The upper

UNITS.

THB METER FO
LENGTH. . .

THE GRAM
\VEIGHT. . .

THE LITER FO
CAPACITY.

Divisions of
o.ooi ; Micro, on

Multiples at
times ; myrta, ic

eter : Micron (/

(g 166).

long distances.

cries, etc.
than the correct

centi, o.oi ; Milli,
too times ; kilo, 1000

ES

>, ooo microns (ju)

Meter (Unit*

39.3704 it , m

Centimeter (cm. )=io millimeters ; 10,000 microns (//) o.oi meter ; 0.3937 (I) inch.
Millimeter (mm. )=i, ooo microns (//) ; o.i cm. ; o.ooi meter ; 0.03937 (-fa) inch.
Micron (u) (Unit of measure in micrometry (g i66)=o.ooi millimeter; one mil-

lionth of a meter ; 0.00003937 ( ^^) inch.
Yard=3 feet ; 36 inches ; 0.91439 meter ; 91.4399 centimeters.
Foot=i2 inches ; 30.4799 centimeters ; 304.799 millimeters.
Inch— y1^ foot ; 3^ yard ; 25.3999 millimeters (2.54 centimeters).
Liter (Unit of capacity )= 1,000 cubic centimeters (milliliters) ; (i quart—.)
Cubic centimeter=o.ooi liter (milliliter) ; (TL cub. inch.)
Fluid ounce (8 fluidrachms)=29.574 cubic centimeters (30 cc. ).
Gram (Unit of weight )= i cc. of water; 15.432 grains.
Kilogram=n, ooo grams ; 2.2046 (2^) Ibs. avoirdupois.
Ounce avoirdupois=437^ grains ; 28.349 grams. 1

Ounce Troy or apothecaries— 480 grains ; 31.103 grams J ^° &ra:ns> approx.

TEMPERATURE

To change Centigrade to Farenheit : (C.X D+32 = F.
the equivalent of 10° Centigrade, C.= io°X f-f 32 = 50° F.

To change Farenheit to Centigrade : (F.— 32°) X f — C. For example to re-
duce 50° Farenheit to Centigrade, F.= 5o°, and (50°— 32°)X f = ioC. ; or — 40

'

For example, to find

Farenheit

.
to Centigrade, F.= — 40° (—40°— 32°)= — 72°,' whence —

sti
foi
an

Tt

Ei
Th
Tt
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S. AGNES SCHOOL
LIBRARY.

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zroscopes

Chicago,
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:roscopes

No...

THE LIBRARY

OF

THE UNIVERSITY
OF CALIFORNIA

PRESENTED BY

PROF. CHARLES A. KOFOID AND
MRS. PRUDENCE W. KOFOID

THE MICROSCOPE

AN

INTRODUCTION TO MICROSCOPIC
METHODS AND TO HISTOLOGY

BY SIMON HENRY GAGE

PROFESSOR OF MICROSCOPY, HISTOL-
OGY AND EMBRYOLOGY IN CORNELL
UNIVERSITY, AND THE NEW YORK
STATE VETERINARY COLLEGE.

NINTH

EDITION

REVISED, ENLARGED AND ILLUSTRATED BY OVER
TWO HUNDRED FIGURES

COMSTOCK PUBLISHING COMPANY

ITHACA, NEW YORK
1904

Copyright, 1903.

BY SIMON HENRY GAGE.

All Rights Reserved.

Press of

THE ITHACA JOURNAL
Ithaca, N. Y.

PREFACE TO THE EIGHTH EDITION

AS the preface to the sixth edition of this work expresses accurately what
should be said to-day it is appended :

"The rapid advance in microscopical knowledge, and the great strides in the
sciences employing the microscope as an indispensable tool, have reacted upon the
microscope itself, and never before were microscopes so excellent, convenient and
cheap. Indeed, the financial reason for not possessing a microscope can no longer
be urged by any high school or academy, or by any person whose profession
demands it.

Naturally, to get the greatest good from instruments, tools, or machines of any
kind, the one who uses them must understand the principles upon which their
action depends, their possibilities and limitations.

That the student may acquire a just comprehension of some of the funda-
mental principles of the microscope, and gain a working acquaintance with it and
its applications, this book has been prepared. It is a growth of the laboratory, and
has been modified from time to time to keep pace with optical improvements and
advancing knowledge. ' '

In rewriting this edition the different chapters have been recast, new figures
added and in most cases the matter considerably increased. A new chapter has
been added upon class demonstrations. The general availability of the constant
electric current, and the improvement in apparatus have made micro-projection
practicable and satisfactory. It has served the writer so well in his teaching of
histology and embryology that it seemed worth while to give the benefit of his
experience to his fellow workers.

It is hoped that the book as it now stands will serve more completely than
ever before the needs of the class-room and of the laboratory.

"Simply reading a work on the microscope, and looking a few times into an
instrument completely adjusted by another, is of very little value in giving real
knowledge. In order that the knowledge shall be made alive, it must become a
part of the student's experience by actual experiments carried out by the student
himself. Consequently, exercises illustrating the principles of the microscope
and the methods of its employment have been made an integral part of the work.

"In considering the real greatness of the microscope, and the truly splendid
service it has rendered, the fact has not been lost sight of that the microscope is,
after all, only an aid to the eye of the observer, only a means of getting a larger
image on the retina than would be possible without it ; but the appreciation of

ivi3484±0

IV

PREFACE

the retinal image, whether it is made with or without the aid of a microscope,
must always depend upon the character and training of the seeing and appreciat-
ing brain behind the eye. The microscope simply aids the eye in furnishing raw
material, so to speak, for the brain to work upon. (From 3d ed.)

Grateful acknowledgment is made to the opticians and instrument makers
for the loan of cuts and for courteous and complete answers to numerous questions ;
to the directors of laboratories in different parts of the country, to his colleagues
in the departments of Physics, Chemistry and Electrical Engineering in Cornell
University ; and finally to his pupils past and present who have given their support
and encouragement.

Inclosing I would like to urge those who are interested in Microscopy to take
some microscopical journal, and if possible to become a member of some micro-
scopical club or society. One can do very little alone, but by helping others
and being helped in return, the workers in any field of human endeavor can
accomplish great things.

SIMON HENRY GAGE,
CORNEI,!, UNIVERSITY,

October /, 1901. ITHACA, N. Y. , U. S. A.

PREFACE TO THE NINTH EDITION

In this edition important changes have been made on pp. 7-8, to the parts
relating to serial sections and to micro-chemistry.

The chapter on the projection microscope has been entirely rewritten and
much more fully illustrated. With the good apparatus now available it is hoped
that teachers will make more use of this excellent method of demonstration.

Due to the courtesy of the manufacturers excellent pictures of the latest
microscopes and apparatus are presented in this edition. It can but be a cause for
rejoicing to the multitudes who now use the microscope to see the beauty of form
which it has attained.

February /,

CONTENTS

CHAPTER I

PAGE

\ i- 59 — The Microscope and its parts i- 33

CHAPTER II

$ 60-128 — Lighting and Focusing ; Manipulation of Dry, Adjustable
and Immersion objectives ; Care of the Microscope and of
the Eyes. Laboratory Microscopes 34- 89

CHAPTER III
$ 129-153 — Interpretation of Appearances 90-102

CHAPTER IV
\ 154-176 — Magnification and Micrometry 103-121

CHAPTER V
\ 177-187 — Drawing with the Microscope 122-133

CHAPTER VI

| 188-233 — The Microspectroscope and Polariscope ; Micro-Chemistry ;

Textile Fibers and Food Products ; Micro-Metallography 134-160

CHAPTER VII

I 234-335 — Slides and Cover-Glasses ; Mounting ; Isolation ; Sectioning
by the Collodion and the Paraffin Methods ; Serial Sections ;
Labeling and Storing Microscopical Preparations ; Reagents
and their Preparation 161-204

CHAPTER VIII

\ 336-392 — Photographing objects with a Vertical Camera ; Photograph-
ing Large Transparent Objects ; Photographing with a Mi-
croscope ; (A) Transparent Objects ; (B) Opaque Objects,
and the Surfaces of Metals and Alloys ; Enlarging Nega-
tives ; Photographing Petri Dishes and Culture Tubes 205-242

CHAPTER IX
$ 393-426 — Class-Room Demonstrations with the Microscope ; With the

Projection Microscope; with the Episcope 243-267

CHAPTER X

I 427-438 — The Abbe Test-Plate and Apertometer ; Equivalent Focus of
Objectives and Oculars ; Drawings for Photo-Engravings ;
Wax Models ; Some Apparatus for Imbedding and Sec-
tioning 268-281

BOOKS AND PERIODICALS 282-288

INDEX 289

THE MICROSCOPE IN SECTION

1. Huygenian ocular (see p. 102 for positive

ocular).

2. Draw-tube by which the tube is length-

ened or shortened

3. Main tube or body containing the draw-

tube, and attached to the pillar by the
arm.

4. Society screw in the lower end of the

draw-tube.

5. Society screw in the lower end of the

tube.
5. Objective in position.

7, Stage, under which is the substage with

the substage condenser.

8. Spring clip for holding the specimen.

g. Screw for centering, and handle for the
iris diaphragm in the achromatic con-
denser (see Fig. 41).

10. Iris diaphragm outside the principal

focus of the condenser for use in cen-
tering (§ 81),

11. Mirror with plane and concave faces.

12. Horse-shoe base.

13 Rack and pinion for the substage con-
denser.

14. Jointed pillar.

15. Part of pillar with spiral spring of fine

adjustment.

16. Screw of fine adjustment.

17. Milled head of coarse adjustment.

THE MICROSCOPE

AND

MICROSCOPICAL METHODS

CHAPTER I

THE MICROSCOPE AND ITS PARTS

APPARATUS AND MATERIAL FOR THIS CHAPTER

A simple microscope ($ 2, u) ; A compound microscope with nose-piece (Figs.
70-80); eye-shade (Fig. 60), achromatic (g 20), apochromatic (^22), dry (§17),
immersion ($ 18), unadjustable and adjustable objectives ($ 23, 24) ; Huygenian or
negative ($38), positive (§37) and compensation oculars ($39) ; stage microme-
ter (Ch. IV) ; homogeneous immersion liquid (§ 18) ; mounted letters or figures
($ 53) ; ground-glass and lens paper (g 53).

A MICROSCOPE

I i. A Microscope is an optical apparatus with which one may obtain a clear
image of a near object, the image being always larger than the object ; that is, it
enables the eye to see an object under a greatly increased visual angle, as if the
object were brought very close to the eye without affecting the distinctness of
vision. Whenever the microscope is use,d for observation, the eye of the observer
forms an integral part of the optical combination (Figs. 16, 21).

$ 2. A Simple Microscope. — With this an enlarged, erect image of an object
may be seen. It always consists of one or more converging lenses or lens-systems
(Figs. 16-20), and the object must be placed within the principal focus (§ n).
The simple microscope may be held in the hand or it may be mounted in some
way to facilitate its use (Figs 17-20).

MICROSCOPE AND ACCESSORIES

[CH. I

FIGS. 1-9, showing the Principal Optic Axis and the Optical Center of various
forms of Lenses.

Axis. The Principal Optic Axis. c-cf . Centers of curvature of the two sur-
faces of the lens, c.l. Optical center of the lens, r-r' '. Radii of curvature of
the two lens surfaces, t-t' '. Tangents in Fig. 4.

$ 3. Principal Optic Axis. — In spherical lenses, i. e., lenses whose surfaces
are spherical, the Axis is a line joining the centers of curvature and indefinitely
extended. In the lens it is the unbroken part of the line c-cf in the figures. In
lenses with one plane surface (Figs. 3, 6, 7) the radius of the plane surface is any
line at right angles to it; but in determining the axis it must be the one which is
continuous with the radius of the curved surface, consequently the axis in such
lenses is on the radius of the curved surface which meets the plane surface at right
angles.

\ 4. Optical Center. — The optical center of a lens is the point through which
rays pass without angular deviation, that is, the emergent ray is parallel to the

\_CH. I

MICROSCOPE AND ACCESSORIES

incident ray. It is determined geometrically by drawing parallel radii of the
curved surfaces, r-r' in Figs. 4-9, and joining the peripheral ends of the radii.
The optical center is the point on the axis cut by the line joining the peripheral
ends of the parallel radii of the two lens surfaces. In Figs. 4-5 it is within the
lens ; in 6-7 it is at the curved surface, and in the meniscus (8, 9) it is wholly out-
side the lens, being situated on the side of the greater curvature.

In determining the center in a lens with a plane surface, the conditions can
be satisfied only by using the radius of the curved surface which is continuous
with the axis of the lens, then any line at right angles to the plane surface will
be parallel with it, and may be considered part of the radius of the plane surface.
(That is, a plane surface may be considered part of a sphere with infinite radius,
hence any line meeting the plane surface at right angles may be considered as the
peripheral part of the radius. ) In Figs. 6, 7, (r'} is the radius of the curved sur-
face and (r) of the plane surface ; and the point where a line joining the ends of
these radii crosses the axis is at the curved surface in each case.

By a study of Fig. 4 it will be seen that if tangents be drawn at the peripheral
ends of the parallel radii, the tangents will also be parallel and a ray incident at
one tangential point and traversing the lens and emerging at the other tangential
point acts as if traversing, and is practically traversing a piece of glass which has
parallel sides at the point of incidence and emergence, therefore the emergent ray
will be parallel with the incident ray. This is true of all rays traversing the center
of the lens.

Thick Lenses. — In all of the diagrams of lenses and the course of rays through
them in this book the lenses are treated as if they were infinitely thin. In thick
lenses like those figured, while there would be no angular deviation for rays trav-
ersing the center of the lens, there would be lateral displacement. This is shown
in Fig. 57 illustrating the effect of the cover-glass.

\ 5. Secondary Axis. — Every ray traversing the center of the lens, except the
principal axis, is a secondary axis ; and every secondary axis is more or less
oblique to the principal axis. In Fig. 14, line (2), is a secondary axis, and in Fig.
15, line (i). See also Fig. 58.

FIGS, ro, ii. — Sectional views of a
concave or diverging and a convex or
converging lens to show that in the con-
cave lens the principal focus is virtual as
indicated by the dotted lines, while with
the convex lens the focus is real and on
the side of the lens opposite to that from
which the light comes.

\ 6. Principal Focus. — This is the point where rays parallel with the axis and
traversing the lens cross the axis ; and the distance from the focus to the center of
the lens measured along the axis is the Principal Focal Distance. In the diagrams,
Fig. 10 is seen to be a diverging lens and the rays cross the axis only by being pro-
jected backward. Such a focus is said to be virtual, as it has no real existence. In

MICROSCOPE AND ACCESSORIES

\CH. I

Fig. 1 1 the rays do cross the axis and the focus is said to be real. If the light
came from the opposite direction it would be seen that there is a principal focus
on the other side, that is there are two principal foci, one on each side of the lens.
These two foci are both principal foci, but they will be equally distant from the
center of the lens only when the curvature of the two lens surfaces are equal.
There may be foci on secondary axes also, each focus on a secondary axis has its
conjugate. In the formation of images the image is the conjugate of the object
and conversely the object is the conjugate of the image.

W A

FIG. 12. — Double Convex Lens, Showing Chromatic Aberration.

The ray of white light (w) is represented as dividing into the short waved, blue
(b) and the long waved, red (r} light. The blue (b) ray comes to a focus nearer
the lens and the red ray ( r} farther from the lens than the principal focus ( J) .
Principal focus (f) for rays very near the axis ; f and f" ', foci of blue and red
light coming from near the edge of the lens. The intermediate wave lengths
would have foci all the way between ff and ff/ .

\ 7. Chromatic Aberration. — This is due to the fact that ordinary light con-
sists of waves of varying length, and as the effect of a lens is to change the direc-
tion of the waves, it changes the direction of the short waves more markedly
than the long waves. Therefore, the short waved, blue light will cross the axis
sooner than the long waved, red light, and there will result a superposition of
colored images, none of which are perfectly distinct (Fig. 12).

FIG. 13. The ray (o) near the
edge of the lens is brought to a
focus nearer the lens than the
ray (i). Both are brought to
a focus sooner than rays very
near the axis, (f) Principal
focus for rays very near the
axis; (f} Focus for the ray
(i), and (f") Focus for the ray

(o). Intermediate rays would
FIG. 13. Double Convex Lens, showing ., ,, ,, ' r

6 cross the axis all the way from
Spherical Aberration. ( ff t f\

% 8. Spherical Aberration.— This is due to the unequal turning of the light
in different zones of a lens. The edge of the lens refracts proportionally too
much and hence the light will cross the axis or come to a focus nearer the lens
than a ray which is nearer the middle of the lens. Thus, in Fig. 13, if the focus

CH.I-]

MICROSCOPE AND ACCESSORIES

of parallel rays very near the axis is at/, rays (o i), nearer the edge, would come to
a focus nearer the lens, the focus of the ray nearest the edge being nearest the lens.

\ 9. Correction of Chromatic and of Spherical Aberration. — Every simple
lens has the defect of both chromatic and spherical aberration, and to overcome
this, kinds of glass of different refractive power and different dispersive power
are combined, concave lenses neutralizing the defects of convex lenses. If the
concave lens is not sufficiently strong to neutralize the aberrations of the convex
lens, the combination is said to be under-corrected, while if it is too strong and
brings the marginal rays or the blue rays to a focus beyond the true principal
focus, the combination is over-corrected.

In Newton's time there was supposed to be a direct proportion between the
refractive power of any transparent medium and its dispersive power ( i. e. its power
to separate the light into colors). If this were true then the contention of Newton
that it would be impossible to do away with the color without al the same time
doing away with the refraction would be true and useful achromatic combinations
would be impossible. It was found by experiment, however, that there is not a
direct ratio between the refractive and dispersive powers for the different colors
in different forms of glass, so that it is possible to do away largely with chromatic
aberration and retain sufficient refraction to make the combination serve for the
production of images. ( See also the discussion under apochromatic objectives \ 22 )

Probably no higher technical skill is used in any art than is requisite in the
preparation of microscopical objectives, oculars and illuminators.

FIGS. 14 AND 15. 14. Convex lens
showing the position of the object ( A-B )
outside the principal focus (F], and
the course of the rays in the formation
of real images. To avoid confusion the
rays are drawn from only one point.

A B. Object outside the principal
focus. Bf Af . Real, enlarged image
on the opposite side of the lens.

Axis. Principal optic axis. 1,2,3.
Rays after traversing the lens. They
are converging, and consequently form
a real image. The dotted line and the
line (2} give the direction of the rays as
if unaffected by the lens. (/^). The
principal focus.

FIG. 15. — Convex lens, showing the
position of the object (A B} within the
principal focus and the course of rays
in the formation of a virtual image.

A B. The object placed between the lens and its focus ; A' B' virtual image
formed by tracing the rays backward. It appears on the same side of the lens as
the object, and is erect (\ //).

Axis. The principal optic axis of the lens. F. The principal focus.

i, 2, j. Rays from the point B of the object. They are diverging after trav-
ersing the lens, but not so divergent as if no lens were present, as is shown by the

MICROSCOPE AND ACCESSORIES

[CH. I

dotted lines. Ray (/) traverses the center of the lens, and is therefore not deflected.
It is a secondary axis ($5).

\ 10. Geometrical Construction of Images. — As shown in Figs. 14-15, for the
determination of any point of an image, or the image being known, to determine
the corresponding part of the object, it is necessary to know the position of the
principal focus (and there is one on each side of the lens, $6), and the optical
center (Figs. 1-9 of the lens). Then a secondary axis (2) in Fig. 14, (i) in Fig.
15, is drawn from the extremity of the object and prolonged indefinitely above the
lens, or below it for virtual images. A second line is drawn from the extremity of
the object, (3) in Fig. 14, (2) in Fig. 15, to the lens parallel with the principal
axis. After traversing the lens it must be drawn through the principal focal point.
If now it is prolonged it will cross the secondary axis above the lens for a real
image and below for a virtual image. The crossing point of these lines determines
the position of the corresponding part of the image. Commencing with any point
of the object the corresponding point of the image may be determined as just
described, and conversely commencing with the image, corresponding points of
the object may be determined.

SIMPLE MICROSCOPE : EXPERIMENTS

§ ii. Employ a tripod or other simple microscope, and for object
a printed page. Hold the eye about two centimeters from the upper
surface of the magnifier, then alternately raise and lower the magnifier
until a clear image may be seen. (This mutual arrangement of micro-
scope and object so that a clear image is seen, is called focusing).
When a clear image is seen, note that the letters appear as with the
unaided eye except that they are larger, and the letters appear erect or
right side up, instead of being inverted, as with the compound
microscope (§12).

FIG. 1 6. Diagram of the simple microscope show-
ing the course of the rays and all the images, and
that the eye forms an integral part of it.

A* B1. The object within the principal focus. A*
£3. The virtual image on the same side of the lens
as the object. It is indicated with dotted lines, as it
has no actual existence.

B2 A2. Retinal image of the object (A1 Bl). The
virtual image is simply a projection of the retinal
image in the field of. vision.

Axis. The principal optic axis of the micro-
scope and of the eye. Cr. Cornea of the eye. L.
Crystalline lens of the eye. R. Ideal refracting
surface at which all the refractions of the eye may
be assumed to take place.

CH. /]

MICROSCOPE AND ACCESSORIES

Hold the simple microscope directly toward the sun and move it
away from and toward a piece of printed paper until the smallest
bright point is obtained. This is the burning point or focus and as
the rays of the sun are nearly parallel, the burning point represents
approximately the principal focus (Fig. n). The above and follow-
ing operations are more easily accomplished if the lens is supported as
in Fig. 20.

Without changing the position of the magnifier or paper look into
the magnifier, holding the eye close to the upper surface and the
letters on the paper may be seen, but they will appear much sharper
to the eyes of most people if the magnifier is brought nearer to the
paper, that is so that the printed paper is within the principal focal
distance (Fig. 15 and 16).

Fig. 1 6 A. Figures of a normal (emme-
tropic], a far sighted (hyperopic) and a short
sighted (myopic] eye to show that when the eye
is at rest the normal eye (E) focuses parallel
rays on the retina while the far-sighted eye (//)
focuses parallel rays beyond the retina. The
short sighted eye (M) focuses parallel rays in
front of the retina. The dotted lines show that
in the hyperopic eye the rays must be converging
to come to a focus on the retina while with the
myopic eye they must be diverging.

After getting as clear an image as possible by focusing the simple
microscope, raise the magnifier until the letters are at a distance a
little greater than the principal focal distance. Look into the magni-
fier and note the clearness of the virtual image, then slowly elevate
the head above the magnifier and when the eye is about 60 to 100
centimeters above the lens a real image can be seen. That is an
image in which the letters are inverted as with the objective of the
compound microscope (see § 53). If the magnifier is raised somewhat
so that the printed letters are markedly without the principal focus the
real image will be seen more clearly especially if the eye is brought
somewhat near the magnifier. The above experiments show two
things.

