125.
Accommodation, 164.
Achromatopsy, 170..
Acid, acetic, 129.
carbolic, 130.
chromic, 131.
hydrochloric, 133.
nitric, 133.
osmic, 133.
picric, 134.
Adjustment, coarse, 62.
fine, 61.
Adulterations, 140.
Bacon, Roger, 6.
Bacteria, 154.
B
mounting of, 154.
Base, 56.
Bastin's microtome, 118.
Bichloride of mercury, 138.
Biot, 5.
Blood corpuscles, 4.
Body, 64.
Bonani, 7.
Bone, sections of, 158.
Brewster, 44.
Brücke, 6.
Burning spheres, 4.
C
Calcium, 9.
1
Air bubbles, 147.
Alcanna, 129.
Alcohol, 129.
Amblyopia, 170.
tobacco, 183.
Ammonia, 130.
Amici, 130.
Aniline blue, 130.
Aperture, 93.
angular, 94, 96.
numerical, 94.
Aplanatic lenses, 47.
Archimedes, 3.
Aristophanes, 4.
Arm, 61.
Astigmatism, 177.
Astrology, 5.
Astronomy, 5.
Camel's hair brushes, 128.
Camera lucida, 80.
Abbé, 82.
Wollaston, 80.
Camera obscura, 162.
Canada balsam, 130.
Carbolic acid, 130.
Carey, 44.
Cedar oil, 131.
Celloidin, 131, 150.
solution of, 151.
Chairs, 115.
Chevalier, 6.
Cherubin, 8.
Chloral hydrate, 131.
Chloride of sodium, 131.
Chlor-iodide of zinc, 131..
Chromatic aberration, 91.
Chromic acid, 131.
Automatic microtome, 119.
185

186
GENERAL INDEX.
Clearing, 143, 149.
Clearing agents, 135.
E
Cloves, oil of, 131.
Coddington, 44.
Coddington lens, 44.
Color blindness, 170.
Coarse adjustment, 62.
Compound microscope, 50.
images by, 51.
mechanism, 52.
optical parts, 66.
Concave mirror, 21.
Condensers, 67.
Conjugate foci, 19.
Correction collar, 8.
Cover-glasses, 121, 122.
Cover-glass gauge, 124.
Crown glass, English, 9.
German, 9.
Current slides, 121.
Emmetropia, 176.
Engravers' lens, 46.
Entoptic phenomena, 167.
Eosin, 132.
Euler, 5.
Evaporating dishes, 128.
Eye, 161.
aberration of, 167.
care of, 178, 182.
exercise of, 181.
refraction, 162.
Eye-glasses, 179.
Eyes, inflammation of, 178.
injuries of, 183.
optical properties, 161.
use of, 109.
Eye-pieces, 68.
Eye-point, 11O.
Eye-shades, 179.
D
F
Dammar, 132.
Definition, 99.
Dehydrating, 142.
agents, 135.
Deodati, 6.
Desk, 115.
Devino, 7.
Diagonal sections, 146.
Diamonds, artificial, 9, 10.
Diaphragms, 59.
dome, 59.
iris, 59, 61.
position of, 60.
wheel, 58.
Diatoms, 87.
Diffused light, 21.
Diplopia, 169.
Dolland, 5.
Double vision, 169.
Drawing prism, 81.
Dissecting microscope, 47, 49.
Dissecting needles, 127.
Farsight, 175.
senile, 177.
Fine adjustment, 61.
Field-lens, 68.
Fixing agents, 134.
Foci, 19, 28, 31.
Focal distance, 97.
Focus, apparent, 20.
real, 20.
Focussing, 107.
Fontana, 6.
Foot, 56.
Formalin, 132.
Frauenhofer lines, 93.
Fuchsin, I32.
G
Galileo, 6.
Gentian violet, 132.
Glass, 9.
Draw-tube, 65.
Drebbel, 6.
Drugs, powdered, 140.
Bohemian, 9.
bottle, 9.
coloration of, II.

GENERAL INDEX.
187
Glass, coloring of, 10.
crown, 9.
history of, 9.
manufacture of, 9, 10.
Glycerine, 132.
Glycerine jelly, 132.
Gold size, 133.
Gauge, 123.
H
Hand sections, 143.
Hardening agents, 134.
Hartnack, 8.
Hasting's lenses, 41.
History, of microscope, 3.
Hooke, R., 4, 7.
Hydrochloric acid, 133.
Hypermetropia, 175.
I
Illumination, 105.
Images, 15.
Imbedding, 150, 152.
Immersion lenses, 95.
Infiltrating, 150, 152.
Lenses, cylindrical, 180.
foci of, 28.
forms of, 26.
image by, 30.
immersion, 4, 95.
optic center, 30.
rock crystal, 3.
Life slides, 121.
Light, diffuse, 21.
intensity of, 35, 36.
reflection of, 12.
12/
refraction of, 22.
Lieberkühn, 7.
Linen tester, 44.
Leeuwenhock, 4.