8

MICROSCOPE AND ACCESSORIES

\_CH. I

1 i ) That every convex or converging lens or lens system can
serve to form either a virtual or a real image, depending upon its
position with reference to the object.

(2) They show also that without changing the position of the
magnifier, if it is slightly further from the object than its principal
focal distance, either a virtual image or a real image may be seen by
many people, depending upon the position of the eye. (a) If the
eye is close to the magnifier an enlarged erect virtual image will be
seen. (b) With the eye at a considerable distance an enlarged
inverted real image may be seen.

Fig. 1 6 B. Figure to show that with a
simple microscope if the object is slightly be-
yond Ihe principal focus -(F) a real image
will be formed at A' which can be seen by an
eye at £, and that if a normal or hyperopic
eye is at Ef a virtual image can be seen
without changing the position of the simple
microscope. The long-sighted eye can see
this image best as it naturally focuses con-
verging rays on the retina. The myopic eye
either sees no image at all, or a mere blur,
depending .upon the amount of myopia. A.
object; A/ real image above the magnifier ;
A." virtual image which can be seen below
the lens by an eye at E> '; E. eye in posi-
tion to see a real image ; E.f eye in position
to see A" a virtual image; F. principal
focus of the magnifier.

FIG. 17. Tripod Magnifier.

While the law is absolute that real images are formed only when
the object is without the principal focal distance, and virtual images
only when the object is within the focus, the above experiments show

CH.I}

MICROSCOPE AND ACCESSORIES

8a

FIG. 18. Lens-holder (The Bausch FIG. 19. The Hastings Aplan-

& Lomb Optical Co.) atic Triplet. (The Bausch & Lomb

Optical Co. )

FIG. 20. Dissecting Microscope. This is simply a device for holding the lens
and the object to be observed. ( The Bausch & Lomb Optical Co. )

8b

MICROSCOPE AND ACCESSORIES

[_CH. I

most conclusively that the eye is a part of the optical arrangement
when the microscope is actually used for observation, and that the
microscope with the eye is a different apparatus from the microscope
considered by itself.

The diagrams, Figs. 16 A, B, are introduced to show under what
conditions both a virtual and a real image may be seen without
changing the position of the magnifier or the object.

FIG. 17 A. Diagrams showing- the formation of real and of virtual images
and of the retinal image in using the simple microscope. See the explanation of
Figs. 14, 75, 16.

Simple microscopes are very convenient when only a small mag-
nification (Ch. IV) is desired, as for dissecting. Achromatic triplets
are excellent and convenient for the pocket. For use in conjunction
with a compound microscope, the tripod magnifier (Fig. 17) is one of
the best forms. For many purposes a special mechanical mounting is
to be preferred.

COMPOUND MICROSCOPE

\ 12. A Compound Microscope. — This enables one to see an enlarged, in-
verted image. It always consists of two optical parts — an objective, to produce an
enlarged, inverted, real image of the object, and an ocular acting in general like
a simple microscope to magnify this real image (Fig. 21). There is also usually
present a mirror, or both a mirror and some form of condenser or illuminator for
lighting the object. The stand of the microscope consists of certain mechanical
arrangements for holding the optical parts and for the more satisfactory use of
them. (See frontispiece. )

\ 13. The Mechanical Parts of a laboratory, compound microscope are shown
in the frontispiece, and are described in the explanation of that figure. The stu-

CH. /]

MICROSCOPE AND ACCESSORIES

dent should study the figure with a microscope before him and become thoroughly
familiar with the names of all the parts. See also the cuts of microscopes at the
end of Ch. II.

OPTICAL PARTS

\ 14. Microscopic Objective. — This
consists of a converging lens or of one
or more converging lens-systems, which
give an enlarged, inverted, real image of
the object ( Figs. 14, 21 ). And as for the
formation of real images in all cases,
the object must be placed outside the
principal focus, instead of within it, as
for the simple microscope. (See |§ n,
53, Figs. 1 6, 21.)

Modern microscopic objectives usu-
ally consist of two or more systems or
combinations of lenses, the one next
the object being called the front com-
bination or lens, the one farthest from
the object and nearest the ocular, the
back combination or system. There may
be also one or more intermediate sys-
tems. Each combination is, in general,
composed of a convex and a concave
lens. The combined action of the sys-
tem serves to produce an image free
from color and from spherical distor-
tion. In the ordinary achromatic ob-J
jectives the convex lenses are of crown
and the concave lenses of flint glass
(Figs. 22,23). .

FIG. 21. Diagram showing the
principle of a compound microscope with
the course of the rays from the object
(A B) through the objective to the real
image (B' A'}, thence through the ocu-
lar and into the eye to the retinal image
(A*B2), and the projection of the retinal
image into the field of vision as the
virtual image (B^A*).

AB. The object. A*B\ The retinal
image of the inverted real image, (BlAl) ,
formed by the objective. B^A*. The
inverted virtual image, a projection of
the retinal image.

•c

IO

MICROSCOPE AND ACCESSORIES

Axis. The principal optic axis of the microscope and of the eye.

Cr. Cornea of the eye. L. Crystalline lens of the eye . R. Single, ideal, re-
fracting surface at which all the refractions of the eye may be assumed to take
place.

F. F. The principal focus of the positive ocular and of the objective.

Mirror. The mirror reflecting parallel rays to the object. The light is central.
See Ch. II.

Pos. Ocular. An ocular in which the real image is formed outside the ocular.
Compare the positive ocular with the simple microscope (Fig. 16).

NOMENCLATURE OR TERMINOLOGY OF OBJECTIVES

\ 15. Equivalent Focus. — In America, England, and sometimes also on the
Continent, objectives are designated by their equivalent focal length. This length
is given either in inches (usually contracted to in. ) or in millimeters (mm.) Thus:
An objective designated T^ in. or 2 mmM indicates that the objective produces a
real image of the same size as is produced by a simple converging lens whose
principal focal distance is T^ inch or 2 millimeters (Fig. u). An objective
marked 3 in. or 75 mm., produces approximately the same sized real image as a
simple converging lens of 3 inches or 75 millimeters focal length. And in accord-
ance with the law that the relative size of object and image vary directly as their
distance from the center of the lens (Figs. 14, 15, see Ch. IV,) it follows that the
less the focal distance of the simple lens or of the equivalent focal distance of the
objective, the greater is the size of the real image, as the tube-length remains con-
stant and the image in all cases is found at about i6oor 250 mm. from the objective.
§ 16. Numbering or Lettering Objectives. — Instead of designating objectives
by their equivalent focus, many Continental opticians use letters or figures for this
purpose. With this method the smaller the number, or the earlier in the alpha-
bet the letter, the lower is the power of the objective. (See further in Ch. IV, for
the power or magnification of objectives). This method is entirely arbitrary and
does not, like the one above, give direct information concerning the objective.

§ 17. Air or Dry Objectives. — These are objectives in which the space be-
tween the front of the objective and the object or cover-glass is filled with air
(Fig. 22). Most objectives of low and medium power (i. e,, \ in. or 3 mm. and
lower powers) are dry.

FIG. 22. Section of a dry objective showing
working distance and lighting by reflected
light.

Axis. The principal optic axis of the ob-
jective.

B C. Back Combination, composed of a
plano-concave lens of flint glass (F}, and a
double convex lens of crown glass (c).
F C. Front Combination.
C, O, si. The cover-glass, object and slide.
Mirror. The mirror is represented as above
the stage, and as reflecting parallel rays from
its plane face upon the object.

Stage. Section of the stage of the microscope.

CH. /]

MICROSCOPE AND ACCESSORIES

n

W. The Working Distance, that is the distance from the front of the objective
to the object when the objective is in focus.

§ 18. Immersion Objectives. — An immersion objective is one with which
there is some liquid placed between the front of the objective and the object or
cover-glass. The most common immersion objectives are those (A) in which
water is used as the immersion fluid, and (B) where some liquid is used having the
same refractive and dispersive power as the front lens of the objective. Such a
liquid is called homogeneous, as it is optically homogeneous with the front glass of
the objective. It may consist of thickened cedar wood oil or of glycerin contain-
ing some salt, as stannous chlorid in solution. When oil is used as the immersion
fluid the objectives are frequently called oil immersion objectives. The disturb-
ing effect of the cover-glass (Fig. 57) is almost wholly eliminated by the use of
homogeneous immersion objectives, as the rays undergo very little or no refraction
on passing from the cover-glass through the immersion medium and into the ob-
jective ; and when the object is mounted in balsam there is practically no refrac-
tion in the ray from the time it leaves the balsam till it enters the objective.

FIG. 23. Sectional view of an Immersion, Ad-
justable Objective, and the object lighted with axial
or central and with oblique light.

Axis. The principal optic axis of the objective.

B C, M C, F C. The back, middle and front
combination of the objective. In this case the
front is not a combination, but a single plano-
convex lens.

A, B. Parallel rays reflected by the mirro*
axially or centrally upon the object.

C. Ray reflected to the object obliquely.

I. Immersion fluid between the front of the
objective and the cover glass or object (O).

Mirror. The mirror of the microscope.

O. Object. It is represented without a cover-
glass. Ordinarily objects are covered whether ex-
amined with immersion or with dry objectives.

Stage. Section of the stage of the microscope.

\ 19. Non- Achromatic Objectives. — These are objectives in which the chro-
matic aberration is not corrected, and the image produced is bordered by colored
fringes. They show also spherical aberration and are used only on very cheap
microscopes. (\\ 7, 8, Figs. 12, 13).

£ 20. Achromatic Objectives. — In these the chromatic and the spherical aber-
ration are both largely eliminated by combining concave and convex lenses of dif-
ferent kinds of glass "so disposed that their opposite aberrations shall correct
each other." All the better forms of objectives are achromatic and also aplanatic.
That is the various spectral colors come to the same focus.

| 21. Aplanatic Objectives, etc.— These are objectives or other pieces of
optical apparatus (oculars, illuminators, etc.), in which the spherical distortion is

12 MICROSCOPE AND ACCESSORIES [CM. I

wholly or nearly eliminated, and the curvatures are so made that the central and
marginal parts of the objective focus rays at the same point or level. Such pieces
of apparatus are usually achromatic also.

§ 22. Apochromatic Objectives. — A term used by Abbe to designate a form of
objective made by combining new kinds of glass with a natural mineral (Calcium
fluorid, Fluorite, or Fluor spar). The name, Apochromatic, is used to indicate
the higher kind of achromatism in which rays of three spectral colors are com-
bined at one focus, instead of rays of two colors as in the ordinary achromatic ob-
jectives. At the present time (1901) several opticians make apochrornatic ob-
jectives without using the fluorite. Some of the early apochromatics deteriorated
rather quickly in hot moist climates. Those now made are quite permanent.

The special characteristics of these objectives, when used with the "compen-
sating oculars" are as follows :

(1) Three rays of different color are brought to one focus, leaving a small ter-
tiary spectrum only, while with objectives as formerly made from crown and flint
glass, only two different colors could be brought to the same focus.

(2) In these objectives the correction of the spherical aberration is obtained
for two different colors in the brightest part of the spectrum, and the objective
shows the same degree of chromatic correction for the marginal as for the central
part of the aperture. In the old objectives, correction of the spherical aberration
was confined to rays of one color, the correction being made for the central part of
the spectrum, the objective remaining under-corrected spherically for the red rays
and <?zw-corrected for the blue rays ($9).

(3) The optical and chemical foci are identical, and the image formed by
the chemical rays is much more perfect than with the old objectives, hence the
new objectives are well adapted to photography.

(4) These objectives admit of the use of very high oculars, and seem to be a
considerable improvement over those made in the old way with crown and flint
glass. According to Dippel (Z. w. M. 1886, p. 300) dry apochrornatic objectives
give as clear images as the same power water immersion objectives of the old form.

§ 23. Non-Adjustable or Unadjustable Objectives. — Objectives in which the
lenses or lens systems are permanently fixed in their mounting so that their rela-
tive position always remains the same. Low power objectives and those with
homogenous immersion are mostly non-adjustable. For beginners and those un-
skilled in manipulating adjustable objectives ($ 24), non- adjustable ones are more
satisfactory, as the optician has put the lenses in such a position that the most
satisfactory results may be obtained when the proper thickness of cover-glass and
tube-length are employed. (See table of tube-length and thickness of cover-glass
below, p. 14.)

§ 24. Adjustable Objectives.— An adjustable objective is one in which the dis-
tance between the systems of lenses (usually the front and the back systems) may
be changed by the observer at pleasure. The object of this adjustment is to cor-
rect or compensate for the displacement of the rays of light produced by the
mounting medium and the cover-glass after the rays have left the object. It is
also to compensate for variations in "tube-length." See $29. As the displace-
ment of the rays by the cover-glass is the most constant and important, these ob-
jectives are usually designated as having cover-glass adjustment or correction.
(Fig. 23. See also practical work with adjustable objectives, Ch. II).

CH. 7] MICROSCOPE AND ACCESSORIES 13

\ 25. Parachromatic, Pantachromatic and Semi-apochromatic Objectives.
These are trade names for objectives, most of them containing one or more lenses
of the new glass (§22). They are said to approximate much more closely to the
apochromatics than to the ordinary objectives.

\ 26. Variable Objective. — This is a low power objective of 36 to 26 mm.
equivalent focus, depending upon the position of the combinations. By means of
a screw collar the combinations may be separated, diminishing the power, or
approximated and thereby increasing it.

$ 27. Projection Objectives. — These are designed especially for projecting
an image on a screen and for photo-micrography. They are characterized by hav-
ing a flat, sharp field brilliantly lighted. (See Ch. IV, IX. )

\ 28. Illuminating or Vertical Illuminating Objectives. — These are designed
for the study of opaque objects with good reflecting surfaces, like the rulings on
metal bars and broken or polished and etched surfaces of metals employed in
micro- metallography. The light enters the side of the tube or objective and is
reflected vertically downward through the objective and thereby is concentrated
upon the object. The object reflects part of the light back into the microscope
thus enabling one to see a clear image.

$ 29. Tube-Length and Thickness of Cover-Glasses. — "In the construction
of microscopic objectives, the corrections must be made for the formation of the
image at a definite distance, or in other words the tube of the microscope on
which the objective is to be used must have a definite length. Consequently the
microscopist must know and use this distance or 'microscopical tube-length' to
obtain the best results in using any objective in practical work." Unfortunately
different opticians have selected different tube-lengths and also different points
between which the distance is measured, so that one must know what is meant by
the tube-length of each optician whose objectives are used. See table.

The thickness of cover-glass used on an object (See Ch. VII, on mounting),
except with homogeneous immersion objectives, has a marked effect on the light
passing from the object (Fig. 57). To compensate for this the position of the sys-
tems composing the objective are closer together than they would be if the object
were uncovered. Consequently, in non-adjustable objectives some standard
thickness of cover-glass is chosen by each optician and the position of the systems
arranged accordingly. With such an objective the image of an uncovered object
would be less distinct than a covered one, and the same result would follow the
use of a cover-glass much too thick.

• *The information contained in the tables on the following page was very
kindly furnished by the opticians named, or obtained by consulting catalogs. In
most of the later catalogs the information is definite, and many makers now
not only put their names and the equivalent focal length on their objectives,
but they add the numerical aperture (§ 31) and the tube-length for which
the objective is corrected. This is in accordance with the recommendations
of the author in the original paper on "tube-length," (Proc. Amer. Soc.
Micr., Vol. IX., p. 168, also by Bausch, Vol. XII, p. 43). If the table in
this edition is compared with the original table or with that in the pre-
vious editions of this book some differences will be noted, the changes being

MICROSCOPE AND ACCESSORIES

\_CH. I

"Tube-length" in
Millimeters.

In the following tables tube-length b-d of the diagram greatly preponderates,
and a large majority of unadjustable objectives are corrected for a thickness of cover-
glass falling between fifteen and twenty hundredths of a millimeter (o. 15-0. 20 mm. ) .
Length in Millimeters and Parts included in the '"Tube-Length" by

Various Opticians.
Pts. included
in "Tube-
length. ' '
See Diagram.

fChas. Baker, London, England 150 or 250 mm.

The Bausch & Lomb Optical Co. ,

Rochester, N. Y 160 or 216 mm.

R. & J. Beck, London, England 160 or 220 mm.

Bezu, Hausser & Cie, Paris, France 180 mm.

Klonne und Miiller, Berlin, Germany 160 or 250 mm.

Queen & Co., Incorporated, Phila , Pa 170 mm.

Ross, Ltd, London, England 160 or 254 mm.

W. und H. Seibert, Wetzlar, Germany 170 mm.

Swift & Son, London, England 160 or 228 mm.

Watson & Sons, London, England 160 or 250 mm.

R. Winkel, Goettingen, Germany 192 mm.

[Carl Zeiss, Jena, Germany 160 or 250 mm.

f Ernst Leitz, Wetzlar, Germany 170 mm.

| Nachet et Fils, Paris, France 160 mm.

j Powell & Lealand, London, England 254mm.

I C. Reichert, Vienna, Austria 160-180 mm.

| Spencer Lens Company, Buffalo, N. Y 160 mm.

[ W. Wales, New York 254 mm.

The Gundlach Opt. Co., Rochester, N. Y. 254 mm.

E. Hartnack, Potsdam, Germany 160 mm.

Dollond & Co., London, England 165, 240 mm.

Ve"rick (Stiassnie) Paris, France 160-200 mm.

P. Wachter, Berlin-Friedenau, Germany 160 mm.

J. Zentmayer, Philadelphia, Pa, i6oor235tnm.

Cover-Glass for Which Non-Adjustable Objectives are Corrected by

Various Opticians.

f The Bausch & Lomb Optical Co., Rochester, N. Y.
J Klonne und Miiller, Berlin, Germany,
j Queen & Co., Incorporated, Philadelphia, Pa.
[The Spencer Lens Co., Buffalo, N. Y.
f Ernst Leitz, Wetzlar, Germany.
•I P. Wachter, Berlin-Friedenau, Germany.
I R. Winkel, Goettingen, Germany.

(Chas. Baker, London, England.
R. & J. Beck, Ltd., London, England.
Gundlach Optical Co., Rochester, N. Y.
W. und H. Seibert, Wetzlar, Germany.
E. Hartnack, Potsdam, Germany.
C. Reichert, Vienna, Austria.
Ross, Ltd., London, England.
• Verick (Stiassnie), Paris, France.
( Carl Zeiss, Jena, Germany.

J. Zentmayer, Philadelphia, Pa.
J Dollond & Co., London, England.
\ Nachet et Fils, Paris, France.

Be"zu Hausser & Cie, Paris, France,
f Powell & Lealand, London, England.
\ Swift & Son, London, England.
Watson & Sons, London, England.
W. Wales, New York.

FIG. 24.
Thickness of

o. 18 mm.
0.17 mm.

0.15 mm.

0.15-0.18 mm. \

0.15-0.20 mm

0.12-0.17 mm
0.10-015 mm.
0.10-0.12 mm
o.io mm.

0.20 mm.
0.25 mm.

CH. /]

MICROSCOPE AND ACCESSORIES

§ 30. Aperture of Objectives. — The angular aperture or angle
of aperture of an objective is the "angle contained, in each case, be-
tween the most diverging of the rays issuing from the axial point of an
object [/. £., a point in the object situated on the optic axis of the
microscope], that can enter the objective and take part in the formation
of an image. ' ' (Carpenter) .

in the direction of uniformity and in general in the direction recommended by the
writer and Mr. Bausch and the committee of the American Microscopical Society.
The recommendations of the committee, published in the Proceedings, Vol. XII.,
p. 250, are as follows :

"Believing in the desirability of a uniform tube-length for microscopes, we
unanimously recommend : i. That the parts of the microscope included in the
tube-length should be the same by all opticians, and that the parts included should
be those between the upper end of the tube where the ocular is inserted and the
lower end of the tube where the objective is inserted.

2. That the actual extent of tube
length as denned in section i — Be, for the
short or continental tube, 160 mm., or 6.3
inches, and 216 mm., or 8^ inches, for
the long tube, and that the draw tube of
the microscope possess two special
marks indicating these standard lengths.

3. That oculars be made par-focal,
and that the par-focal plane be coincident
with that of the upper end of the tube.

4. That the mounting of all object-
ives of 6 mm. (% inch) and shorter focus
should be such as to bring the optical
center of the objective \% inches below
the shoulder, and that all objectives be
marked with the tube-length for which
they are corrected.

5. That non-adjustable objectives be
corrected for cover-glass from ^ to T20°¥
mm. (Iy^ to T|^ inch) in thickness.

These recommendations give a dis-
tance of 10 inches (254 mm.) between the
par-focal plane of the ocular and the op-
tical center of the objective for the long
tube, and are essentially in accord with
the actual practice of opticians.

At the request of the committee, a
joint conference was held with the opti-
cians belonging to the Society and present at the meeting. They expressed their
belief in the entire practicability of the above recommendations and a willingness
to adopt them."

(Signed) SIMON H. GAGE,

A. CLIFFORD MERCER,
CHARLES E. BARR.

FIG. 25. The tube of a microscope with
ocular micrometer and nose piece in
position to show that in measuring
tube-length one must measure from
the eye lens to the place where the ob-
jective is attached. (Zeiss1 Catalog.}

1 6 MICROSCOPE AND ACCESSORIES \CH. I

In general the angle increases with the size of the lenses forming the objective
and the shortness of the equivalent focal distance (\ 15). If all objectives were
dry or all water or all homogeneous immersion a comparison of the angular aper-
ture would give one a good idea of the relative number of image forming rays

FIG. 26. Diagram illustrating the angular aperture of
a microscopic objective. Only the front lens of the objective
is shown.

Axis. The principal optic axis of the objective.

B A, B C, the most divergent rays that can enter the
objective, they mark the angular aperture. A B D or C B
D half the angular aperture. This is designated by u in
making Numerical Aperture computations. See the table, $ 33.

transmitted by different objectives ; but as some are dry,
others water and still others homogeneous immersion, one
can see at a glance that, other things being equal, the dry
objective (Fig. 27) receives less light than the water immersion, and the water im-
mersion (Fig. 28) less than the homogeneous immersion (Fig. 29). In order to
render comparison accurate between different kinds of objectives, Professor
Abbe takes into consideration the rays actually passing from the back combi-
nation of the objectives to form the real image ; he thus takes into account the
medium in front of the objective as well as the angular aperture. The term
"Numerical Aperture," (N. A.} was introduced by Abbe to indicate the capacity
of an optical instrument "for receiving rays from the object and transmitting
them to the image.

\ 31. Numerical Aperture (abbreviated N. A.), as now employed for micro-
scope objectives, is the ratio of the semi-diameter of the emergent pencil to the
focal length of the lens. Or as the factors are more readily obtainable it is sim-
pler to utilize the relationship shown in the La Grange- Helmholtz- Abbe formula,
and indicate the aperture by the expression : N. A.=« sin u. In this formula n
is the index of refraction of the medium in front of the objective (air, water or
homogeneous liquid) , and sin u is the sine of half the angle of aperture ( Fig. 26,
DBA). For the mathematical discussion showing that the expressions

semi-diameter of emergent pencil . .

— . n , -: 7-7? — ; — — = n sin u, the student is referred to the journal

focal length of the lens

of the Royal Microscopical Society , i88i,pp. 392-395, 1898, p. 363.

For example, take three objectives each of 3 mm. equivalent focus, one being
a dry, one a water immersion, and one a homogeneous immersion. Suppose that
the dry objective has an angular aperture of 106°, the water immersion of 94° and
the homogeneous immersion of 90°. Simply compared as to their angular aper-
ture, without regard to the medium in front of the objective, it would look as if
the dry objective would actually take in and transmit a wider pencil of light than
either of the others. However, if the medium in front of the objective is con-
sidered, that is to say, if the numerical instead of the angular apertures are
compared, the results would be as follows : Numerical Aperture of a dry objective
of 106°, N. A.=« sin u. In the case of dry objectives the medium in front of the
objective being air, the index of refraction is unity, whence «— 1. Half the
angular aperture is ^ °=53°. By consulting a table of natural sines it will be
found that the sine of 53° is 0.799, whence N. A.=« or 1 X sin u or 0.799—0.799.*

*\ 32. Interpolation. — In practice, as in solving problems similar to those
on the following pages and those in refraction if one cannot find a sine exactly

CH. /.]

MICROSCOPE AND ACCESSORIES

FIGS. 27-29 are somewhat modified from
Ellenberger, and are introduced to illustrate
the relative amount of utilized light, with dry,
water immersion and homogeneous immer-

27 sion objectives of the same equivalent focus.
The point from which the rays emanate is in
air in each case. If Canada balsam were be-
neath the cover-glass in place of the air there
would be practically no refraction of the rays
on entering the cover glass (§ /£).

FIG. 27. Showing the course of the rays

28 passing through a cover glass from an axial
point of the object, and the number that
finally enter the front of a dry objective.

FIG. 28. Rays from the axial point of
the object traversing a cover of the same thick-
ness as in Fig. 27, and entering the front
lens of a water immersion objective.
FIG. 29. Rays from an axial point of the

29 object traversing a cover glass and entering
the front of a homogeneous immersion
objective.

With the water immersion objective the medium in front is
water, and its index of refraction is 1.33, whence n= 1.33. Half
the angular aperture is -9^ °= 47°, and by the table the sine of 47° is
found to be 0.731, i. e., sin 2^=0.731, whence N. A.=n or i.33Xsin u
or 0.731=0.972.

\\\\

SAll//

j*-^^^g^

//C

corresponding to a given angle ; or if one has an angle which does not correspond
to any sine or angle given in the table, the sine or angle may be closely approxi-
mated by the method of interpolation, as follows : Find the sine in the table nearest
the sine whose angle is to be determined. Get the difference of the sines of the
angles greater and less than the sine whose angle is to be determined. That will give
the increase of sine for that region of the arc for 15 minutes. Divide this increase
by 15 and it will give with approximate accuracy the increase for I minute. Now
get the difference between the sine whose angle is to be determined and the sine
just below it in value. Divide this difference by the amount found necessary for
an increase in angle of i minute and the quotient will give the number of minutes
the sine is greater than the next lower sine whose angle is known. Add this num-
ber of minutes to the angle of the next lower sine and the sum will represent the
desired angle of the sine. Or if the sine whose angle is to be found is nearer in
size to the sine just greater, proceed exactly as before, getting the difference in the
sines, but subtract the number of minutes of difference and the result will give the
angle sought. For example take the case in Section 97 where the sine of the
angle of 28° 54' is given as 0.48327. If one consults the table the nearest sines
found are 0.48099, the sine of 28° 45', and 0.48461, the sine of 29°. Evidently

1 8 MICROSCOPE AND ACCESSORIES {_CH. I

With the oil immersion in the same way N. A.= n sin u ; n or the
index of refraction of the homogeneous fluid in front of the objective
is 1.52, and the semi-angle of aperture is ^-0—4.$°. The sine of 45°
is 0.707, whence N. A.=n or 152 x sin u or 0.707=1.074.

By comparing these numerical apertures : Dry 0.799, water 0.972,
homogeneous immersion 1.074, tne same idea of the real light efficiency
and image power of the different objectives is obtained, as in the graphic
representations shown in Figs. 27-29.

If one knows the numerical aperture (N. A.) of an objective the
angular aperture is readily determined from the formula ; and one
can determine the equivalent angles of objectives used in different
media (i. e., dry or immersion). For example, suppose each of three
objectives has a numerical aperture (N. A.) of 0.80, what is the an-
gular aperture of each ? Using the formula of N. A.=w sin u, one has
N. A. = 0.80 for all the objectives.
For the dry objective n = i (Refractive index of air).

" water immersion objective 77=1.33 (Refractive index of water).
" homogeneous immersion objective n= 1.52 (Refractive index
of homogeneous liquid). And 2 u is to be found in each case.

For the dry objective, substituting the known values the formula
becomes 0.80= i sin u, or sin u = 0.80. By inspecting the table of
natural sines (3d page of cover) it will be found that 0.80 is the sine
of 53 degrees and 8 minutes. As this is half the angle the entire
angular aperture of the dry objective must be 53° 8'x 2 = 106° 16'.

For the water immersion objective, substituting the known values

in the formula as before : 0.80 = 1.33 sin u, or sin u= — = 0.6015.

* *3O

Consulting the table of sines as before, it will be found that 0.6015 is
the sine of 36° 59' whence the angular aperture (water angle) is 36°
59'X2 = 73058'.

For the homogeneous immersion objective, substituting the known
values, the formula becomes: 0.80 = 1.52 sin u whence sin *u =
0.80

1-52

= 0.5263. And by consulting the table of sines it will be found

then the angle sought must lie between 28° 45', and 29°. If the difference between
0.48481 and 0.48099 be obtained, 0.48481 — 0.48099 = 0.00382, and if this increase for
15' be divided by 15 it will give the increase for i minute ; 0.00382 -f- 15 =0.000254.
Now the difference between the sine whose angle is to be found and the next
lower sine is 0.48327—0.48099 = 0.00228. If this difference be divided by the
amount found necessary for i minute it will give the total minutes above 28° 45';
o. 002 28 -5- 0.000254 = 9. That is, the angle sought is 9 minutes greater than

\_CH. I

MICROSCOPE AND ACCESSORIES

that this is the sine of 31° 45^' whence 2 u or the entire angle (balsam
or oil angle) is 63° 31'.

That is, three objectives of equal resolving powers, each with a
numerical aperture of 0.80 would have an angular aperture of 106° 16'
in air, 73° 58' in water and 63° 31' in homogeneous immersion liquid.

For the apparatus and method of determining aperture, see
appendix.

§ 33. Table of a Group of Objectives with the Numerical
Aperture (N. A) and the method of obtaining it. Half the angular
aperture is designated by u and the index of refraction of the medium in
front of the objective by n. For dry objectives this is air and n = i, fot
water immersions n = f.jj, and for homogeneous immersions n = 1.
(For a table of natural sines, see third page of cover. )

OBJECTIVE.

!P

Index of

NATURAL SINE Refraction
of half the angular of the medi-
. ._ um in front
aperture oftheobjec-
(sinu. ) tive (n).

NUMERICAL APERTURE
(N. A.) =/zsin u

25 mm.

(Dry.)

20°

Sin — = 0.1736 j n = i N.A.=

i Xo. 1736 =0.173

25 mm.

(Dry.)

40°

40
Sin -^— = 0.3420 » — i

N.A.=

i X 0.3420 =0.342

12^2 mm.
(Dry.)

42°

Sin ^- = 0.3584 n=l

N.A.=

i Xo. 3583 =0.358

12^ mm.
(Dry.)

100°

Sin ^° = 0.7660 « = i N.A.=

i X 0.7660 = 0.766

6 mm.

(Dry.)

75°

Sin— - = 0.6087 n = i

N.A.=

i Xo. 6087 =0.609

6 mm.
(Dry.)

136°

Sin -*r = 0.9272 n = i

N. A.=

1X0.9272=0.927

3 mm.
(Dry.)

115°

Sin ^=0.8434 n = l

N. A.=

1X0.8434 = 0.843

3 mm.
(Dry.)

163°

Sin ^ = 0.9890 n=^1

N. A.=

1X0.9890 = 0.989

2 mm.
Water.
Immersion.

96° 1 2'

N. A.=i.33X 0.7443 = 0.99

oin — 0.7443 '* L'6o

2 mm.
Homogeneous
Immersion.

no°38/

-inIIO°38/ n^' *«!«

C5in — 0.0^^3 '* ^-o-^

2 mm.
Homogeneous
Immersion.

I34°io/

•

N. A.=

1.52X0.9210 = 1.40

20 MICROSCOPE AND ACCESSORIES \_CH. I

§ 34. Significance of Aperture. — As to the real significance of
aperture in microscopic objectives, it is now an accepted doctrine that —
the corrections in spherical and chromatic aberration being the same —
(i) Objectives vary directly as their numerical aperture in their ability
to define or make clearly visible minute details (resolving power). For
example an objective of 4 mm. equivalent focus and a numerical aper-
ture of 0.50 would define or resolve only half as many lines to the
millimeter or inch as a similar objective of i.oo N.A. So also an
objective of 2 mm. focus and 1.40 N.A. would resolve only twice as
many lines to the millimeter as a 4 mm. objective of 0.70 N.A. Thus
it is seen that defining power is not a result of magnification but of
aperture, otherwise the 2 mm. objective would resolve far more than
twice as many lines as the 4 mm. objective.

Taking the results of the researches of Abbe as a guide to visibility
with the microscope, one has the general formula 2\xN.A. That is
twice the number of wave lengths of the light used multiplied by the
numerical aperture of the objective. From this general statement it will
be seen that the shorter the wave lengths of the light, the more there
will be in an inch or centimeter and therefore the greater the number of
lines visible in a given space. That is the kind of light used is one ele-
ment and the objective the other in determining the number of lines
visible under the microscope.

Following Mr. E. M. Nelson (Jour. Roy. Micr. Soc., 1893, P- J5)
it is believed that not more than ^ths of the numerical aperture of an
objective is really available for microscopic study, with a central, solid
cone of light. To determine the number of lines visible in a given space
with a given light the formula would become 2\ X 3/^ths N.A. — 3/2/lN. A.
To determine the working-resolving power of any objective it is only
necessary to know the number of light waves in a given space, say an
inch or a centimeter and to multiply this number by 3/2 N. A. For
example suppose one uses ordinary daylight and assumes the average
wavelength is 1/46666 in., then there must be 46,666 per inch and
46,666x3/2 = 70,000 approximately. If the N. A. is i, then the
objective will resolve or make visible 70,000 lines to the inch, or ap-
proximately 28,000 to the centimeter. If blue light were used, the
number would be 32,000 per centimeter, or 80,000 per inch. It will
be seen that the number of lines here given is smaller than that in the
table of Carpenter-Dallinger, because in the latter the full aperture
is supposed to be employed and the light is of the greatest available
obliquity.

CH. /] MICROSCOPE AND ACCESSORIES 21

(2) The illuminating power of an objective of a given focus is
found to vary directly as the square of the numerical aperture (N. A. )2.
Thus if two 4 mm. objectives of N.A. 0.20 and N.A. 0.40 were compared
as to their illuminating power it would be found from the above that
they would vary as o.2o2:o.402= 0.0400:0.1600 or 1:4. That is the
objective of 0.20 N.A. would have but ^th the illuminating power of
the one of 0.40 N.A.

(3) The penetrating power, that is the power to see more than one

plane, is found to vary as the reciprocal of the numerical aperture

so that in an objective of a given focus the greater the aperture the
less the penetrating power.

Of course when equivalent focus and numerical aperture both differ
the problem becomes more complex.

While all microscopists are agreed that the fineness of detail which
can be seen depends directly on the numerical aperture of the objective
used, the general theory of microscopic vision has two interpretations :

(A.) That it is as with the unaided eye, the telescope and the
photographic camera. This is the original view and the one which many
are favoring at the present day (see Mercer, Proceedings of the Amer.
Micr. Soc. 1896, pp. 321-396).

(B) The other view originated with Professor Abbe, and in the
words of Carpenter-Dallinger, pp. 62, 43 : ''What this is becomes ex-
plicable by the researches of Abbe. It is demonstrated that micro-
scopic vision is sui generis. There is and can be, no comparison between
microscopic and macroscopic vision. The images of minute objects
are not delineated microscopically by means of the ordinary laws of
refraction ; they are not dioptrical results, but depend entirely on the
laws of diffraction. These come within the scope of and demonstrate
the undulatory theory of light, and involve a characteristic change
which material particles or fine structural details, in proportion to their
minuteness, effect in transmitted rays of light. The change consists
generally in the breaking up of an incident ray into a group of rays
with large angular dispersion within the range of which periodic alter-
nations of dark and light occur. ' '

For a consideration of the aperture question, its history and sig-
nificance, see J. D. Cox, Proc. Amer. Micr. Soc., 1884, pp. 5-39 ;
Jour. Roy. Micr. Soc., 1881, pp. 303, 348, 365, 388 ; 1882, pp. 300,
460 ; 1883, p. 790 ; 1884, p. 20 ; 1896, p. 681 ; 1897, p. 71 ; 1898, pp.
354, 362, 592 ; Mercer, Proceedings Amer. Micr. Soc., 1896, pp. 321-

22

MICROSCOPE AND ACCESSORIES

[CH. I

396 ; Lewis Wright, Philos. Mag., June, 1898, pp. 480-503 ; Carpen-
ter-Dallinger, Chapter II ; Nelson, Jour. Quekett Micr. Club, VI, pp.
14-38.

THE OCULAR

g 35. A Microscopic Ocular or Eye-Piece consists of one or more converging
lenses or lens systems, the combined action of which is, like that of a simple
microscope, to magnify the real image formed by the objective.

FIG. 30. Sectional view of a Huygenian ocular to show
the formation of the Eye-Point.

Axis .Optic axis of the ocular. D. Diaphragm of the
ocular. E. L. Eye-Lens. F. L. Field-Lens.

E. P. Eye-point. As seen in section, it appears some-
thing like an hour-glass. When seen as looking into the
ocular, i. <?., in transection, it appears as a circle of light. It
is at the point where the most rays cross.

Depending upon the relation and action of the different
lenses forming oculars, they are divided into two great
groups, negative and. positive.

36. Negative Oculars are those in which the real, inverted image is formed
within the ocular, the lower or field-lens serving to collect the image-forming rays
somewhat, so that the real image is smaller than as if the field-lens were absent
(Fig. 21 ). As the field-lens of the ocular aids in the formation of the real image
it is considered by some to form a part of the objective rather than of the ocular.
The upper or eye-lens of the ocular magnifies the real image.

§ 37. Positive Oculars are those in which the real, inverted image of the
object is formed outside the ocular, and the entire system of ocular lenses magnifies
the real image like a simple microscope (Fig. 16).

Positive and negative oculars may be readily distinguished, as a positive ocular
may be used as a simple microscope, while a negative ocular cannot be so used
when its field-lens is in the natural position toward the object. By turning the
eye-lens toward the object and looking into the field-lens an image may be seen,
however.

In works and catalogs concerning the microscope and microscopic apparatus,
and in articles upon the microscope in periodicals, various forms of oculars or eye-
pieces are so frequently mentioned, without explanation or definition, that it
seems worth while to give a list, with the French and German equivalents, and a
brief statement of their character.

Achromatic Ocular ; Fr. Oculaire achromatique ; Ger. achromatisches Okular.
Oculars in which chromatic aberration is wholly or nearly eliminated. — Aplanatic
Ocular ; Fr. Oculaire aplanatique ; Ger. aplanatisches Okular (see \ 21). — Binocu-
lar, stereoscopic Ocular ; Fr. Oculaire binoculaire stereoscopique ; Ger. stereosko-
pisches Doppel-Okular. An ocular consisting of two oculars about as far apart as
the two eyes. These are connected with a single tube which fits a monocular mi-
croscope. By an arrangement of prisms the image forming rays are divided, half

CH. /] MICROSCOPE AND ACCESSORIES 23

being sent to each eye. The most satisfactory form was worked out by Tolles and
is constructed on true stereotomic principles, both fields being equally illuminated.
His ocular is also erecting. — Campani^s Ocular (see Huygenian Ocular). — Com-
pound Ocular ; Fr. Oculaire compose ; Ger. zusammengesetztes Okular. An ocu-
lar of two or more lenses, e. g., the Huygenian (see Fig. 30). — Continental Ocular.
An ocular mounted in a tube of uniform diameter as in Fig. ^i.—Deep Ocular,
see high ocular. — Erecting Ocular ; Fr. Oculaire redresseur ; Ger. bildumkeh-
rendes Okular. An ocular with which an erecting prism is connected so that the
image is erect as with the simple microscope. Such oculars are most common on
dissecting microscopes. — Filar micrometer Ocular; Screw m. o., Cobweb m. o.,
Ger. Okular-Schraubenmikrometer. A modification of Ramsden's Telescopic Cob-
web micrometer ocular. — Goniometer Ocular ; Fr. Oculaire a goniometre ; Ger.
Goniometer-Okular. An ocular with goniometer for measuring the angles of minute
crystals. — High Ocular, sometimes called a deep ocular. One that magnifies
the real image considerably, i. e., 10 to 20 fold. — Huygenian Ocular, Huygens' O.,
Campani's O., Airy's O.; Fr. Oculaire d'Huygens, o. de Campani ; Ger. Huy-
gens'sches Okular, Campaniches Okular, see \ 38. — Index Ocular ; Ger. Spitzen-
O. An ocular with a minute pointer or two pointers at the level of the real image.
The points are movable and serve for indicators and also, although not satisfac-
torily, for micrometry. — Kellner's Ocular, see orthoscopic ocular — Low ocular,
also called shallow ocular. An ocular which magnifies the real image only moder-
ately, i. e. , 2 to 8 fold. — Micrometer or micrometric Ocular ; Fr. Oculaire microme-
trique ou a micrometre ; Ger. Mikrometer-Okular, Mess Okular, Beneches O.,
Jackson m. o., see \ 41. — Microscopic Ocular ; Fr. Oculaire microscopique ; Ger. mi-
kroskopisches Okular. An ocular for the microscope instead of one for a telescope.
— Negative Ocular, see \ 36. — Nelson's screw-micrometer ocular. A modification of
the Ramsden's screw or cob-web micrometer in which positive compensating ocu-
lars may be used. — Orthoscopic Oculars; also called Kellner's Ocular ; Fr. Ocu-
laire orthoscopique ; Ger. Kellner'sches oder orthoskopisches Okular. An ocular
with an eye-lens like one of the combinations of an objective (Figs. 22, 23) and a
double convex field lens. The field-lens is in the focus of the eye-lens and there
is no diaphragm present. The field is large and flat. — Par-focal Oculars, a series
of oculars so arranged that the microscope remains in focus when the oculars are
interchanged (Pennock, Micr. Bulletin, vol. iii, p. 9, 31). — Periscopic Ocular ; Fr.
Oculaire periscopique ; Ger. periskopisches Okular. A positive ocular devised by
Gundlach. It consists of a double convex field-lens and a triplet eye-lens. It
gives a large, flat field. — Positive Ocular, see \ 37. — Projection Ocular ; Fr. Ocu-
laire de projection ; Ger. Projections-Okular, see \ 40. — Ramsden's Ocular ; Fr.
Oculaire de Ramsden ; Ger. Ramsden'sches Okular. A positive ocular devised by
Ramsden. It consists of two plano-convex lenses placed close together with the
convex surfaces facing each other. Only the central part of the field is clear.
Searching Ocular ; Fr. Oculaire d'orientation ; Ger. Sucher-Okular, see § 39,
Shallow Ocular, see low ocular. — Solid Ocular, holosteric O. ; Fr. Oculaire holo-
stere ; Ger. holosterisches Okular, Vollglass-Okular. A negative eye-piece de-
vised by Tolles. It consists of a solid piece of glass with a moderate curvature at
one end for a field-lens, and the other end with a much greater curvature for an
eye-lens. For a diaphragm, a groove is cut at the proper level and filled with
black pigment. It is especially excellent where a high ocular is desired. — Spectral

24 MICROSCOPE AND ACCESSORIES {CH. I

or spectroscopic Ocular ; Fr. Oculaire spectroscopique ; Ger, Spectral-Okular, see
Microspectroscope, Ch. VI. — Statiroscopic Ocular ; Fr. Oculaire Stauroscopique.
Ger. Stauroskop-Okular. An ocular with a Bertrand's quartz plate for mineralog-
ical purposes. — Working Ocular; Fr. Oculaire de travail; Ger. Arbeits-Okular,
see § 39.

g 38. Huygenian Ocular — A negative ocular designed by Huygens for the
telescope, but adapted also to the microscope. It is the one now most commonly
employed. It consists of a field-lens or collective (Fig. 30), aiding the objective
in forming the real image, and an eye-lens which magnifies the real image. While

Ocular lo. 2

FIG. 31. Compensating Oculars of Zeiss, with section removed to show the con-
struction. The line A- A is at the level of the upper end of the tube of the micro-
scope while B-B represents the lower focal points. It will be seen that the mount-
ing is so arranged that the lower focal points in all are in the same plane and
therefore the microscope remains in focus upon changing oculars. ( The oculars are
par-focal}. The lower oculars, 2, 4 and 6 are negative, and the higher ones, 8, 12,
18, are positive. The numbers 2, 4, 6, 8, 12, 18, indicate the magnification of the
ocular. From Zeiss' Catalog. )

the field-lens aids- the objective in the formation of the real, inverted image, and
increases the field of view, it also combines with the eye-lens in rendering the
image achromatic. (See $46).

$ 39. Compensating Oculars. — These are oculars specially constructed for
use with the apochromatic objectives. They compensate for aberrations outside
the axis which could not be so readily eliminated in the objective itself. An ocu-
lar of this kind, magnifying but twice, is made for use with high powers, for the
sake of the large field in finding objects ; it is called a searching ocular ; those
ordinarily used for observation are in contradistinction called working oculars.
Part of the compensating oculars are positive and part negative. (Fig. 31. )

\ 40. Projection Oculars.— These are oculars especially designed for project-
ing a microscopic image on the screen for class demonstrations, or for photo-
graphing with the microscope. While they are specially adapted for use with
apochromatic objectives, they may also be used, with ordinary achromatic
objectives of large numerical aperture.