M
Magnification, IOI.
of lenses, 41.
Magnifiers, 42.
Stanhope, 44.
tripod, 48.
Magnifying power, 100.
Marjoram, oil of, 133.
Material, 115.
Media, mounting, 136.
Mercury, 133.
celloidin, 148.
direct, 148.
paraffine, 151.
special, 154.
Iodine, 133.
Iridescence, 10.
J
Methods, 138.
James I, 6.
Jansens, 6.
Japanese paper, 128.
K
Kepler, 6.
L
Labels, 128.
Lavender, oil of, 133.
Layard, 3.
Lead, 9.
Lemon, oil of, 133.
Lenses, achromatic, 92.
aplanatic, 46.
best form of, 39.
Coddington, 44.
Methyl violet, 133.
Micrometers, 77.
eye-piece, 77.
stage, 77.
Microphotography, 84.
Microscope, binocular, 75.
chemical, 74.
care of, IIO.
chairs, 115.
compound, 50.
demonstration, 115.
desk, 115.
dissecting, 47.

188
GENERAL INDEX.
Microscope, inverted, 74.
Oculars, 68.
continental, 69.
manipulation of, 104.
petrographical, 73.
portable, 75.
position of, 104.
purchase of, 112.
simple, 37.
tables, 115.
Micro-technique, I, 115.
Miroscopy, 1, 3.
Microtomes, 117.
automatic, 119.
Bastin, 118.
simple, 117.
Mirrors, 12, 66.
concave, 14, 17.
plane, 13, 17.
spherical, 14.
Mounting, 143.
Mounting media, 136.
Mounts, entire objects, 138.
permanent, 148.
temporary, 138.
Myopia, 174.
Nearsight, 174.
N
Needle preparations, 142.
Newton, 5, 7.
Nitric acid, 133.
Nimrod, 3.
Nineveh, 3.
Nobert plate, 80.
Nose-piece, 64.
double, 64.
triple, 65.
Numerical aperture, 94.
Objectives, 71.
O
construction of, 72.
immersion, 95.
high power, 73.
low power, 73.
English, 69.
Huyghenian, 68.
negative, 68.
orthoscopic, 71.
positive, 68.
solid, 70.
Oil, of cedar, 131.
of cloves, 131.
of lavender, 133.
of lemon, 133.
of marjoram, 133.
Optic center, 30.
Osmic acid, 133.
Paraffine, 133.
Penetration, 98.
Phenol, 134.
P.
Phenomena, entoptic, 16.
optical, 168.
Phloroglucin, 136.
Photography, 84.
Picric acid, 134.
Pillar, 56.
Pincers, 127.
Plutarch, 3.
Polybius, 3.
Porta, 161.
Potash, 134.
Potassium, 9.
Powders, 140.
Powers, of lenses, 41.
Presbyopia, 177.
Preserving fluids, 137.
Prisms, 25.
Q
Quartz, 9.
R
magnification of, 101.
medium power, 73.
systems, 72.
Radial sections, 146.
Razor, 144.
handling of, 144.
sharpening of, 144.
GENERAL INDEX.
189
dehydrating, 135.
Reading glass, 42.
Reagents, 129.
clearing, 135.
fixing, 134.
hardening, 134.
softening, 135.
Reflectors, 80.
micro-chemical, 136.
Refraction, 22.
index of, 22, 23.
of eye, 162.
Resolving power, 99.
Riddle, 8.
Ross, 8.
Rotifers, 4.
Safranin, 134.
Sand, 10.
Satan's tears, 10.
Sectioning, 151.
S
by hand, 143.
of bone, 158.
Section knife, 117, 144.
Section lifters, 126.
Sections, diagonal, 146..
kinds of, 145.
longitudinal, 146.
mounting of, 147.
tangential, 146.
transverse, 146.
Seneca, 4.
Senile farsight, 177.
Shades, 179.
Shellac, 134.
Sight, defective, 169.
Siphon slides, 121.
Slide boxes, 127.
Slide centerer, 125.
Slips, concave, 120.
glass, 120.
Slow adjustment, 61.
Sodium, 9.
Sodium chloride, 131.
Softening agents, 135.
Spectacles, 179.
Spencer, 8.
Spherical aberration, 89.
Squinting, 171.
Stage, 56.
Stage aquaria, 121.
Staining, 149, 153.
Stains, 136.
Stanhope magnifiers, 44.
'Steinheil's lenses, 47.
Strabismus, 171.
convergent, 172.
divergent, 172.
Substage, 57.
Syracuse glasses, 125.

Tables, 115.
T
Tangential sections, 146.
Tannin, 135.
Telescopes, 5, 6.
Test objects, 87.
Textile fabrics, 142.
Transverse sections, 145.
Trichinæ, 156.
Trichinosis, 156.
Tube, 64.
Tube-length, 54.
Turn table, 124.
Turpentine, 134.
V
Vision, binocular, 165.
defective, 169.
double, 169.
stereoscopic, 166.
weak, 170.
W
Watch glasses, 125.
Watchmaker's lens, 45.
Weak eyes, 170.
Wollaston, 5.
Working distance, 97.
X
Xylol, 134.
Z
Zinc, chlor-iodide of, 131.
NOV 1 5 1917
UNIVERSITY OF MICHIGAN
3 9015 07505 6575