CH. /]

MICROSCOPE AND ACCESSORIES

FIG. 32. Projection Oculars with section re-
moved to show the construction. Below are
shown the upper ends with graduated circle to
indicate the amount of rotation found necessary
to focus the diaphragm on the screen. No. 2,
No. 4. The numbers indicate the amount the
ocular magnifies the image formed by the
objective as with the compensation oculars.
(Zeis? Catalog.}

| 41. Micrometer Ocular. — This is an
ocular connected with an ocular micrometer.
The micrometer may be removable, or it may
be permanently in connection with the ocular,
and arranged with a spring and screw, by which
it may be moved back and forth across the
field. (SeeCh. IV.)

No. 2.

FIG. 33 FIG. 34

FIGS. 33-34- Ocular Micrometer and movable scale. Fig. 33 is a side view of
the ocular while Fig. 34 gives a sectional end view, and shows the ocular micrometer
in position. In both the screw which moves the micrometer is shown at the left.
( From Bausch & Lomb Opt. Co. )

I 42. Spectral or Spectroscopic Ocular.— (See Micro-Spectroscope, Ch. VI).

DESIGNATION OF OCULARS

\ 43. Equivalent Focus. — As with objectives, some opticians designate the
oculars by their equivalent focus ( \ 15 ). With this method the power of the ocular,
as with objectives, varies inversely as the equivalent focal length, and therefore
the greater the equivalent focal length the less the magnification. This seems as
desirable a mode for oculars as for objectives and is coming more and more into
use by the most progressive opticians. It is the method of designation advo-
cated by Dr. R. H. Ward for many years, and was recommended by the committee
of the American Microscopical Society, (Proc. Amer. Micr. Soc., 1883, p. 175, 1884,
p. 228).

26

MICROSCOPE AND ACCESSORIES

\_CH.I

FIG. 35. Ocular Screw- Micrometer with
compensation ocular 6. The upper figure
shows a sectional view of the ocular and the
screw for moving the micrometer at the right.
At the left is shown a clamping screw to
fasten the ocular to the upper part of the mi-
croscope tube. Below is a face view, showing
the graduation on the wheel. An ocular
micrometer like this is in general like the
cob-web micrometer and may be used for
measuring objects of varying sizes very accu-
rately. With the ordinary ocular micrometer
very small objects frequently fill but a part of
an interval of the micrometer, but with this
the movable cross lines traverse the object (or
rather its real image] regardless of the minute-
ness of the object. (Zeiss^ Catalog}.

\ 44. Numbering and Lettering. — Oculars like objectives may be numbered or
lettered arbitrarily. When so designated, the smaller the number, or the earlier
the letter in the alphabet, the lower the power of the ocular.

$ 45. Magnification. — The compensating oculars are marked with the amount
they magnify the real image. Thus an ocular marked X 4, indicates that the real
image of the objective is magnified four fold by the ocular.

The projection oculars are designated simply by the amount they multiply the
real image of the objective. Thus for the short or 160 mm. tube-length they are,
X2, X4 ; and for the long or 250 mm. tube, they are X3 and X6. That is, the
final image on the screen or the ground glass of the photographic camera will be
2, 3, 4, or 6 times greater than it would be if no ocular were used. See Ch. VIII.
§ 46. Standard Size Oculars. — The Royal Microscopical Society of London
took a very important step (Dec. 20, 1899) in establishing standard sizes for ocu-
lars and sub-stage condensers. To quote from the Journal of the Royal Micro-
scopical Society for 1900, p. 147 :

Resolved, "That the standard size for the inside diameter of the substage fit-
ting be 1.527 in. =38. 786 mm. That the gauges for standardizing eye-pieces be
the internal diameters of the draw-tubes, the tightness of the fit being left to the
discretion of the manufacturers. ' '

The sizes for oculars are four in number, I and 2 being most common.
( i ) 0.9173 inch = 23.300 mm. This is the Continental size.

This is the size used by the English Opticians

for student and small microscopes.
Medium size binoculars (English.)
Long tube binoculars.
For the history of the Huygenian Ocular, and a discussion of formulae for its
construction, see Nelson, J. R. M. S., 1900, p. 162-169.

EXPERIMENTS

§ 47. Putting an Objective in Position and Removing it. —
Elevate the tube of the microscope by means of the coarse adjustment

(2) 1.04 inch = 26.416 mm.

(3)
(4)

1.27
1.41

inch =32.258 mm.
= 35.814 mm.

CH. /] MICROSCOPE AND ACCESSORIES 27

(frontispiece) so that there may be plenty of room between its lower
end and the stage. Grasp the objective lightly near its lower end with
two fingers of the left hand, and hold it against the nut at the lower
end of the tube. With two fingers of the right hand take hold of the
milled ring near the back or upper end of the objective and screw it
into the tube of the microscope. Reverse this operation for removing
the objective. By following this method the danger of dropping the
objective will be avoided.

§ 48. Putting an Ocular in Position and Removing it. — Ele-
vate the body of the microscope with the coarse adjustment so that the
objective will be 2 cm. or more from the object — grasp the ocular by
the milled ring next the eye-lens (Fig. 21), and the coarse adjustment
or the tube of the microscope and gently force the ocular into position.
In removing the ocular, reverse the operation. If the above precau-
tions are not taken, and the oculars fit snugly, there is danger in in-
serting them of forcing the tube of the microscope downward and the
objective upon the object.

§ 49. Putting an Object under the Microscope. — This is so
placing an object under the simple microscope, or on the stage of the
compound microscope, that it will be in the field of view when the
microscope is in focus (§ 50).

With low powers, it is not difficult to get an object under the
microscope. The difficulty increases, however, with the power of the
microscope and the smallness of the object. It is usually necessary to
move the object in various directions while looking into the micro-
scope, in order to get it into the field. Time is usually saved by get-
ting the object in the center of the field with a low objective before
putting the high objective in position. This is greatly facilitated by
using a nose-piece, or revolver. (See Figs. 36-363, and the pictures of
microscopes, Ch. II.)

FIG. 36. Triple nose-piece or revol- FIG. 363. Triple nose-piece or re-

ver for quickly changing objectives. ( The volver for quickly changing objectives.
Spencer Lens Co. ) ( The Bausch & Lomb Optical Co. )

28 MICROSCOPE AND ACCESSORIES [CH. I

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

In general, the greater the magnification of the entire microscope,
whether the magnification is produced mainly by the objective, the
ocular, or by increasing the tube length, or by a combination of all
three (see Ch. IV, under magnification), the smaller is the field.

The size of the field is also dependent, in part, without regard to
magnification, upon the size of the opening in the ocular diaphragm.
Some oculars, as the orthoscopic and periscopic, are so constructed as
to eliminate the ocular diaphragm, and in consequence, although this
is not the sole cause, the field is considerably increased. The exact
size of the field may be read off directly by putting a stage micrometer
under the microscope and noting the number of spaces required to
measure the diameter of the light circle.

4 5

17

85 mm

FIG. 37. Figures showing approximately the actual size of the field with ob-
jectives 0/85 mm., 45 mm., 77 mm., 3 mm., and 2 mm., equivalent focus, and
ocular of 3jY2 mm., equivalent focus in each case. This figure shows graphically
what is also very clearly indicated in the table ( \ 52}.

§ 51. The size of the field of the microscope as projected into the
field of vision of the normal human eye (i. e., the virtual image) may
be determined by the use of the camera lucida with the drawing surface
placed at the standard distance of 250 millimeters (Ch. IV.)

§ 52. Table showing the actual size in millimeters of the field of a
group of commonly used objectives and oculars. Compare with the graphic
representation in Fig. 37. See also § 50.

CH.r\

MICROSCOPE AND ACCESSORIES

29

Equivalent i Diameter
**»*•$* ! of Field
Objective in mm.

Equivalent
Focus of
Ocular

Kind of
Ocular

15-4
85 mm j 10.6

37 X mm.
25 "

Huygenian

! 8'3

I2tf "'

7-o
45 mm 5.0
4.0

37 yz mm.
i2>£ "

Huygenian

3-o
17 mm. 2.0

37>£ mm.
25

Huygenian

1.6

J« ••

5-7

N. A. =0.25 J;4

0.97

180 mm.
45 "

;s "

Compensation

0.541

5 mm. °-37i

37^ mm.
25

Huygenian

0.290

12^ "

0.850

N. A. = 0.92 °;^°0
0.173

180 mm.
45 "
15 "

10

Compensation

0.270
2 mm al86

37^ mm.
25 "

Huygenian

0.147

12^ '«

0.450
0.251
N. A. = 1.25 1 0.125

0.088

j

180 mm.
45
15

10

Compensation

FUNCTION OF AN OBJECTIVE

§ 53. Put a 50 mm. objective on the microscope or screw off the
front combination of a 16 mm.,(^-in.), and put the back combination
on the microscope for a low objective.

Place some printed letters or figures under the microscope, and
light well. In place of an ocular put a screen of ground glass, or a
piece of lens paper, over the upper end of the tube of the microscope.*

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

30 MICROSCOPE AND ACCESSORIES \_CH. I

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

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

The letters will appear as if printed on the ground glass or paper,
but will be inverted ( Fig. 21).

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

To demonstrate that the object must be outside the principal focus
with the compound microscope, remove the screen and turn the tube of
the microscope directly toward the sun. Move the tube of the micro-
scope with the coarse adjustment until the burning or focal point is
found (§ 6). Measure the distance from the paper object on the stage
to the objective, and it will represent approximately the principal
focal distance (Figs. 10, n). Replace the screen over the top of the
tube, no image can be seen. Slowly raise the tube of the microscope
and the image will finally appear. If the distance between the object
and the objective is now taken, it will be found considerably greater
than the principal focal distance (compare § n).

§ 54. Aerial Image. — After seeing the real image on the ground-
glass, or paper, use the lens paper over about half of the opening of
the tube of the microscope. Hold the eye about 250 mm. from the
microscope as before and shade the top of the tube by holding the hand
between it and the light, or in some other way. The real image can
be seen in part as if on the paper and in part in the air. Move the
paper so that the image of half a letter will be on the paper and half
in the air. Another striking experiment is to have a small hole in the
paper placed over the center of the tube opening, then if a printed word
extends entirely across the diameter of the tube its central part may be
seen in the air, the lateral parts on the paper. The advantage of the
paper over part of the opening is to enable one to accommodate the

CH. /] MICROSCOPE AND ACCESSORIES 31

eyes for the right distance. If the paper is absent the eyes adjust
themselves for the light circle at the back of the objective, and the
aerial image appears low in the tube. Furthermore it is more difficult
to see the aerial image in space than to see the image on the ground-
glass or paper, for the eye must be held in the right position to receive
the rays projected from the real image, while the granular surface of
the glass and the delicate fibres of the paper reflect the rays irregularly,
so that the image may be seen at almost any angle, as if the letters
were actually printed on the paper or glass.

§ 55. The Function of an Objective, as seen from these experi-
ments, is to form an enlarged, inverted, real image of an object, this
image being formed on the opposite side of the objective from the
object (Fig. 21).

FUNCTION OF AN OCULAR

§ 56. Using the same objective as for § 53, get as clear an image
of the letters as possible on the lens paper screen. Look at the image
with a simple microscope (Fig. 17 or 1 8) as if the image were an object.

Observe that the image seen through the simple microscope is
merely an enlargement of the one on the screen, and that the letters
remain inverted, that is they appear as with the naked eye (§ n).
Remove the screen and observe the aerial image with the tripod.

Put a 50 mm. (A, No. i or 2 in.), ocular i. e., an ocular of low
magnification) in position (§ 48). Hold the eye about 10 to 20 milli-
meters from the eye-lens and look into the microscope. The letters
will appear as when the simple microscope was used (see above), the
image will become more distinct by slightly raising the tube of the
microscope with the coarse adjustment.

§ 57. The Function of the Ocular, as seen from the above, is
that of a simple microscope, viz. : It magnifies the real image formed
by the objective as if that image were an object. Compare the image
formed by the ocular (Fig. 21), and that formed by a simple microscope

(Fig- 38).

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

MICROSCOPE AND ACCESSORIES

\CH. I

in order to see the aerial image with the unaided eye, the simple micro-
scope, ocular or eye must be in the path of the rays (Fig. 21.)

FIG. 38. Diagram of the simple microscope
showing the course of the rays and all the images,
and that the eye forms an integral part of it.

A1 B*. The object within the principal focus.
A* B*. The virtual image on the same side of
the lens as the object. It is indicated by dotted
lines, as it has no actual existence.

B2 A2. Retinal image of the object (A1 S1}.
The virtual image is simply a projection of the
retinal image into the field of vision.

Axis. The principal optic axis of the micro-
scope and of the eye. Cr. Cornea of the eye. L.
Crystalline lens of the eye. R. Ideal refracting
surface at which all the refractions of the eye may
be assumed to take place. .5*. .- , .^ 3

§ 58. The field-lens of a Huygenian ocular makes the real
image smaller and consequently increases the size of the field ; it also
makes the image brighter by contracting the area of the real image.
(Fig. 30.) Demonstrate this by screwing off the field-lens and using
the eye-lens alone as an ocular, refocusing if necessary. Note also that
the image is bordered by a colored haze (§7).

When looking into the ocular with the field-lens removed, the eye
should not be held so close to the ocular, as the eye-point is consider-
ably farther away than when the field-lens is in place.

§ 59. The eye-point. — This is the point above the ocular or
simple microscope where the greatest number of emerging rays cross.
Seen in profile, it may be likened to the narrowest part of an hour
glass. Seen in section (Fig. 30), it is the smallest and brightest
light circle above the ocular. This is called the eye-point, for if the
pupil of the eye is placed at this level, it will receive the greatest
number of rays from the microscope, and consequently see the largest
field.

Demonstrate the eye-point by having in position an objective and
ocular as above (§ 53). Light the object brightly, focus the micro-
scope, shade the ocular, then hold some ground-glass or a piece of the
lens paper above the ocular and slowly raise and lower it until the
smallest circle of light is found. By using different oculars it will be

CH. /.] MICROSCOPE AND ACCESSORIES 33

seen that the eye-point is nearer the eye-lens in high than in low ocu-
lars, that is the eye-point is nearer the eye-lens for an ocular of small
equivalent focus than for one of greater focal length.

REFERENCES FOR CHAPTER I

In chapter X will be given a bibliography, with full titles, of the works and
periodicals referred to.

For the subjects considered in this chapter, general works on the microscope
may be consulted with great advantage for different or more exhaustive treatment.
The most satisfactory work in English is Carpenter-Dallinger, 8th Ed. For the
history of the microscope, MayaH's Cantor Lectures on the microscope are very
satisfactory. For a continuation of the history begun by Mayall in the Cantor
Lectures see Nelson, Journal of the Queckett Micr. Club, and the Jour. Roy.
Micr. Soc., 1897-1901 — . Carpenter-Dallinger, 8th Ed. Petri, Das Mikroskop.

The following special articles in periodicals may be examined with advantage :

Apochromatic Objectives, etc. Dippel in Zeit. wiss. Mikr., 1886, p. 303 ; also
in the Jour. Roy. Micr. Soc., 1886, pp. 316, 849, mo, ; same, 1890, p. 480 ; Zeit. f..
Instrumentenk. , 1890, pp. 1-6 ; Micr. Built., 1891, pp. 6-7.

Tube-length, etc. Gage, Proc. Amer. Soc. Micrs., 1887, pp. 168-172 ; also in
the Microscope, the Jour. Roy. Micr. Soc., and in Zeit, wiss. Mikr., 1887-8.
Bausch, Proc. Amer. Soc. Micrs., 1890, pp. 43-49 ; also in the Microscope, 1890,
pp. 289-296.

Aperture. J. D. Cox, Presidential Address, Proc. Amer. Soc. Micrs., 1884, pp,
5-39, Jour. Roy. Micr. Soc., iSSi, pp. 303, 348, 365, 388 ; 1882, pp. 300, 460 ; 1883,
p. 790 ; 1884, p. 20. Czapski, Theorie der optischen Instrumente nach Abbe.
See also references in \ 34.

The Barnes Dissecting Microscope ( The Bausch & Lomb Optical Company).

CHAPTER II

LIGHTING AND FOCUSING; MANIPULATION OF DRY,

ADJUSTABLE AND IMMERSION OBJECTIVES ; CARE

OF THE MICROSCOPE AND OF THE EYES ;

LABORATORY MICROSCOPES

APPARATUS AND MATERIAL FOR THIS CHAPTER

Microscope supplied with plane and concave mirror, Achromatic and Abbe
condensers, dry, adjustable and immersion objectives, oculars, triple nose-piece.
Microscope lamp and movable condenser (bull's eye or other form, Fig. 53),
Homogeneous immersion liquid ; Benzin, alcohol, distilled water ; Mounted prep-
aration of fly's wing($ 70); Mounted preparation of Pleurosigma ($ 77, 78). Stage
or ocular micrometer (\ 92); Glass slides and cover-glasses (Ch. VII); 10 per ct.
solution of salicylic acid in 95 per ct. alcohol (\ 92); Preparation of stained bac-
teria ($ 108); Vial of equal parts olive or cotton seed oil or liquid vaselin and
benzin (\ 112): Double eye shade (Fig. 60); Screen for whole microscope (Fig. 59).

FOCUSING

$ 60. Focusing is mutually arranging an object and the microscope so that a
clear image may be seen.

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

In general, the higher the power of the whole microscope whether simple or
compound, the nearer together must the object and objective be brought. With
the compound microscope, the higher the objective, and the longer the tube of
the microscope, the nearer together must the object and the objective be brought.
If the oculars are not par-focal, the higher the magnification of the ocular, the
nearer must the object and objective be brought.

$ 61. Working Distance. — By this is meant the space between the simple mi-
croscope and the object, or between the front lens of the compound microscope and
the object, when the microscope is in focus. This working distance is alwa3?s con-
siderably less than the equivalent focal length of the objective. For example,
the front-lens of a 6 mm. or %ih in. objective would not be 6 millimeters or j^th
inch from the object when the microscope is -in focus, but considerably less than
that distance. If there were no other reason than the limited working distance of
high objectives, it would be necessary to use a very thin cover-glass over the

CH. 77]

LIGHTING AND FOCUSING

35

object. (See \ 24, 29. ) If too thick covers are used it may be impossible to get
an objective near enough an object to get it in focus. For objects that admit of
examination with high powers it is always better to use thin covers.

LIGHTING WITH DAYLIGHT

\ 62. Unmodified sunlight should not be employed except in special cases.
North light is best and most uniform. When the sky is covered with white clouds
the light is most favorable. To avoid the shadows produced by the hands in
manipulating the mirror, etc. , it is better to face the light ; but to protect the
eyes and to shade the stage of the microscope some kind of screen should be
used. The one figured in (Fig. 62) is cheap and efficient. If one dislikes to face
the window or lamp it is better to sit so that the light will come from the left as in
reading.

It is of the greatest importance and advantage for one who is to use the
microscope for serious work that he should comprehend and appreciate thoroughly
the various methods of illumination, and the special appearances due to different
kinds of illumination.

Depending on -whether the light illuminating an object traverses the object or
is reflected upon it, and also whether the object is symmetricallj* lighted, or
lighted more on one side than the other, light used in microscopy is designated as
reflected and transmitted, axial and oblique.

39
FIGS. 39-40. For full explanation see Figs. 22 and 23.

\ 63. Reflected, Incident or Direct Light. — By this is meant light reflected
upon the object in some way and then irregularly reflected from the object to the
microscope. By this kind of light objects are ordinarily seen by the unaided eye,
and the objects are mostly opaque. In Vertebrate Histology, reflected light is but

36 LIGHTING AND FOCUSING \_CH. II

little used ; but in the study of opaque objects, like whole insects, etc., it is used
a great deal. For low powers, ordinary daylight that naturally falls upon the
object, OF is reflected or condensed upon it with a mirror or condensing lens,
answers very well. For high powers and for special purposes, special illuminating
apparatus has been devised (g 28). (See also Carpenter-Dallinger, Ch. IV).

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

Almost all objects studied in Vertebrate Histology are lighted by transmitted
light, and they are in some way rendered transparent or semi-transparent. The
light traversing and serving to illuminate the object in working with a compound
microscope is usually reflected from a plane or concave mirror, or from a mirror to
a condenser (| 88), and thence transmitted to the object from below (Figs. 48-51).

\ 65. Axial or Central Light. — By this is understood light reaching the object,
the rays of light being parallel to each other and to the optic axis of the micro-
scope, or a diverging or converging cone of light whose axial ray is coincident with
the optic axis of the microscope. In either case the object is symmetrically
illuminated.

\ 66. Oblique Light. — This is light in which parallel rays from a plane mirror
form an angle with the optic axis of the microscope (Fig. 40). Or if a concave
mirror or a condenser is used, the light is oblique when the axial ray of the cone
of light forms an angle with the optic axis (Fig. 40).

DIAPHRAGMS

$ 67. Diaphragms and their Proper Employment. — Diaphragms are opaque
disks with openings of various sizes, which are placed between the source of light
or mirror and the object. In some cases an iris diaphragm is used, and then the
same one is capable of giving a large range of openings. The object of a dia-
phragm in general, is to cut off all adventitious light and thus enable one to light
the object in such a way that the light finally reaching the microscope shall all
come from the object or its immediate vicinity. The diaphragms of a condenser
serve to vary its aperture to the needs of each object and each objective.

$ 68. Size and Position of Diaphragm Opening. — When no condenser is used
the size of the opening in the diaphragm should be about that of the front lens
of the objective. For some objects and some objectives this rule may be quite
widely departed from ; one must learn by trial.

When lighting with a mirror the diaphragm should be as close as possible to
the object in order, (a) that it may exclude all adventitious light from the object ;
(b ) that it may not interfere with the most efficient illumination from the mirror
by cutting off a part of the illuminating pencil. If the diaphragm is a considera-
ble distance below the object, ( i ) it allows considerable adventitious light to reach
the object and thus injures the distinctness of the microscope image ; (2) it pre-
vents the use of very oblique light unless it swings with the mirror ; ( 3 ) it cuts off
a part of the illuminating cone from a concave mirror. On the other hand, even
with a small diaphragm, the whole field will be lighted.

CH. //] LIGHTING AND FOCUSING 37

With an illuminator or condenser (Figs. 41, 48) , the diaphragm serves to narrow
the pencil to be transmitted through the condenser, and thus to limit the aperture
(see \ 84). Furthermore, by making the diaphragm opening eccentric, oblique
light may be used, or by using a diaphragm with a slit around the edge ( central
stop diaphragm), the center remaining opaque, the object may be lighted with a
hollow cone of light, all of the rays having great obliquity. In this way the so-
called dark -ground illumination may be produced (| 92 ; Fig. 51).

ARTIFICIAL ILLUMINATION

| 69. For evening work and for certain special purposes, artificial illumina-
tion is employed. A good petroleum (kerosene) lamp with flat wick has been
found very satisfactory, also an incandescent electric or Welsbach light, but for
brilliancy and for the actinic power necessary for very rapid photo-micrography
(see Ch. VIII) the electric arc lamp or an acetylene lamp serves well. Whatever
source of artificial light is employed, the light should be brilliant and steady.

LIGHTING AND FOCUSING I EXPERIMENTS

§ 70. Lighting with a Mirror. — As the following experiments
are for mirror lighting only, remove the substage condenser if present
(see § 79, for condenser). Place a mounted fly's wing under the
microscope, put the 16 mm.(^j in.) or other low objective in position,
also a low ocular. With the coarse adjustment lower the tube of the
microscope to within about i cm. of the object. Use an opening in
the diaphragm about as large as the front lens of the objective ;
then with the plane mirror try to reflect light up through the diaphragm
upon the object. One can tell when the field (§ 50) is illuminated, by
looking at the object on the stage, but more satisfactorily by looking
into the microscope. It sometimes requires considerable manipulation
to light the field well. After using the plane side of the mirror turn
the concave side into position and light the field with it. As the con-
cave mirror condenses the light, the field will look brighter with it than
with the plane mirror. It is especially desirable to remember that the
excellence of lighting depends in part on the position of the diaphragm
(§ 68). If the greatest illumination is to be obtained from the concave
mirror, its position must be such that its focus will be at the level of
the object. This distance can be very easily determined by finding the
focal point of the mirror in full sunlight.

§ 71. Use of the Plane and of the Concave Mirror. — The mir-
ror should be freely movable, and have a plane and a concave face. The
concave face is used when a large amount of light is needed, the plane
face wrhen a moderate amount is needed or when it is necessary to have
parallel rays or to know the direction of the rays.

38 LIGHTING AND FOCUSING [CH. II

§ 72. Focusing with Low Objectives. — Place a mounted fly's
wing under the microscope ; put the 16 mm. (^i in.) objective in
position, and also the lowest ocular. Select the proper opening in the
diaphragm and light the object well with transmitted light (§ 64, 68).

Hold the head at about the level of the stage, look toward the
window, and between the object and the front of the objective ; with
the coarse adjustment lower the tube until the objective is within
about half a centimeter of the object. Then look into the microscope
and slowly elevate the tube with the coarse adjustment. The image
will appear dimly at first, but will become very distinct by raising the
tube still higher. If the tube is raised too high the image will become
indistinct, and finally disappear. It will again appear if the tube is
lowered the proper distance.

When the microscope is well focused try both the concave and the
plane mirrors in various positions and note the effect. Put a high
ocular in place of the low one (§43). If the oculars are not par-
focal it will be necessary to lower the tube somewhat to get the micro-
scope in focus.*

Pull out the draw-tube 4-6 cm., thus lengthening the body of the
microscope ; it will be found necessary to lower the tube of the micro-
scope somewhat. (For reason, see Fig. 58.)

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

§ 74. Focusing with High Objectives. — Employ the same
object as before, elevate the tube of the microscope and, if no revolving
nose-piece is present, remove the 16 mm. (^ in.) objective as indi-
cated. Put the 3 mm.(^ in.) or a higher objective in place, and use
a low ocular.

*Par-focal oculars are so constructed, or so mounted, that those of different
powers may be interchanged without the microscopic image becoming wholly out
of focus (Fig. 31). When high objectives are used, while the image may be
seen after changing oculars, the instrument nearly always needs slight focusing.
With low powers this may not be necessary.

Objectives are also now commonly mounted in the triple or double revolving
nose-pieces (.Figs. 36, 36 a) so that if one of the objectives is in focus either of the
others will be approximately in focus when turned into position. This is a very
great convenience.

CH. //] LIGHTING AND FOCUSING 39

Light well, and employ the proper opening in the diaphragm, etc.
(§ 68). Look between the front of the objective and the object as
before (§72), and lower the tube with the coarse adjustment till the
objective almost touches the cover-glass over the object. Look into
the microscope, and with the coarse adjustment, raise the tube very
slowly until the image begins to appear, then turn the milled head of
the fine adjustment (frontispiece), first one way and then the other, if
necessary, until the image is sharply defined.

In practice it is found of great advantage to move the preparation
slightly while focusing. This enables one to determine the approach
to the focal point either from the shadow or the color, if the object is col-
ored. With high powers and scattered objects there might be no object
in the small field (see § 50, Fig. 37 for size of field). By moving the
preparation an object will be moved across the field and its shadow
gives one the hint that the objective is approaching the focal point. It
is sometimes desirable to focus on the edge of the cement ring or on
the little ring made by the marker (see Figs. 61-66).

Note that this high objective must be brought nearer the object
than the low one, and that by changing to a higher ocular (if the ocu-
lars are not par-focal) or lengthening the tube of the microscope it
will be found necessary to bring the objective still nearer the object, as
with the low objective. (For reason see Fig. 58.)

§ 75. Always Focus Up, as directed above. If one lowers the
tube only when looking at the end of the objective as directed above,
there will be no danger of bringing the objective in contact with the
object, as may be done if one looks into the microscope and focuses
down.

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

§ 76. Determination of Working Distance. — As stated in § 61,
this is the distance between the front lens of the objective and the
object when the objective is in focus. It is always less than the equiv-
lent focal length of the objective.

Make a wooden wedge 10 cm. long which shall be exceedingly thin
at one end and about 20 mm. thick at the other. Place a slide on the
stage and some dust on the slide. Do not use a cover-glass. Focus the
dust carefully first with the low then with the high objective. When

40 LIGHTING AND FOCUSING [CH. II

the objective is in focus push the wedge under the objective on the
slide until it touches the objective. Mark the place of contact with a
pencil and then measure the thickness of the wedge with a rule
opposite the point of contact. This thickness will represent very
closely the working distance. For measuring the thickness of the
wedge at the point of contact for the high objective use a steel scale
ruled in ^ths mm. and the tripod to see the divisions. Or one may
use a cover-glass measure (Ch. VIII) for determining the thickness of
the wedge.

For the higher powers if one has a microscope in which the fine ad-
justment is graduated, the working distance may be readily determined
when the thickness of the cover-glass over the specimen is known, as
follows : Get the object in focus, lower the tube of the microscope, un-
til the front of the objective just touches the cover-glass. Note the
position of the micrometer screw and slowly focus up with the fine
adjustment until the object is in focus. The distance the objective was
raised plus the thickness of the cover-glass represents the working dis-
tance. For example, a 3 mm. objective after being brought in contact
with the cover- glass was raised by the fine adjustment a distance repre-
sented by 1 6 of the divisions on the head of the micrometer screw.
Each division represented .01 mm., consequently the objective was
raised . 16 mm. As the cover-glass on the specimen used was . 15 mm.
the total working distance is .16 +.15 =-31 mm.

| 76a. Free Working Distance. — In the microscope catalog of Zeiss there is
given a table of the size of the field and also of the "free working-distance." This
free working-distance is the space between the lower end of the objective and the
cover glass of Ty^mm. thickness, when the objective is in focus on an object imme-
diately under the cover. This is exceedingly practical information for a possessor
of a microscope, and it is hoped that the other opticians will adopt the suggestion.
Naturally, however, the free working-distance for each optician should be reckoned
from the top of the cover for which his unadjustable objectives are corrected. If,
for example, the thickness of cover for which an objective is corrected is -^ mm.
then the free working-distance should be that between the top of this and the
objective when the objective is in focus on an object under the cover. (See the
table of cover-glass thickness, p. 14).

CENTRAL AND OBLIQUE LIGHT WITH A MIRROR

§ 77. Axial or Central Light (§ 65).— Remove the condenser
or any diaphragm from the substage, then place a preparation contain-
ing minute air bubbles under the microscope. The preparation may
be easily made by beating a drop of mucilage on a slide and covering

CH. //] LIGHTING AND FOCUSING 41

it (see Ch. III). Use a 3 mm.,(^ in.) or No. 7 objective and a medium
ocular. Focus the microscope and select a very small bubble, one
whose image appears about i mm. in diameter, then arrange the plane
mirror so that the light spot in the bubble appears exactly in the
center. Without changing the position of the mirror in the least,
replace the air bubble preparation by one of Pleiirosigma angulatum or
some other finely marked diatom. Study the appearance very carefully.

§ 78. Oblique Light (§ 66). — Swing the mirror far to one side
so that the rays reaching the object may be very oblique to the optic
axis of the microscope. Study carefully the appearance of the diatom
with the oblique light. Compare the appearance with that where central
light is used. The effect of oblique light is not so striking with histo-
logical preparations as with diatoms.

It should be especially noted in §§ 77, 78, that one cannot deter-
mine the exact direction of the rays by the position of the mirror.
This is especially true for axial light ( §77). To be certain the light
is axial some such test as that given in § 77 should be applied. (See
also Ch. Ill, under Air-bubbles.)

CONDENSERS OR ILLUMINATORS*

§ 79. These are lenses or lens-systems for the purpose of illuminat-
ing with transmitted light the object to be studied with the microscope.

For the highest kind of investigation their value cannot be over-
estimated. They may be used either with natural or artificial light,
and should be of sufficient numerical aperture to satisfy objectives of
the widest angle.

*No one has stated more cleaily, or appreciated more truly the value of cor-
rect illumination and the methods of obtaining it than Sir David Brewster, 1820,
1831. He says of illumination in general : "The art of illuminating microscopic
objects is not of less importance than that of preparing them for observation. "
"The eye should be protected from all extraneous light, and should not receive any
of the light which proceeds from the illuminating center, excepting that portion
of it which is transmitted through or reflected from the object." So likewise the
value and character of the substage condenser was thoroughly understood and
pointed out by him as follows : "I have no hesitation in saying that the apparatus
for illumination requires to be as perfect as the apparatus for vision, and on this
account I would recommend that the illuminating lens should be perfectly free of
chromatic and spherical aberration, and the greatest care be taken to exclude all
extraneous light both from the object and from the eye of the observer." See Sir
David Brewster's treatise on the Microscope, 1837, pp. 136, 138, 146, and the
Edinburgh Journal of Science, new series, No. n (1831) p. 83.

42 LIGHTING AND POCUSING {_CH. II

It is of the greatest advantage to have the sub-stage condenser
mounted so that it may be easily moved up or down under the stage.
The iris diaphragm is so convenient that it should be furnished in all
cases, and there should be marks indicating the N. A. of the condenser
utilized with different openings. Finally the condenser should be
supplied with central stops for dark-ground illumination (§ 92) and
with blue and neutral tint glasses to soften the glare when artificial
light is used (§ 89, 93).

Condensers or Illuminators fall into two great groups, the
Achromatic, giving a large aplanatic cone, and Non-achromatic,
giving much light, but a relatively small aplanatic cone of light.

§ 80. Achromatic -Condenser. — It is still believed by all expert
microscopists that the contention of Brewster was right, and the con-
denser to give the greatest aid in elucidating microscopic structure
must approach in excellence the best objectives. That is, it should be
as free as possible from spherical and chromatic aberration, and there-
fore would transmit to the object a very large aplanatic cone of light.
Such condensers are especially recommended for photo-micrography by
all, and those who believe in getting the best possible image in every
case are equally strenuous that achromatic condensers should be used
for all work. Unfortunately good condensers like good objectives are
expensive, and student microscopes as well as many others are usually
supplied with the non-achromatic condensers or with none.

Many excellent achromatic condensers have been made, but the
most perfect of all seems to be the apochromatic of Powell and Lealand
(Carpenter-Dallinger, p. 302). To attain the best that was possible
many workers have adopted the plan of using objectives as condensers.
A special substage fitting is provided with the proper screw and the
objective is put into position, the front lens being next the object. As
will be seen below (§ 83-84), the full aperture of an objective can
rarely be used, and for histological preparations perhaps never, so that
an objective of greater equivalent focus, i. <?. , lower power is used for
the condenser than the one on the microscope. It is much more con-
venient, however, to have a special condenser with iris diaphragm or
special diaphragms so that one may use any aperture at will, and thus
satisfy the conditions necessary for lighting different objects for the
same objective and for lighting with objectives of different apertures.
An excellent condenser of this form has been produced by Zeiss (Fig.
41). It has a total numerical aperture of i.oo, and an aplanatic aper-
ture of 0.65.

CH. //] LIGHTING AND FOCUSING 43

FIG. 41. Zeiss' Achromatic Conden-
ser, c. s. c. s. Centering screws for
changing the position of the condenser
and making its axis continuous with
that of the microscope. A segment of
the condenser is cut away to show the
combinations of lenses. For very low
powers the upper lens is sometimes
screwed off. There is an iris dia-
phragm between the middle and lower
combinations. ( Zeiss ' Catalog ) .

§ 81. Centering the Condenser. — To get the best possible
illumination for bringing out in the clearest manner the minute details
of a microscopic object two conditions are necessary, viz.: The princi-
pal optic axis of the condenser must be continuous with that of the
microscope (see frontispiece) and the object must be in the focus of the
condenser, i e. , at the apex of the cone of light given by the condenser.

The centering is most conveniently accomplished as follows
although daylight may be used with almost equal facility. A very
small diaphragm is put below the condenser. (If the Zeiss achromatic
condenser is used, the diaphragm of the Abbe illuminator serves for
this. If there is no pin-hole diaphragm one can be made of stiff,
black paper. Care must be taken, however, to make the opening ex-
actly central. This is best accomplished by putting the paper disc over
the iris or metal diaphragms and then making the hole in the center of
the small circle uncovered by the metal diaphragm. For the hole a fine
needle is best). Light well and lower the objective so that it is at
about its working-distance from the top of the condenser. If now the
condenser is lowered or racked away from the objective the image of
the diaphragm will appear. If the opening is not central it should be
made so by using the centering screws of the condenser.

A better plan than to lower the condenser to focus the image of the
diaphragm, is to raise the body of the microscope slowly with the coarse
adjustment. It is almost impossible to make apparatus so accurate that
two parts like the body of the microscope and the substage, each work-
ing on different sliding surfaces, shall continue in exactly the same
plane. So one will find that if the condenser be accurately centered
with the condenser lowered, and then the condenser be racked up close
to "the stage and the image of the diaphragm opening brought again
into focus by racking up the body of the microscope, it will not be

44

LIGHTING AND FOCUSING

\_CH.

accurately centered in most cases. For this reason it is advised that
the condenser be left in position close to the stage and the tube of the
microscope be used to focus the diaphragm exactly as in ordinary
work.

FIG. 42 . Shows that the optic axis of
the condenser does not coincide with that
of the microscope. ( D) . Image of the
diaphragm of the condenser shown at
one side of the field of view.

FIG. 43. Shows the image of the
diaphragm (D] in the center of the field
of the microscope, and thus the coin-
cidence of the axis of the condenser with
that of the microscope.

.Exc

FIG. 42

FIG. 43

§ 82. Centering the Image of the Source of Illumination.—
For the best results it is not only necessary that the condenser be pro-
perly centered, but that the object to be studied should be in the image
of the source of illumination and that this should also be centered
(Figs. 44, 45). After the condenser itself is centered the iris diaphragm
is opened to its full extent or the diaphragm carrier turned wholly
aside. A transparent specimen like the fly's wing is put under the
microscope and focused. The condenser is then turned up and down
until the image of the flame is apparently on the specimen. If this
cannot be accomplished the relative position of the lamp and condenser
is not correct and should be so changed that the image of the edge of
the flame is sharply defined. This image must also be centered. This
is easily accomplished by manipulation of the mirror or, if a lamp is
used, by changing the position of the lamp or of the bull's eye

(Fig- 53).

§83. Proper Numerical Aperture of the Condenser. — As

stated above, the aperture of the condenser should have a range by
means of properly selected diaphragms to meet the requirements of all
objectives from the lowest to those of the highest aperture. It is
found in practice that for diatoms, etc., the best images are obtained
when the object is lighted with a cone which fills about three- fourths
of the diameter of the back lens of the objective with light but for
histological and other preparations of lower refractive power only one-
half or one-third the aperture often gives the most satisfactory images
(§ 34).

CH. 77]

LIGHTING AND FOCUSING

45

FIG. 44. Shows the image of the
flame (Ft.) in the center (€} of the
field of the microscope and illuminat-
ing the object.

FIG. 45. Shows the image of the
flame (Fl.} at one side of the centre
(Exc.} and not properly illuminating
the object.

Exc

FIG. 44.

FIG. 45. *

To determine this in any case focus upon some very transparent
object, take out the ocular, look down the tube at the back lens. If less
than three-fourths of the back lens is lighted, increase the opening in
the diaphragm— if more than three-fourths diminish it. For some
objects it is advantageous to use less than three-fourths of the aper-
ture. Experience will teach the best lighting for special cases.

Obj

O

Obj

Til

Ilium

FIG. 46.

FIG. 47.

FIGS. 46-47. Figures showing the dependence of the objective upon the ilium
inating cone of the condenser (Nelson}.

FIG. 46 (A). The illuminating cone from the condenser {I Hum}. This is
seen to be just sufficient to fill the objective (Obj}.

(B}. The back lens of the objective entirely filled with light, showing that the
numerical aperture of' the illuminator is equal to that of the objective.

FIG. 47 (A}. In this figure the illuminating conefrome the cotidenser (Ilium.}
is seen to be insufficient to fill the objective (Obj}.

(B}. The back lens of the objective only partly filled with light, due to the
restricted aperture of the illuminator.

§ 84. Aperture of the Illuminating Cone and the Field.— It
is to be remarked that with a very small source of light the entire aper-
ture of the objective may be filled if a proper illuminator or condenser
is used. The aperture depends on the diaphragm used with the con-
denser. And the size of the diaphragm must be directly as the aper-
ture of the objective. That is, it is just the reverse of the rule for
diaphragms where no condenser is used (§ 67) ; for there the diaphragm

46 LIGHTING AND FOCUSING [CH. II

is made large for low powers, and consequently low apertures, while
with the condenser the diaphragm is made small for low and large for
high powers as the aperture is greater in the high powers of a given
series of objectives. It is very instructive to demonstrate this by using
a 1 6 mm. objective and opening the diaphragm of the condenser till the
back lens is just filled with light. Then if one uses a 3 or 4 mm. ob-
jective it will be seen that the back lens of the higher objective is only
partly filled with light and to fill it the diaphragm must be much more
widely opened.

With a condenser, then, the diaphragm has simply to regulate the
aperture of the illuminating cone, and has nothing to do with lighting
a large or a small field.

With the condenser there are two conditions that must be fulfilled,
— the proper aperture must be used, and that is determined by the dia-
phragm, and secondly the whole field must be lighted. The latter is
accomplished by using a larger source of light, as the face instead of
the edge of a lamp flame, or by lowering or raising the condenser so
that the object is not in the focus of the condenser, but above or below
it, and therefore lighted by a converging or diverging beam where the
Tight is spread over a greater area (Figs. 48-51, § 88).

§ 85. Non- Achromatic Condenser. — Of the non-achromatic
condensers or illuminators, the Abbe condenser or illuminator is the
one most generally used. From its cheapness it is also much more com-
monly used than the achromatic condenser. It consists of two or three
very large lenses and transmits a cone of light of 1.20 N. A. to 1.40 N.
A., but the aberrations, both spherical and chromatic, are very great in
both forms. Indeed, so great are they that in the best form of three
lenses with an illuminating cone of 1.40 N. A., the aplanatic cone
transmitted is only 0.5, and it is the aplanatic cone^ which is of real use
in microscopic illumination where details are to be studied. There is
no doubt, however, that the results obtained with a non-achromatic
condenser like the Abbe are much more satisfactory than with no con-
denser. The highest results cannot be attained with it, however.
(Carpenter-Dallinger, p. 309).

§ 86. Arrangement of the Condenser. — The proper position of
the illuminator for high objectives is one in which the beam of light
traversing it is brought to a focus on the object. If parallel rays are
reflected from the plane mirror to it, they will be focused only a few
millimeters above the upper lens of the condenser ; consequently the
illuminator should be about on the level of the top of the stage and

CH. 77] LIGHTING AND FOCUSING 47

therefore almost in contact with the lower surface of the slide. For
some purposes when it is desirable to avoid the loss of light by reflec-
tion or refraction, a drop of water or homogeneous immersion fluid is
put between the slide and condenser, forming the so-called immersion
illuminator. This is necessary only with objectives of high power
and large aperture or for dark-ground illumination.

§87. Centering the Condenser. — The illuminator should be
centered to the optic axis of the microscope, that is the optic axis of
the condenser and of the microscope should coincide. Unfortunately
there is extreme difficulty in determining when the Abbe illuminator is
centered. Centering is approximated as follows : Put a pin-hole dia-
phragm— that is a diaphragm with a small central hole — over the end
of the condenser (Fig. 52), the central opening should appear to be in
the middle of the field of the microscope. If it does not the condenser
should be moved from side to side by loosening the centering screws
until it is in the center of the field. In case no pin-hole diaphragm
accompanies the condenser, one may put a very small drop of ink, as
from a pen-point, on the center of the upper lens and look at it with
the microscope to see if it is in the center of the field. If it is not,
the condenser should be adjusted until it is. When the condenser is
centered as nearly as possible remove the pin-hole diaphragm or the
spot of ink. The microscope and illuminator axes may not be entirely
coincident even when the center of the upper lens appears in the cen-
ter of the field, as there may be some lateral tilting of the condenser, but
the above is the best the ordinary worker can do, and unless the
mechanical arrangements of the illuminator are very deficient, it will
be very nearly centered.

It is to be hoped that the opticians will devise some kind of
mounting for this the most cemmonly used condenser whereby it may
be centered as described for the achromatic condenser instead of by the
crude methods described above. If the condenser mounting regularly
possessed centering screws as in the microscope of Watson & Sons and
there were a centering diaphragm in the proper position so that its im-
age could be projected into the field of view, the operation would be
very simple. If, further, the condensers of Powell and Lealand were
selected as models the condensers need not be so bulky, and would still
retain all their efficiency.

Fortunately the Royal Microscopical Society of London which has
done so much toward standardizing microscopical apparatus has recently
proposed as a standard size for the substage fitting for the condenser of
1.527 in.= 38.786 mm. (see § 46).

4^ LIGHTING AND FOCUSING \CH. II

§ 88. Mirror and Light for the Abbe Condenser. — It is best to
use light with parallel rays. The rays of daylight are practically par-
allel ; it is best therefore to employ the plane mirror for all but the
lowest powers. If low powers are used the whole field might not be
illuminated with the plane mirror when the condenser is close to the
object ; furthermore, the image of the window frame, objects outside
the building, as trees, etc. , would appear with unpleasant distinctness
in the field of the microscope. To overcome these defects one can
lower the condenser and thus light the object with a diverging cone of
light, or use the concave mirror and attain the same end when the con-
denser is close to the object (Fig. 48).

§ 89. Artificial Light. — If one uses lamp light, it is recommend-
ed that a large bull's eye be placed in such a position between the
light and the mirror that parallel rays fall upon the mirror or in some
cases an image of the lamp flame. If one does not have a bull's eye
the concave mirror may be used to render the rays less divergent. It
may be necessary to lower the illuminator somewhat in order to illum-
inate the object in its focus.

ABBE CONDENSER : EXPERIMENTS

§ 90. Abbe Condenser, Axial and Oblique Light. — Use a dia-
phragm a little larger than the front lens of the 3 mm. (^ in.) objec-
tive, have the illuminator on the level, or nearly on the level of the
upper surface of the stage, and use the plane mirror. Be sure that
the diaphragm carrier is in the notch indicating that it is central in
position. Use the Pleurosigma as object. Study carefully the appear-
ance of the diatom with this central light, then make the diaphragm
eccentric so as to light with oblique light (§78). The differences in
appearance will probably be even more striking than with the mirror
alone.

§ 91. Lateral Swaying of the Image. — Frequently in study-
ing an object, especially with a high power, it will appear to sway
from side to side in focusing up or down. A glass stage micrometer or
fly's wing is an excellent object. Make the light central or axial and
focus up and down and notice that the lines simply disappear or grow
dim. Now make the light oblique, either by making the diaphragm
opening eccentric or if simply a mirror is used, by swinging the mirror
sidewise. On focusing up and down, the lines will sway from side to
side. What is the direction of apparent movement in focusing down

CH. //]

LIGHTING AND FOCUSING

49

with reference to the illuminating ray ? What in focusing up ? If one
understands the experiment it may sometimes save a great deal of con-
fusion. (See under testing the microscope for swaying with central
light § 119.)

§ 92. Dark-Ground Illumination. — When an object is lighted
with rays of a greater obliquity than can get into the front lens of the
objective, the field will appear dark (Fig. 51). If now the object is

FIGS. 48-51. Sectional views of the Abbe Illuminator of 1.20 N. A. showing
various methods of illumination (\ oo) Fig. 48, axial light with parallel rays,
Fig. 49, oblique light. Fig. 50, axial light with converging beam. Fig. 51, dark-
ground illumination with a central stop diaphragm.

Axis. The optic axis of the illuminator and of the microscope. The illumi-
nator is centered, that is its optic axis is a prolongation of the optic axis of the
microscope.

S. Axis. Secondary axis. In oblique light the central ray passes along a
secondary axis of the illuminator, and is therefore oblique to the principal axis.

D. D. Diaphragms. These are placed in sectional and in face views. The
diaphragm is placed between the mirror and the illuminator. In Fig. 49 the open-
ing is eccentric for oblique light, and in Fig. 51 the opening is a narrow ring, the
central part being stopped out, thus giving rise to dark-ground illumination ($ 92).

Obj. Obj. The front of the objective.

composed of fine particles, or is semi-transparent, it will refract or
reflect the light which meets it, in such a way that a part of the very

50 LIGHTING AND FOCUSING [CH. II

oblique rays will pass into the objective, hence as light reaches the
objective only from the object, all the surrounding field will be dark
and the object will appear like a self-luminous one on a dark back-

FIG. 52. An Abbe Condenser in its mounting
{The Bausch & Lomb Optical Company}.

ground. This form of illumination is most
successful with low powers. It is well to
make the illuminator immersion for this
experiment, (see § 105).

(A) With the Mirror— Remove all the
diaphragms so that very oblique light may
be used, employ a stage micrometer in
which the lines have been filled with graph-
ite, use a 1 6 mm. (^ in.) objective, and when the light is sufficiently
oblique the lines will appear something like streaks of silver on a
black back-ground. A specimen like that described below in (B) may
also be used.

(B) With the Abbe Condenser. — Have the illuminator so that the
light is focused on the object (see § 86) and use a diaphragm with
the annular opening (Fig. 51); employ the same objective as in
(A). For object place a drop of 10 % solution of salicylic acid in 95 %
alcohol on the middle of a slide ; it will crystallize. The crystals will
appear brilliantly lighted on a dark back-ground. Put in an ordinary
diaphragm and make the light oblique by making the diaphragm
eccentric. The same specimen may also be tried with a mirror and
oblique light. In order to appreciate the difference between this dark-
ground and ordinary transmitted-light illumination, use an ordinary
diaphragm and observe the crystals.

A very striking and instructive experiment may be made by add-
ing a very small drop of the solution to the dried preparation, putting
it under the microscope quickly, lighting for dark-ground illumination
and then watching the crystallization.

ARTIFICIAL ILLUMINATION

§ 93. For evening work and for regions where daylight is not
sufficiently brilliant, artificial illumination must be employed. Fur-
thermore, for the most critical investigation of bodies with fine mark-
ings like diatoms, artificial light has'been found superior to daylight.

A petroleum (kerosene) lamp with flat wick gives a satisfactory
light. It is recommended that instead of the ordinary glass chimney,

CH. //]

LIGHTING AND FOCUSING

one made of metal with a slit- opening covered with an oblong cover-
glass is more satisfactory, as the source of light is more restricted.
Very excellent results may be obtained, however, with the ordinary
bed-room lamp furnished with the usual glass chimney.

The new acetylene light promises to be excellent for micro-
scopic observation and for photo-micrography. (See under photo-
micrography.)

FIG. 53. i.
separate stand.

Lamp with slit-opening in metal chimney.
3. Screen showing image of flame.

2. BuWs eye on

Whenever possible the edge of the flame is turned toward the
microscope, the advantage of this arrangement is the great brilliancy,
due to the greater thickness of the flame in this direction.

§ 94. Mutual Arrangement of Lamp, Bull's Eye and Micro-
scope.— To fulfill the conditions given above, namely, that the object
be illuminated by the image of the source of illumination the lamp
must be in such a position that the condenser projects a sharp image
of the flame upon the object (Fig. 53), and only by trial can this posi-
tion be determined. In some cases it is found advantageous to discard
the mirror and allow the light from the bull's eye to pass directly into
the condenser. This method is especially excellent in photomicro-
graphy (see Ch. VIII).

§ 95. Illuminating the Entire Field. — With low objectives
and large objects, the entire object might not be illuminated if the
above method were strictly followed ; in this case turn the lamp so
that the flame is oblique, or if that is not sufficient, continue to turn
the lamp until the full width of the flame is used. If necessary the

LIGHTING AND FOCUSING

\CH. II

condenser may be lowered, and the concave mirror used. (See
also §84.)

REFRACTION AND COLOR IMAGES

\ 96. Refraction Images are those mostly seen in studying microscopic
objects. They are the appearances produced by the refraction of the light on
entering and on leaving an object. They therefore depend (a) on the form of the
object, (b) on the relative refractive powers of object and mounting medium.
With such images the diaphragm should not be too large (see §83).

If the color and refractive index of the object were exactly like the mount-
ing medium it could not be seen. In most cases both refractive index and color
differ somewhat, there is then a combination of color and refraction images which
is a great advantage. This combination is generally taken advantage of in histol-
ogy. The air bubble in $ 77 is an example of a purely refractive image.

54

N'

FIGS. 54-56. Diagrams illustrating refraction in different media and at plane
and curved surfaces. In each case the denser medium is represented by line shad-
ing and the perpendicular or normal to the refracting surface is represented by the
dotted line N-N' , the refracted ray by the bent line A C.

\ 97 . Refraction. — Lying at the basis of microscopical optics is refraction , which
is illustrated by the above figures. It means that light passing from one medium
to another is bent in its course. Thus in Fig. 54 light passing from air into water
does not continue in a straight line but is bent toward the normal N-NX, the
bending taking place at the point of contact of the air and water ; that is, the ray
of light A B entering the water at B is bent out of its course, extending to C
instead of Cx.

Conversely, if the ray of light is passing from water into air, on reaching the
air it is bent_/r0w the normal, the ray C B passing to A and not in a straight line
to Cx/. By comparing Figs. 55, 56 in which the denser medium is crown glass in-
stead of water, the bending of the rays is seen to be greater as crown glass is
denser than water.

It has been found by physicists that there is a constant relation between the
angle taken by the ray in the rarer medium and that taken by the ray in the
denser medium. The relationship is expressed thus : Sine of the angle of inci-

CH. 77] LIGHTING AND FOCUSING 53

dence divided by the sine of the angle of refraction equals the index of refraction.

In the figures, \ ° .7 = index of refraction. Worked out completely in

oin C lj J\

Fig. 54,

the index of refraction from air to water is 1.33. (See $ 33.) In Figs. 55-56,
illustrating refraction in crown glass, the angles being given, the problem is easily
solved as just illustrated. (For table of natural sines see third page of cover ; for
interpolation, | 32).

| 98. Absolute Index of Refraction. — This is the index of refraction obtained
when the incident ray passes from a vacuum into a given medium. As the index
of the vacuum is taken as unity, the absolute index of any substance is always
greater than unity. For many purposes, as for the object of this book,
air is treated as if it were a vacuum, and its index is called unity, but in reality
the index of refraction of air is about 3 ten-thousandths greater than unity.
Whenever the refractive index of a substance is given, the absolute index is
meant unless otherwise stated. For example, when the index of refraction of
water is said to be 1.33, and of crown glass 1.52, etc., these figures represent the
absolute index, and the incident ray is supposed to be in a vacuum.

\ 99. Relative Index of Refraction. — This is the index of refraction between
two contiguous media, as for example between glass and diamond, water and
glass, etc. It is obtained by dividing the absolute index of refraction of the sub-
stance containing the refracted ray, by the absolute index of the substance trans-
mitting the incident ray. For example, the relative index from water to glass is
1.52 divided by 1.33. If the light passed from glass to water it would be, 1.33
divided by 1.52.

By a study of the figures showing refraction, it will be seen that the greater the
refraction the less the angle and consequently the less the sine of the angle, and as
the refracti on between two media is the ratio of the sines of the angles of incidence

and refraction ( - ) , it will be seen that whenever the sine of the angle of refrac-
Vsin rj\

tion is increased by being in a less refractive medium, the index of refraction will
show a corresponding decrease and vice versa. That is ihe ratio of the sines of the
angles of incidence and refraction of any two contiguous substances is inversely as
the refractive indices of those substances. The formula is :

/ Sine of angle of incident ray \ / Index of refraction of refracting medium \
\ Sine of angle of refracted ray / \ Index of refraction of incident medium /

Abbreviated ( - - | = | r -. ) • BV means of this general formula one can

\sm rj \index ij

solve any problem in refraction whenever three factors of the problem are known.

The universality of the law may be illustrated by the following cases :

(A) Light incident in a vacuum or in air, and entering some denser medium,

as water, glass, diamond, etc.

/ Sine of angle made by the ray in air \ _ /Index of ref.of denser med. \
\ Sine of angle made by ray in denser medium / \ Index of ref. of air ( i) /

If the dense substance were glass ( - - ) — ( — ^— J . If the two media were

\sin r)~ V i /

54 LIGHTING AND FOCUSING [CH. II

water and glass, the incident light being in water the formula would be :

( —. ) = ( ) • If the incident ray were in glass and the refracted ray in

\sm r) V 1.33 /

water : ( — ) — ( -£s- } - And similarly for any two media ; and as stated

Vsin r) V 1.52 /

above if any three of the factors are given the fourth may be readily found.

§ ico. Critical Angle and Total Reflection. — In order to understand the Wol-
laston camera lucida (Ch. IV) and other totally reflecting apparatus, it is necessary
briefly to consider the critical angle.

The critical angle is the greatest angle that a ray of light in the denser of two
contiguous media can make with the normal and still emerge into the less refrac-
tive medium. On emerging it will form an angle of 90° with the normal, and if
the substances are liquids, the refracted ray will be parallel with the surface of the
denser medium.

Total Reflection. — In case the incident ray in the denser medium is at an angle
with the normal greater than the critical angle^ it will be totally reflected at the
surface of the denser medium, that surface acting as a perfect mirror. By consult-
ing the figures it will be seen that there is no such thing as a critical angle and
total reflection in the rarer of two contiguous media.

To find the critical angle in the denser of two contiguous media : —

Make the angle of refraction (z. e. , the angle in the rarer of the two media)

/ sin i \ / index r \

90 and solve the general equation : ( —. I = ( -. — , r I . Let the two sub-

\sin r/ \index z /

stances be water and air, then the sine of r (90°) is i, the index of air is i, that of
water 1.33, whence (- - 1 = 1 - - j or sin z' — 75 1 -j- . This is the sine of

48° + , and whenever the ray in the water is at an angle of more than 48° it will
not emerge into the air, but be totally reflected back into the water.

The case of a ray passing from crown glass into the water :

/ _ sin i \ _ / index water (1.33) \ / sin i \ / 1.33 \

\sin r (sin 90°= i)/ \index glass (i. 52)7 °r \ i / \ 1.527'
whence sin z =.875 sine of critical angle in glass covered with water. The
corresponding angle is approximately 61°.

| 101. Color Images. — These are images of objects which are strongly col-
ored and lighted with so wide an aperture that the refraction images are drowned
in the light. Such images are obtained by removing the diaphragm or by using a
larger opening. This method of illumination is especially applicable to the study
of deeply stained bacteria. (See below. § 108).

ADJUSTABLE WATER AND HOMOGENEOUS OBJECTIVES
EXPERIMENTS

§ 102. Adjustment for Objectives. — As stated above ($ 24), the aberration
produced by the cover-glass (Fig. 57), is compensated for by giving the combina-
tions in the objective a different relative position than they would have if the
objective were to be used on uncovered objects. Although this relative position
cannot be changed in unadjustable objectives, one can secure the best results .of

CH. //]

LIGHTING AND FOCUSING

55

which the objective is capable by selecting covers of the thickness for which the
objective was corrected. (See table p. 14.) Adjustment maybe made also by
increasing the tube-length for covers thinner than the standard and by shortening
the tube-length for covers thicker than the standard (Fig. 58).

FIG. 57. Effect of the cover-glass
on the rays from the object to the
objective (Ross}.

Axis. The projection of the optic
axis of the microscope.

F. Focal or axial point of the
objective.

F' and F" . Points on the axis
where rays 2 and j appear to originate
if traced backward after emerging
from the upper side of the cover-glass.

In learning to adjust objectives, it is best for the student to choose some
object whose structure is well agreed upon , and then to practice lighting it, shad-
ing the stage and adjusting the objective, until the proper appearance is obtained.
The adjustment is made by turning a ring or collar which acts on a screw and
increases or diminishes the distance between the systems of lenses, usually the
front and the back systems (Fig. 40).

§ 103. General Directions. — (A) The thinner the cover-glass, the
further must the system of lenses be separated, i. e., the ad justing collar
is turned nearer the zero or the mark "uncovered," and conversely; (B)
the thicker the cover-glass the closer together are the systems brought
by turning the adjusting collar from the zero mark. This also increases
the magnification of the objective (Ch. IV).

The following specific directions for making the cover-glass adjust-
ment are given by Mr. Wenham (Carpenter, yth Ed., p. 166). "Select
any dark speck or opaque portion of the object, and bring the outline
into perfect focus ; then lay the finger on the milled-head of the fine
motion, and move it briskly backwards and forwards in both directions
from the first position. Observe the expansion of the dark outline of
the object, both when within and when without the focus. If the
greater expansion or coma is when the object is without the focus, or
farthest from the objective \i. e., in focusing up], the lenses must be
placed further asunder, or toward the mark uncovered [the adjusting
collar is turned toward the zero mark as the cover-glass is too thin for
the present adjustment]. If the greater expansion is when the object
is within the focus, or nearest the objective [z. e., in focusing down],
the lenses must be brought closer together, or toward the mark covered
[i. e., the adjusting collar should be turned away from the zero mark,

56 LIGHTING AND FOCUSING {_CH. II

the cover-glass being too thick for the present adjustment] ." In most
objectives the collar is gradiiated arbitrarily, the zero (O} mark represent-
ing the position for uncovered objects. Other objectives have the collar
graduated to correspond to the various thicknesses of cover-glasses for which
the objective may be adjtisted. This seems to be an admirable plan ; then
if one knows the thickness of the cover-glass on the preparation (Ch. VIII)
the adjusting collar may be set at a corresponding mark, and one will feel
confident that the adjustment will be approximately correct. It is then
only necessary for the observer to make the slight adjustment to compensate
for the mounting medium or any variation from the standard length of the
tube of the microscope. In adjusting for variations of the length of
the tube from the standard it should be remembered that : (A) If the
tube of the microscope is longer than the standard for which the ob-
jective was corrected, the effect is approximately the same as thicken-
ing the cover-glass, and therefore the system of the objective must be
brought closer together, i. e., the adjusting collar must be turned away
from the zero mark. (B) If the tube is shorter than the standard for
which the objective is corrected, the effect is approximately the same as
diminishing the thickness of the cover-glass, and the systems must
therefore be separated (Fig. 40).

In using the tube-length for cover correction Shorten the tube for
too thick covers and Lengthen the tube for too thin covers.

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

§ 104. Water Immersion Objectives. — Put a water immersion
objective in position (§ 47) and the fly's wing for object under the
microscope. Place a drop of distilled water on the cover-glass, and
with the coarse adjustment lower the tube till the objective dips into
the water, then light the field well and turn the fine adjustment one
way and another till the image is clear. Water immersions are exceed-
ingly convenient in studying the circulation of the blood, and for

CH. 77]

LIGHTING AND FOCUSFNG

57

many other purposes where aqueous liquids are liable to get on the
cover-glass. If the objective is adjustable, follow the directions given
in § 103.

FIG. 58. Figure to show that in lengthening the tube of the microscope the ob-
ject must be brought nearer the principal focus or center of the lens. It will be seen
by consulting the figure that in shortening the tube of the microscope the object
must be removed farther from the center of the lens. By consulting the figure
showing the effect of the cover-glass (Fig. 57) it will be seen that the effect of the
cover-glass is to bring the object nearer the objective, and the thicker the cover the
nearer is the object brought to the objective. As shortening the tube serves to remove
the object, it neutralizes the effect of the thick cover, and if the cover is so thin that
it does not elevate the object enough for the corrections of the objective, then an in-
crease in the tube-length will correct the defect.

58 LIGHTING AND FOCUSING \_CH. II

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

HOMOGENEOUS IMMERSION OBJECTIVES : EXPERIMENTS

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

§ 106. Tester for Homogeneous Liquid. — In order that full
advantage be derived from the homogeneous immersion principle, the
liquid employed must be truly homogeneous. To be sure that such is
the case, one may use a tester like that constructed by the Gundlach
Optical Co. , then if the liquid is too dense it may be properly diluted
and vice versa. For the cedar oil immersion liquid, the density may
be diminished by the addition of pure cedar wood oil. The density
may be increased by allowing it to thicken by evaporation. (See H.
L. Smith, Proc. Amer. Soc. Micr., 1885, p. 83, and Ch. X).

§ 107. Refraction Images. — Put a 2 mm.(T^-th in.) homogeneous
immersion objective in position, employ an illuminator. Use some
histological specimen like a muscular fiber as object, make the dia-
phragm opening about 3 mm. in diameter, add a drop of the homo-
geneous immersion liquid and focus as directed in § 74. The object
will be clearly seen in all details by the unequal refraction of the light
traversing it. The difference in color between it and the surrounding
medium will also increase the sharpness of the outline. If an air bub-
ble preparation (§ 77) were used, one would get pure, refraction
images. •

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

CH. //] LIGHTING AND FOCUSING 59

jo cm

FIG. 59. Screen for shading the microscope and
the face of the observer. This is very readily con-
structed as shown in the figure by supporting a wire
in a disc of lead, iron, or heavy wood. The screen is
then completed by hanging over the bent wire, cloth or
manilla paper 30x40 cm. The lower edge of the
screen should be a little below the stage of the micro-
scope and the upper edge high enough to screen the
eyes of the observer.

§ 109. Shading the Object.— To get the
clearest image of an object no light should
reach the eye except from the object. A hand-
kerchief or a dark cloth wound around the

objective will serve the purpose. Often the proper effect may be ob-
tained by simply shading the top of the stage with the hand or with a
piece of bristol board. Unless one has a very favorable light the shading
of the object is of the greatest advantage, especially with homogeneous
immersion objectives. The screen (Fig. 59) is the most satisfactory
means for this purpose, as the entire microscope above the illuminating
apparatus is shaded.

§ no. Cleaning Homogeneous Objectives. — After one is
through with a homogeneous objective, it should be carefully cleaned as
follows : Wipe off the homogeneous liquid with a piece of the lens
paper (§ 114), then if the fluid is cedar oil, wet one corner of a fresh
piece in benzin or chloroform and wipe the front lens with it. Imme-
diately afterward wipe with a dry part of the paper. The cover-glass
of the preparation can be cleaned in the same way. If the homogen-
eous liquid is a glycerin mixture proceed as above, but use water to
remove the last traces of glycerin.

CARE OF THE MICROSCOPE

§111. The microscope should be handled carefully and kept per-
fectly clean. The oculars and objectives should never be allowed to
fall.

When not in use keep it in a place as free as possible from dust.

All parts of the microscope should be kept free from liquids,
especially from acids, alkalies, alcohol, benzin, turpentine and
chloroform.

§ 112. Care of the Mechanical Parts. — To clean the mechan-
ical parts put a small quantity of some fine oil (olive oil or liquid vas-

60 LIGHTING AND FOCUSING \_Cff. II

elin and benzin, equal parts), on a piece of chamois leather or on the
lens paper, and rub the parts well, then with a clean dry piece of the
chamois or paper wipe off most of the oil. If the mechanical parts
are kept clean in this way a lubricator is rarely needed. Where op-
posed brass surfaces "cut," i. <?., when from the introduction of some
gritty material, minute grooves are worn in the opposing surfaces, giv-
ing a harsh movement, the opposing parts should be separated, care-
fully cleaned as described above and any ridges or prominences scraped
down with a knife. Where the tendency to "cut" is marked, a very
slight application of equal parts of beeswax and tallow, well melted
together, serves a good purpose.

In cleaning lacquered parts, benzin alone answers well, but it
should be quickly wiped off with a clean piece of the lens paper. Do
not use alcohol as it dissolves the lacquer.

§ 113. Care of the Optical Parts. — These must be kept scrupu-
lously clean in order that the best results may be obtained.

Glass surfaces should never be touched with the fingers, for that
will soil them.

The glass of which the lenses are made is quite soft, consequently
it is necessary that only soft, clean cloth or paper be used in wiping
them.

Whenever an objective is left in position on a microscope, or when
several are attached by means of a revolving nose-piece, an ocular
should be left in the upper end of the tube to prevent dust from falling
down upon the back lens of the objective.

§ 114. Lens Paper. — The so-called Japanese filter paper, which
from its use with the microscope, I have designated lens paper, has
been used in the author's laboratory for the last sixteen years for clean-
ing the lenses of oculars and objectives, and especially for removing
the fluid used with immersion objectives. Whenever a piece is used
once it is thrown away. It has proved more satisfactory than cloth or
chamois, because dust or sand is not present ; and from its bibulous
character it is very efficient in removing liquid or semi-liquid substances.

§ 115. Dust may be removed with a camel's hair brush, or by
wiping with the lens paper.

Cloudiness may be removed from the glass surfaces by breathing
on them, then wiping quickly with a soft cloth or the lens paper.

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

CH. //] LIGHTING AND FOCUSING 61

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

Water may be removed with soft cloth or the paper.

Glycerin may be removed with cloth or paper saturated with dis-
tilled water ; remove the water as above.

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

Canada Balsam, damar, paraffin, or any oily substance may be re-
moved with a cloth or paper wet with chloroform, benzin or xylene.
The application of these liquids and their removal with a soft dry cloth
or paper should be as rapid as possible, so that none of the liquid will
have time to soften the setting of the lenses.

Shellac Cement may be removed by the paper or a cloth moistened
in 95 per cent, alcohol.

Brunswick Black, Gold Size, and all other substances soluble in
chloroform, etc., maybe removed as directed for balsam and damar.

In general, use a solvent of the substance on the glass and wipe it
off quickly with a fresh piece of the lens paper.

It frequently happens that the upper surface of the back combina-
tion of the objective becomes dusty. This may be removed in part by
a brush, but more satisfactorily by using a piece of the soft paper loosely
twisted. When most of the dust is removed some of the paper may be
put over the end of a pine stick (like a match stick) and the glass sur-
face carefully wiped.

CARE OF THE EYES

§ 1 1 6. Keep both eyes open, using the eye-screen if necessary (Figs.
60, 6oa); and divide the labor between the two eyes, i. e., use one eye
for observing the image awhile and then the other. In the begin-
ning it is not advisable to look into the microscope continuously for
more than half an hour at a time. One never should work with the
microscope after the eyes feel fatigued. After one becomes accustomed
to microscopic observation he can work for several hours with the
microscope without fatiguing the eyes. This is due to the fact that
the eyes become inured to labor like the other organs of the body by
judicious exercise. It is also due to the fact that but very slight ac-
commodation is required of the eyes, the eyes remaining nearly in a
condition of rest as for distant objects. The fatigue incident upon

62

LIGHTING AND FOCUSING

{_CH.

using the microscope at first is due partly at least to the constant effort
on the part of the observer to remedy the defects of focusing the
microscope by accommodation of the eyes. This should be avoided
and the fine adjustment of the microscope used instead of the muscles
of accommodation. With a microscope of the best quality, and suita-
ble light — that is light which is steady and not so bright as to dazzle
the eyes nor so dim as to strain them in determining details — micro-
scopic work should improve rather than injure the sight.

7 X 44 cm.

FIG. 60. Double Eye Shade. This is
readily made by taking some thick bris-
tol board 7x14 centimeters and making
an oblong opening with rounded ends
(o-o) and of such a diameter that it goes
readily over the tube of the microscope.
This is then covered on both sides with
velveteen and a central slit (s) made in

^ </ the cloth. This admits the tube of the

microscope and holds the screen in posi-
tion. It may readily be pulled from side to side and thus serves for either eye, or
for the use of the eyes alternately.

FIG. 60 a. Adjusting Eye-Shade. This is prepared like the preceding by cov-
ering a card about 6 x 12 centimeters with black velveteen. A copper wire about
1 mm.(l/& in. ) and of the right length is curved as shown in the figure. Its ends are
rounded, and finally it is put under the cloth and sewed carefully all around. The
card and cloth are then cut as shown. The flexible wire makes it possible to put
the screen on the tube at any level.

§ 117. Position and Character of the 'Work-Table. — The
work-table should be very firm and large (60 x 120 cm.; 24x48 in.),
so that the necessary apparatus and material for work may not be too
crowded. The table should also be of the right height to make work
by it comfortable. An adjustable stool, something like a piano stool is
convenient, then one may vary the height corresponding to the neces-

CH. If] LIGHTING AND FOCUSING 63

sities of special cases. It is a great advantage to sit facing the window
if daylight is used, then the hands do not constantly interfere with
the illumination. To avoid the discomfort of facing the light a screen
like that shown in Fig. 59 is very useful (see also under lighting,

§62).

TESTING THE MICROSCOPE

| 118. Testing the Microscope. — To be of real value this must be accom-
plished by a person with both theoretical and practical knowledge, and also with
an unprejudiced mind. Such a person is not common, and when found does not
show over anxiety to pass judgment. Those most ready to offer advice should
as a rule be avoided, for in most cases they simply "have an ax to grind," and are
sure to commend only those instruments that conform to the "fad" of the day.
From the writer's experience is seems safe to say that the inexperienced can do no
better than to state clearly what he wishes to do with a microscope and then trust
to the judgment of one of the optical companies. The makers of microscopes and
objectives guard with jealous care the excellence of both the mechanical and
optical part of their work, and send out only instruments that have been carefully
tested and found to conform to the standard. This would be done' as a matter of
business prudence on their part, but it is believed by the writer that microscope
makers are artists first and take an artist's pride in their work, they therefore have
a stimulus to excellence greater than business prudence alone could give.

2 119. Mechanical Parts. — All of the parts should be firm, and not too easily
shaken. Bearings should work smoothly. The mirror should remain in any
position in which it is placed.

Focusing Adjustments. — The coarse or rapid adjustment should be by rack
and pinion, and work so smoothly that even the highest power can be easily focused
with it. In no case should it work so easily that the body of the microscope is
liable to run down and plunge the objective into the object. If any of the above
defects appear in a microscope that has been used for some time, a person with
modjerate mechanical instinct will be able to tighten the proper screw, etc.

The Fine Adjustment is more difficult to deal with. From the nature of its
purpose unless it is approximately perfect, it would be better off the microscope
entirely. It has been much improved recently.

It should work smoothly and be so balanced that one cannot tell by the feel-
ing when using it whether the screw is going up or down. Then there should be
absolutely no motion except in the direction of the optic axis, otherwise the
image will appear to sway even with central light. Compare the appearance
when using the coarse and when using the fine adjustment. There should be no
swaying of the image with either if the light is central ($ 77).

| 120. Testing the Optical Parts. — As stated in the beginning, this can be
done satisfactorily only by an expert judge. It would be of very great advantage
to the student if he could have the help of such a person. In no case is a micro-
scope to be condemned by an inexperienced person. If the beginner will bear in
mind that his failures are due mostly to his own lack of knowledge and lack of
skill ; and will truly endeavor to learn and apply the principles laid down in this
and in the standard works referred to, he will learn after a while to estimate at
their true value all the pieces of his microscope. (See Ch. X).

64 LA BORA TOR Y MICROSCOPES \_CH. II

LABORATORY AND HIGH-SCHOOL COMPOUND
MICROSCOPES

$ 121. Optical Parts. — A great deal of beginning work with the microscope in
biological laboratories is done with simple and inexpensive apparatus. Indeed if
one contemplates the large classes in the high schools, the universities and med-
ical schools, it can be readily understood that microscopes costing from $25-50 each
and magnifying from 25 to 500 diameters, are all that can be expected. But for
the purpose of modern histological investigation and of advanced microscopical
work in general, a microscope should have something like the following character :
Its optical outfit should comprise, (a) dry objectives of 50 mm. (2 in.), 16-18 mm.
( % in. ) and 3 mm. ( % in. ) equivalent focus. There should be present also a
2 mm. (^2 in. ) °r :-5 nitn. (T^ in. ) homogeneous immersion objective. Of oculars
there should be several of different power. A centering substage condenser,
and an Abbe camera lucida are also necessities, and a micro-spectroscope and a
micro-polarizer are very desirable.

Even in case all the optical parts cannot be obtained in the beginning, it is
wise to secure a stand upon which all may be used when they are finally secured.

As to the objectives. The best that can be afforded should be obtained. Cer-
tainly at the present, the apochromatics stand at the head, although the best
achromatic objectives approach them very closely.

| 122. Mechanical Parts or Stand. — The stand should be low enongh so that
it can be used in a vertical position on an ordinary table without inconvenience ;
it should have a jointed (flexible) pillar for inclination at any angle to the hori-
zontal. The adjustments for focusing should be two, — a coarse adjustment or
rapid movement with rack and pinion, and a fine adjustment by means of a mi-
crometer screw. Both adjustments should move the entire tube of the microscope.
The body or tube should be short enough for objectives corrected for the short or
160 millimeter tube-length. It is an advantage to have the draw-tube graduated
in centimeters and millimeters. The lower end of the draw tube and of the tube
should each possess a standard screw for objectives (frontispiece). The stage
should be quite large for the examination of slides with serial sections and other
large objects. The substage fittings should be so arranged as to enable one to use
the .condenser or to dispense entirely with diaphragms. The condenser mount-
ing should allow up and down motion.

STANDARD SIZES RECOMMENDED BY THE ROYAL
MICROSCOPICAL SOCIETY

§ 123. Society Screw. — Owing to the lack of uniformity in screws for micro-
scope objectives, the Royal Microscopical Society of London, in 1857, made an
earnest effort to introduce a standard size. The specifications of this standard are

CH. //] LABORATORY MICROSCOPES 65

as follows : "Whitworth thread, i. e., a V shaped thread, sides of thread inclined
to angle of 55° to each other, one-sixth of the V depth of the thread being
rounded off at the top of the thread, and one-sixth of the thread being rounded off
at the bottom of the thread. Pitch of screw, 36 to the inch ; length of thread on
object-glass, 0.125 inch ; plain fitting above thread of object-glass, 0.15 inch long,
to be about the size of the bottom of male thread ; length of thread of nose-piece
[on the lower end of the tube of the microscope] , not less than o. 25 inch ; diam-
eter of the object-glass screw at the bottom of the screw, 0.7626 inch ; diameter
of the nose-piece screw at the bottom of the thread, 0.8 inch."

In order to facilitate the introduction of this universal screw, or as it soon
came to be called " The Society Screw," the Royal Microscopical Society undertook
to supply standard taps. From the mechanical difficulty in making these taps
perfect there soon came to be considerable difference in the "Society Screws, " and
the object of the society in providing a universal screw was partly defeated.

In 1884 the American Microscopical Society appointed Mr. Edward Bausch
and Prof. William A. Rogers upon a committee to correspond with the Royal
Microscopical Society, with a view to perfecting the standard "Society Screw,"
or of adopting another standard and of perfecting methods by which the screws of
all makers might be truly uniform. Although this matter was earnestly consid-
ered at the time by the Royal Microscopical Society, the mechanical difficulties
were so great that the improvements were abandoned.

Fortunately, however, during the year (1896) that society again took hold of
the matter in earnest, and the "Society Screw" is now accurate, and facilities for
obtaining the standard are so good that there is a reasonable certainty that the
universal screw for microscopic objectives may be realized. It is astonishing to
see how widely the "Society Screw" has been adopted. Indeed there is not a
maker of first-class microscopes in the world who does not supply the objectives
and stands with the "Society Screw, " and an objective in England or America which
does not have this screw should be looked upon with suspicion. That is, it is
either old, cheap, or not the product of one of the great opticians. For the Stand-
ard, or "Society Screw," see : Trans. Roy. Micr. Soc. , 1857, pp. 39-41 ; 1859, pp.
92-97 ; 1860, pp. 103-104. (All to be found in Quar. Jour. Micr. Sci., o. s., vols.
VI, VII and VIII). Proc. Amer. Micr. Soc. 1884, p. 274; 1886, p. 199 ; 1893, p.
38. Journal of the Royal Microscopical Society, August, 1896.

In this last paper of four pages the matter is very carefully gone over and full
specifications of the new screw given. It conforms almost exactly with the orig-
inal standard adopted by the society, but means have been devised by which it
may be kept standard.

§124. Standard Size Oculars and Substage Condensers. — For a considera-
tion of these, with measurements, see f 46, 87.

MARKERS AND MECHANICAL STAGES

Markers are devices to facilitate the finding of some object or part which it is
especially desired to refer to again or to demonstrate to a class. The mechanical
stage makes it much easier to follow out a series of objects, to move the slide
when using high powers, and for complete exploration of a preparation. Most of
the mechanical stages have scales or scales and verniers by which an object once
recorded may be readily found again.

66

LABORATORY MICROSCOPES

{_CH.

\ 125. Marker for Preparations. (Figs. 61-66). — This instrument consists of an
objective-like attachment which may be screwed into the nose-piece of the micro-
scope. It bears on its lower end (Figs. 61-3) a small brush and the brush can be
made more or less eccentric and can be rotated, thus making a larger or smaller
circle. In using the marker the brush is dipped in colored shellac or other cement
and when the part of the preparation to be marked is found and put exactly in the
middle of the field the objective is turned aside and the marker turned into posi-
tion. The brush is brought carefully in contact with the cover-glass and rotated.
This will make a delicate ring of the colored cement around the object. Within
this very small area the desired object can be easily found on any microscope.
The brush of the marker should be cleaned with 95% alcohol after it is used.
(Proc. Amer. Micr. Soc., 1894, pp. 112-118).

61.

62.

63-

FIGS. 61-63. Sectional Views of the two Forms of the Marker.

FIG. 61. The simplest form of marker. It consists of the part SS with the
milled edge (M}. This part bears the society or objective screw for attaching the
marker to the microscope. R. Rotating part of the marker. This bears the eccen-
tric brush (B} at its lower end. The brush is on a wire ( W). This wire is eccen-
tric, and may be made more or less so by bending the wire. The central dotted
line coincides with the axis of the microscope. The revolving part is connected
with the ''Society Screw" by the small screw (S }.

FIG. 62. 61S, R, and B. All parts same as with Fig. 6s, except that the brush
is carried by a sliding cylinder the end view being indicated in Fig. <5j.

CH. //]

LABORATORY MICROSCOPES

67

66

FIGS. 64, 65, 66. Specimens Showing the Use of the Marker.

In Fig. 64 a section of a series is marked to indicate that this section shows some-
thing especially well. In Fig. 65 some blood corpuscles showing ingested carbon
very satisfactorily are surrounded by a minute ring, and in Fig. 66 the lines of a
micrometer are ringed to facilitate finding the lines.

\ 126. Pointer in the Ocular. — The Germans have a pointer ocular (Spitzen-
Okular), an ocular with one or two delicate rods or pointers at the level of the
real image, that is, at the level of the diaphragm (Figs. 21, 30 D). For the pur-
poses of demonstrating any particular structure or object in the field, a temporary
pointer may be easily inserted in any ocular as follows : Remove the eye-lens and
with a little mucilage or Canada Balsam fasten an eye-lash (cilium) to the diaphragm
(Fig. 30 D) so that it will project about half way across the opening. If one uses
this ocular, the pointer will appear in the field and one can place the specimen so
that the pointer indicates it exactly, as in using a pointer on a diagram or on the
black-board. It is not known to the author wrho devised this method. It is cer-
tainly of the greatest advantage in demonstrating objects like amoebas or white
blood corpuscles to persons not familiar with them, as the field is liable to have in
it many other objects which are more easily seen.

\ 127. Mechanical Stage. — For High School and ordinary laboratory work a
mechanical stage is not needed ; but for much work, especially where high objec-
tives are used a mechanical stage is of great advantage. It is also advantageous if
the mechanical stage can be easily removed (see Figs. 67 to 70). The one found
on the most expensive American and English microscopes for the last twenty years
and the one now present on the larger continental microscopes, is excellent for
high powers and preparations of moderate dimensions, but for the study of serial
sections and large sections or preparations in general, mechanical stages like those
shown in Figs. 68-69 are more useful. This form of mechanical stage has the
advantage of giving great lateral and forward and backward motion. It is a mod-
ification of the mechanical stage of Tolles. The modification consists in doing
away with the thin plate and having a clamp to catch the ends of the glass slide.
The slide is then moved on the face of the stage proper. This modification was
first made by Mayall. It has since been modified by Reichert, Zeiss, Leitz, and
others in Europe and by the Bausch & Lomb Optical Co. , Queen & Co. , and the
Spencer Lens Co., in America. — Jour. Roy. Micr. Soc., 1885, p. 122. See also
Zeit. Wiss. Mikroskopie ( II) , 1885, pp. 289-295 ; 1887 (IV, pp. 25-30).

Those figured below have the great advantage of ready removal from the stage
of the microscope, thus leaving it free. They have also the very excellent feature
that with them one can explore an entire slide full of serial sections, as the sec-
tions are ordinarily mounted, i. e., under a cover-glass 24X50 mm.

68

LA BORA TOR Y MICROSCOPES

\CH. II

FIG

FIGS. 67, 6ya. The removable mechanical stage of the Bausch & Lomb Opti-
cal Company. In the upper figure it is in position on the stage of the microscope ;
in the lower figure only the clamping part is in position, the rest having been
removed to leave the stage of the microscope free.

CH. II]

LA BORA TOR Y MICROSCOPES

69

FIG. 68.

FIG. 68a.

FIG. 68, 68a. Two forms of removable mechanical stage by Leitz. 68 is some-
what more complex and expensive. Both have the desirable features mentioned
in \ 127.

yo

LABORATORY MICROSCOPES

[CH. II

FIG. 69. The removable mechanical stage of the Spencer Lens Co. It is1 a
modification of a form devised by Winkel. Besides the general features mentioned
in \. 127 it has the advantage of fitting any square stage. It is fastened to the stage
by the clamps shown at the right. Another form, is made having one screw on
the side.

CH. //] LABORATORY MICROSCOPES 71

FIG. 70. Krauss1 Method of
Marking Objectives on a Revolving
Nose- Piece.

As seen in the figure, the equiv-
alent focus of the objective is en-
graved on the diaphragm above the
back lens and may be very readily
seen in rotating the nose-piece. This
is of great advantage, as one can see
what objective is coming into place
without trouble. It is also an ad-
vantage in showing where each ob-
jective belongs when the microscope
comes from the ma n ufacturers. The
method is coming into general use.

FIGURES OF LABORATORY AND HIGH SCHOOL
MICROSCOPES

In order that teachers and students may get a good general idea of the appear-
ance of microscopes of various makers for high school and advanced laboratory
work a few pictures are appended of the microscopes most used in the United
States. This has been rendered possible by the courtesy of the manufacturers or
importers. The microscopes are arranged in alphabetical order of the makers.

Laboratory microscopes which will answer nearly all the requirements for
work in Biology, including Histology, Embryology, Pathology and Bacteriology,
are listed in the makers catalogs at about $100.00. The less expensive micro-
scopes shown are listed at $25 to $45. There is usually a discount of 10% or more
from these prices. Fortunately in the State of New York the State pays half for
high school apparatus, so that there is no reason why every high school should
not be properly equipped with microscopes of a good grade. To avoid misunder-
standing it should be added that the quality of the oculars and objectives on the
high school microscopes figured is the same as for the best laboratory micro-
scopes. The mechanical work also is of excellent quality.

During the last ten years great vigor has been shown in the microscopical
world. This has been stimulated largely by the activity in biological science and
the widespread appreciation of the microscope, not only as a desirable, but as a
necessary instrument for study and research. The production of the new kinds
of glass, (Jena glass), and the apochromatic objectives has been a no less potent
factor in promoting progress. The student is advised to write to one or more of
the opticians for complete catalogs. (See list, p. 2 of cover).

LABORATORY MICROSCOPES

[CJL II

PATENT APPLIED FOR

FIG. 71. The new cone movement, fine adjustment for the Photo-Micrographic
and the Nos. 20 and 35 Microscopes of the Spencer Lens Co. See the figures of those
microscopes pp. 82-87.

FIG. 71 a. Capped balsam bottle of WhitalL Tatum & Co. This is far more
satisfactory than the small spirit lamp figured on p. 172.

Bausch & Lomb Continental Type Microscope BB.

Fig. //. — The most common type in American University Laboratories.

Bausch & Lomb Continental Type Microscope CA.

Fig. 72. — Especially adapted for Bacteriological and
other work requiring large stage room.

Bausch & Lomb Continental Type Microscope DD.

f& 73- — This instrument is adapted for every kind of work, especially
critical Cytological. Bacteriological and Photomicrographic studies.

Bausch & Lomb Petrographical Microscope LA.

Fig. /jvz. — Specially designed throughout for Petrography.

Bausch & Lomb Continental Microscope AC.

Fig. fjb. — A simple construction, very durable and low in price.

Bausch & Lomb Continental Type Microscope B.

A type adapted to High School work when a low-priced instrument

is required.

CH. //]

LABORATORY MICROSCOPES

75

FIG. 74. R. & J. Beck's New Continental Microscope, No. 1125 ( Williams,
Brown & Earle, Philadelphia}.

76

LABOR A TOR Y MICROSCOPES

[_CH.

FIG. 75. E. Leitz Microscope II C. ( Wm. Krafft, New York}.

FIG. 75 a. The new microscope of Leitz -with large stage space, large tube
and new form fine adjustment (see over}.

FIGS. 75, b, c. New Fine Adjustment of Leitz. The movement is produced
by a cam rotating on a fixed axis (/). The cam is moved by a worm wheel (d)
which engages the thread of an endless screw (a] from two sides (Fig. 75).

One complete turn of the endless screw (a] produces an adjustment of i-ioth
mm. By means of the graduated drum (r), on the axis of the screw, one can
easily read i-iooth of a revolution and thus determine an adjustment of i-ioooth
mm. Cuts loaned by Wm. Krafft, N. Y.

CH. II ]

LABORATORY MICROSCOPES

77

FIG. 76. Leitz Microscope HE. It will be noticed that this microscope has no
joint for inclination ( Wm. Krafft, N. Y. ).

LABORATORY MICROSCOPES

[CfJ. II

FIG. 77. NacheVs Microscope 6 bis. Old model. Nachet is a successor to
Hartnack who introduced the present "Continental Model'1'1 May all, Cantor Lec-
tures, p. 68 (Franklin Laboratory Supply Co., Boston}.

CH. //]

LABORATORY MICROSCOPES

79

FIG. 78. NacheVs Microscope, No. u (Franklin Laboratory Supply Co., Boston).

This microscope has no joint for inclination, and no rack and pinion for coarse
adjustment. For coarse adjustment, the tube is pulled up and down with the hands.
This kind of coarse adjustment was much more common ten years ago than now.
(See also Fig. 82. )

8o

LABOR A TOR Y MICROSCOPES

[CH. II

FIG. 79. A. Queen & Co's Continental Microscope , No. II. B. Dust-prooj,
triple nose-piece. The difference between this and the ordinary form can be seen
by comparing with Fig. 36. This form of revolving nose-piece has been made for
many years by Winkel of Goettingen.

CH. 77]

LABORATORY MICROSCOPES

81

FIG. 80. Queen & Co's Acme Microscope for Schools.

82

LABORATORY MICROSCOPES

\_CH.

FIG. 81. Reichert's New Mi-
croscope, No. Ill B. (Richards
& Co., N. Y. and Chicago}.

Fig. 82. The Spencer Lens Company's Microscope No. 1.

1903 Model.

Fig. 83. The Spencer L,ens Company's Microscope No. 2.
general science work in college and high school
laboratories. Latest model.

For

Fig. 84. The Spencer Lens Company's Microscope No. 5. For
professional and laboratory use.

Fig. 85. The Spencer Lens Company's Microscope No. 6.
daily designed for photo-micrography, but equally
well suited for ordinary work.

Espe-

The Spencer Lens Company's New No. 4 Stand, with special fine
adjustment and handle for carrying.

Patented June 24, 1902.

Fig. 85a. The Spencer L,ens Company's New Attachable
Mechanical Stage. Both movements may be operated by the
fingers of one hand, the milled heads being placed together at one
end with coincident axes. At the other end is a milled head for
the lateral movement so that either hand may be used therefor.
The center guide screw furnishes a stop for the slide carrier so
that the objective cannot be injured,

CH. //]

LABORATORY MICROSCOPES

FIG. 86. Zeiss Microscope /a with Mechanical Stage. This figure from Zeiss'
Catalog No. 30, represents the Continental Model of Microscope in its most perfect
form.

K. Milled head of the screw for the lateral movements of the stage.

L. Screw for fixing the laterally moving mechanism of the stage. By un-
screwing this the laterally moving part may be removed, leaving the plain stage.
W. Screw for moving the stage forward and backward.

88

LABOR A TOR Y MICROSCOPES

[CH. II

FIG. 87, Zcntmeyer's Microscope, A7o. V.

CH. //]

LA BORA TOR Y MICROSCOPES

89

FIG. 88. Zentmayer's Microscope, Xo. IV.

CHAPTER III

INTERPRETATION OF APPEARANCES

APPARATUS AND MATERIAL FOR CHAPTER III

A laboratory, compound microscope ( \ 121 ); Preparation of fly's wing ; 50 per
cent, glycerin; Slides and covers; Preparation of letters in stairs (Fig. 89);
Mucilage for air-bubbles and olive or clove oil for oil-globules (\ 136-139). Solid
glass rod, and glass tube (\ 144-146); Collodion ($ 146); Carmine, India ink, or
lamp black (§ 148-150); Frog, castor oil and micro-polariscope (§ 152).

INTERPRETATION OF APPEARANCES UNDER THE MICROSCOPE

§ 129. General Remarks. — The experiments in this chapter are
given secondarily for drill in manipulation, but primarily so that the
student may not be led into error or be puzzled by appearances which
are constantly met with in microscopical investigation. Anyone can
look into a microscope, but it is quite another matter to interpret cor-
rectly the meaning of the appearances seen.

It is especially important to remember that the more of the relations
of any object are known, the truer is the comprehension of the object.
In microscopical investigation every object should be scrutinized from
all sides and under all conditions in which it is likely. to occur in nature
and in microscopical investigation. It is best also to begin with objects
of considerable size whose character is well known, to look at them
carefully with the unaided eye so as to see them as wholes and in their
natural setting ; then a low power is used, and so on, step by step until
the highest power available has been employed. One will in this way
see less and less of the object as a whole, but every increase in magnifi-
cation will give increased prominence to detail, detail which might be
meaningless when taken alone and independent of the object as a
whole. The pertinence of this advice will be appreciated when the
student undertakes to solve the problems of histology ; for even after all
the years of incessant labor spent in trying to make out the structure
of man and the lower animals, many details are still in doubt, the
same visual appearances being quite differently interpreted by eminent
observers.

CH. Ill] INTERPRETATION OF APPEARANCES 91

Appearances which seem perfectly unmistakable with a low power
may be found erroneous or very inadequate, for details of structure that
were indistinguishable with the low power may become perfectly evi-
dent with a higher power or a more perfect objective. Indeed the prob-
lems of microscopic structure appear to become ever more complex, for
difficulties overcome by improvements in the microscope simply give
place to new difficulties, which in some cases render the subject more
obscure than it appeared to be with the less perfect appliances.

The need of the most careful observation and constant watchful-
ness lest the appearances may be deceptive are thus admirably stated
by Dallinger (see Carpenter-Dallinger, p. 427): "The correctness of
the conclusions which the microscopist will draw regarding the nature
of any object from the visual appearances which it presents to him
when examined in the various modes now specified will necessarily
depend in a great degree upon his previous experience in microscopic
observation and upon his knowledge of the class of bodies to which
the particular specimen may belong. Not only are observations of
any kind liable to certain fallacies arising out of the previous notions
which the observer may entertain in regard to the constitution of the
objects or the nature of the actions to which his attention is directed,
but even the most practiced observer is apt to take no note of such
phenomena as his mind is not prepared to appreciate. Errors and im-
perfections of this kind can only be corrected, it is obvious, by general
advance in scientific knowledge ; but the history of them affords a use-
ful warning against hasty conclusions drawn from a too cursory exam-
ination. If the history of almost any scientific investigation were
fully made known it would generally appear that the stability and
completeness of the conclusions finally arrived at had been only
attained after many modifications, or even entire alterations, of doctrine.
And it is therefore of such great importance as to be almost essential
to the correctness of our conclusions that they should not be finally
formed and announced until they have been tested in every conceivable
mode. It is due to science that it should be burdened with as few false
facts [artifacts] and false doctrines as possible. It is due to other
truth-seekers that they should not be misled, to the great waste of
their time and pains, by our errors. And it is due to ourselves that
we should not commit our reputation to the chance of impairment by
the premature formation and publication of conclusions which may be
at once reversed by other observers better informed than ourselves, or
may be proved fallacious at some future time, perhaps even by our

92 INTERPRETATION OF APPEARANCES [Cff. Ill

own more extended and careful researches. The suspension of the judg-
ment whenever there seems room for doubt is a lesson inculcated by all
those philosophers who have gained the highest repute for practical
wisdom ; and it is one which the microscopist cannot too soon learn or
too constantly practice."

For these experiments no condenser is to be used except where
specifically indicated.

§ 130. Dust or Cloudiness on the Ocular. — Employ the 16
mm. (2/i in.) objective, low ocular, and fly's wing as object.

Unscrew the field-lens and put some particles of lint from dark
cloth on its upper surface. Replace the field-lens and put the ocular
in position (§ 48). Light the field well and focus sharply. The im-
age will be clear, but part of the field will be obscured by the irregular
outline of the particles of lint. Move the object to make sure this
appearance is not due to it.

Grasp the ocular by the milled ring, just above the tube of the
microscope, and rotate it. The irregular objects will rotate with the
ocular. Cloudiness or particles of dust on any part of the ocular may
be detected in this way.

§ 131. Dust or Cloudiness on the Objective. — Employ the
same ocular and objective as before and the fly's wing as object. Focus
and light well, and observe carefully the appearance. Rub glycerin
on one side of a slide near the end. Hold the clean side of this end
close against the objective. The image will be obscured, and cannot
be made clear by focusing. Then use a clean slide and the image may
be made clear by elevating the tube slightly. The obscurity produced
in this way is like that caused by clouding the front-lens of the objec-
tive. Dust would make a dark patch on the image that would remain
stationary while the object or ocular is moved.

If a small diaphragm is employed and it is close to the object,
only the central part of the field will be illuminated, and around the
small light circle will be seen a dark ring (Fig. 42). If the diaphragm
is lowered or a sufficiently large one employed the entire field will be
lighted.

§ 132. Relative Position of Objects or parts of the same
object. The general rule is that objects highest up come into focus
last in focusing up,y?r5/ in focusing down.

§ 133. Objects having Plane or Irregular Outlines. — As object
use three printed letters in stairs mounted in Canada balsam (Fig. 89).
The first letter is placed directly upon the slide, and covered with a

CH. Ill] INTERPRETA TION OF APPEARANCES 93

small piece of glass about as thick as a slide. The second letter is
placed upon this and covered in like manner. The third letter is placed
upon the second thick cover and covered with an ordinary cover- glass.
The letters should be as near together as possible, but not over-lapping.
Employ the same ocular and objective as above (§ 130).

• FIG. 89. Letters mounted in stairs to

show the order of coming into focus.

d _

; \ A g. . a, 6, c, d. The various letters indi-

cated by the oblique row of black marks in
sectional view. Slide. The glass slide on which the letters are mounted.

Lower the tube till the objective almost touches the top letter, then
look into the microscope, and slowly focus up. The lowest letter will
first appear, and then, as it disappears, the middle one will appear, and
so on. Focus down, and the top letter will first appear, then the mid-
dle one, etc. The relative position of objects is determined exactly in
this way in practical work.

For example, if one has a micrometer ruled on a cover-glass 15-25
hundredths mm. thick, it is not easy to determine with the naked eye
which is the ruled surface. But if one puts the micrometer under a
microscope and uses a 3 mm.(*^ in.) objective, it is easily determined.
The cover should be laid on a slide and focused till the lines are sharp.
Now, without changing the focus in the least turn the cover over. If
it is necessary to focus up to get the lines of the micrometer sharp, the
lines are on the upper side. If one must focus down, the lines are on
the under surface. With a thin cover and delicate lines this method of
determining the position of the rulings is of considerable practical
importance.

§ 134. Determination of the Form of Objects. — The procedure
is exactly as for the determination of the form of large objects. That
is, one must examine the various aspects. For example, if one were
placed in front of a wall of some kind he could not tell whether it was
a simple wall or whether it was one side of a building unless in some
way he could see more than the face of the wall. In other words, in
order to get a correct notion of any body, one must examine more than
one dimension, — two for plane surfaces, three for solids. So for micro-
scopic objects, one must in some way examine more than one face. To
do this with small bodies in a liquid the bodies may be made to roll
over by pressing on one edge of the cover-glass. And in rolling over
the various aspects are presented to the observer. With solid bodies,

94

INTERPRETATION OF APPEARANCES

\_CH. Ill

like the various organs, correct notions of the form of the elements can
be determined by studying sections cut at right angles to each other.
The methods of getting the elements to roll over, and of sectioning in
different planes are in constant use in Histology, and the microscopist
who neglects to see all sides of the tissue elements has a very inade-
quate and often a very erroneous conception of their true form.

§ 135. Transparent Objects having Curved Outlines. — The
success of these experiments will depend entirely upon the care and skill
used in preparing the objects, in lighting, and in focusing.

Employ a 3 mm. (^ in.) or higher objective and a high ocular for
all the experiments. It may be necessary to shade the object (§ 109)
to get satisfactory results. When a diaphragm is used the opening
should be small and it should be close to the object.

§ 136. Air Bubbles. — Prepare these by placing a drop of thin
mucilage on the center of a slide and beating it with a scalpel blade
until the mucilage looks milky from the inclusion of air bubbles. Put
on a cover-glass but do not press it down.

FIG. 90. Diagram show-
ing how to place a' cover-
glass upon an object with the
forceps.

§ 137. Air Bubbles with Central Illumination. — Shade the
object ; and with the plane mirror, light the field with central light
(Fig. 23).

Search the preparation until an air bubble is found appearing
about i mm. in diameter, get it into the center of the field, and if the
light is central the air bubble will appear with a wide, dark, circular
margin and a small bright center. If the bright spot is not in the
center, adjust the mirror until it is.

This is one of the simplest and surest methods of telling when the
light is central or axial when no condenser is used (§ 65).

Focus both up and down, noting that, in focusing up, the central
spot becomes very clear and the black ring very sharp. On elevating
the tube of the microscope still more the center becomes dim, and the
whole bubble loses its sharpness of outline.

§ 138. Air Bubbles with Oblique Illumination. — Remove the
sub-stage of the microscope and all the diaphragms. Swing the mirror
so that the rays may be sent very obliquely upon the object (Fig. 2^

CH. Ill} INTERPRE TA TION OF APPEARANCES 95

C). The bright spot will appear no longer in the center but on the
side away from the mirror (Fig. 91).

§ r39- Oil Globules. — Prepare these by beating a small drop of
clove oil with mucilage on a slide and covering as directed for air bub-
bles (§ 137), or use a drop of milk.

§ 140. Oil Globules with Central Illumination. — Use the same
diaphragm and light as above (§137). Find an oil globule appearing
about i mm. in diameter. If the light is central a bright spot will ap-
pear in the center as with air. Focus up and down as with air, and
note that the bright center of the oil globule is clearest last in focus-
ing up.

FIG. 91. Very small Globule of Oil (O) and an Air Bubble A

(A) seen by Oblique Light. The arrow indicates the direction of
the light rays.

§ 141. Oil Globules with Oblique Illumination.—
Remove the sub-stage, etc., as above, and swing the mir-
ror to one side and light with oblique light. The bright
spot will be eccentric, and will appear to be on the same
side as the mirror (Fig. 91).

§ 142. Oil and Air Together. — Make a prepara-
tion exactly as described for air bubbles (§ 136), and add
at one edge a little of the mixture of oil and mucilage
(§ T39) ; cover and examine.

The sub-stage need not be used in this experiment. Search the
preparation until an air bubble and an oil globule, each appearing
about i mm. in diameter, are found in the same field of view. Light
first with central light, and note that, in focusing up, the air bubble
comes into focus first and that the central spot is smaller than that of
the oil globule. Then, of course, the black ring will be wider in the
air bubble than in the oil globule. Make the light oblique. The
bright spot in the air bubble will move away from the mirror while
that in the oil globule will move toward it. See Fig. 91.*

§ 143. Air and Oil by Reflected Light. — Cover the diaphragm
or mirror so that no transmitted light (§ 64) can reach the preparation,
using the same preparation as in § 142. The oil and air will appear
like globules of silver on a dark ground. The part that was darkest in

*It should be remembered that the image in the compound microscope is
inverted (Fig. 21), hence the bright spot really moves toward the mirror for air,
and away from it for oil.

96 INTERPRETATION OF APPEARANCES [C7/. Ill

each will be lightest, and the bright central spot will be somewhat
dark.*

§ 144. Distinctness of Outline. — In refraction images this
depends on the difference between the refractive power of a body and
that of the medium which surrounds it. The oil and air were very
distinct in outline as both differ greatly in refractive power from the
medium which surrounds them, the oil being more refractive than the
mucilage and the air less. (Figs. 54-56.)

Place a fragment of a cover-glass on a clean slide, and cover it
(see under mounting). The outline will be distinct with the unaided
eye. Use it as object and employ the 16 mm. (^i in.) objective and
high ocular. Light with central light. The fragment will be outlined
by a dark band. Put a drop of water at the edge of the cover-glass.
It will run in and immerse the fragment. The outline will remain dis-
tinct, but the dark band will be somewhat narrower. Remove the
cover-glass, wipe it dry, and wipe the fragment and slide dry also.
Put a drop of 50% glycerin on the middle of the slide and mount the
fragment of cover-glass in that. The dark contour will be much nar-
rower than before.

Draw a solid glass rod out to a fine thread. Mount one piece in
air, and the other in 50% glycerin. Put a cover-glass on each. Em-
ploy the same optical arrangement as before. Examine the one in air
first. There will be seen a narrow, bright band, with a wide, dark
band on each side.

The one in glycerin will show a much wider bright central band,
with the dark borders correspondingly narrow (Fig. 92, b). The dark
contour depends also on the numerical aperture of the objective — being
wider with low apertures. This can be readily understood when it is
remembered that the greater the aperture the more oblique the rays of
light that can be received, and the dark band simply represents an
area in which the rays are so greatly bent or refracted (Figs. 54-56)
that they cannot enter the objective and contribute to the formation of
the image ; the edges are dark simply because no light from them
reaches the observer.

*It is possible to distinguish oil and air optically, as described above, only
when quite high powers are used and very small bubbles are selected for observa-
tion. If a 1 6 mm. (% in.) is used instead of a 3 mm. (ft in.) objective, the ap-
pearances will vary considerably from that given above for the higher power. It
is well to use a low as well as a high power. Marked differences will also be
seen in the appearances with objectives of small and of large aperture.

CH. Ill ] INTERPRE TA TION OF APPEARANCES 97

FIG. 92. Solid glass rod showing the
appearance when viewed with transmit-
ted, central light, and with an objective
of medium aperture.

a. Mounted in air. b. Mounted in 50 per cent, glycerin.

If the glass rod or any other object were mounted in a medium of
the same color and refractive power, it could not be distinguished from
the medium.*

A very striking and satisfactory demonstration may be made by
painting a zone or band of eosin or other transparent color on a solid
glass rod, and immersing the rod in a test tube or vial of cedar oil;
clove oil or turpentine. Above the liquid the glass rod is very evident,
as it is also at the colored zone, but at other levels it can hardly be
seen in the liquid.

§ 145. Highly Refractive. — This expression is often used in de-
scribing microscopic objects, (medullated nerve fibers, for example),
and means that the object will appear to be bordered by a wide, dark
margin when it is viewed by transmitted light. And from the above
(§ 144), it would be known that the refractive power of the object, and
the medium in which it was mounted must differ considerably.

§ 146. Doubly Contoured. — This means that the object is
bounded by two, usually parallel dark lines with a lighter band between
them. In other words, the object is bordered by (i) a dark line, (2) a
light band, and (3) a second dark line (Fig. 93).

This may be demonstrated by coating a fine glass rod (§ 144) with
one or more coats of collodion or celloidin and allowing it to dry, and
then mounting in 50% glycerin as above. Employ a 3 mm. (^6 in.) or
higher objective, light with transmitted light, and it will be seen that
where the glycerin touches the collodion coating there is a dark line —
next this is a light band, and finally there is a second dark line where
the collodion is in contact with the
glass rod.* (Fig. 93).

FIG. 93. Solid glass rod coated with col- 1
lodion to show a double contour. Toward I
one end the collodion had gathered in afusi- \
form drop.

*Some of the rods have air bubbles in them, and then there results a capillary
tube when they are drawn out. It is well to draw out a glass tube into a fine
thread and examine it as described. The central cavity makes the experiment
much more complex.

98 INTERPRETATION OF APPEARANCES \_CH. HI

§ 147. Optical Section. — This is the appearance obtained in
examining transparent or nearly transparent objects with a microscope
when some plane below the upper surface of the object is in focus.
The upper part of the object which is out of focus obscures the image
but slightly. By changing the position of the objective or object, a
different plane will be in focus and a different optical section obtained.
The most satisfactory optical sections are obtained with high objectives
having large aperture.

Nearly all the transparent objects studied may be viewed in optical
section. A striking example will be found in studying mammalian
red blood-corpuscles on edge. The experiments with the solid glass
rods (Fig. 92) furnish excellent and striking examples of optical
sections.

§ 148. Currents in Liquids. — Employ the 16 mm. (^ in.) ob-
jective, and as object put a few particles of carmine on the middle of a
slide, and add a drop of water. Grind the carmine well with a scalpel
blade, and then cover it. If the microscope is inclined, a current will
be produced in the water, and the particles of carmine will be carried
along by it. Note that the particles seem to flow up instead of down —
why is this ?

Lamp-black rubbed in water containing a little mucilage answers
well for this experiment.

§ 149. Velocity Under the Microscope. — In studying currents
or the movement of living things under the microscope, one should
not forget that the apparent velocity is as unlike the real velocity as
the apparent size is unlike the real size. If one consults Fig. 37 it
will be seen that the actual size of the field of the microscope with the
different objectives and oculars is inversely as the magnification. That
is, with great magnification only a small area can be seen. The field
appears to be large, however, and if any object moves across the field
it may appear to move with great rapidity, whereas if one measures
the actual distance passed and notes the time, it will be seen that the
actual motion is quite slow. One should keep this in mind in study-
ing the circulation of the blood. The truth of what has just been
said can be easily demonstrated in studying the circulation in the gills

*The collodion used is a 6% solution of gun cotton in equal parts of sulphuric
ether and 95% alcohol. It is well to dip the rod two or three times in the collo-
dion and to hold it vertically while drying. The collodion will gather in drops,
and one will see the difference between a thick and a thin membranous covering.
(Fig. 93).

CH. Ill} INTERPRETATION OF APPEARANCES 99

of Necturus, or in the frog's foot, by using first a low power in which
the field is actually of considerable diameter (Fig. 37, Table, § 51) and
then using a high power. With the high power the apparent motion
will appear much more rapid. For spiral, serpentine and other forms
of motion, see Carpenter-Dallinger, p. 433.

§ 150. Pedesis or Brownian Movement. — Employ the same
object as above, but a 3 mm. (^i in.) or higher objective in place of
the 1 6 mm. Make the body of the microscope vertical, so that there
may be no currents produced. Use a small diaphragm and light the
field well. Focus and there will be seen in the field large motionless
masses, and between them small masses in constant motion. This is
an indefinite, dancing or oscillating motion.

This indefinite but continuous motion of small particles in a liquid
is called Pe-de'sis or Brownian movement. Also, but improperly, molec-
ular movement, from the smallness of the particles.

The motion is increased by adding a little gum arabic solution or
a slight amount of silicate of soda or soap ; sulphuric acid and various
saline compounds retard or check the motion. One of the best objects
is lamp-black ground up with a little gum arabic. Carmine prepared
in the same way, or simply in water, is excellent ; and very finely
powdered pumice-stone in water has for many years been a favorite
object.

Pedesis is exhibited by all solid matter if it is finely enough di-
vided and in a suitable liquid. In the minds of most, no adequate
explanation has yet been offered. See Carpenter-Dallinger, p. 431 ;
Beale, p. 195 ; Jevons in Quart. Jour. Science, n. s., Vol. VIII (1878),
p. 167. In 1894, Meade Bache published a paper in the Proc. Amer.
Philos. Soc., Vol. XXXIII, pp. 163-167, entitled "The Secret of
the Brownian Movement." This paper is suggestive if not very
satisfactory.

For the orginal account of this see Robert Brown, "Botanical
appendix to Captain King's voyage to Australia," Vol. II, p. 534.
(1826).

See also Dr. C. Aug. Sigm. Schultze, "Mikroskopische Unter-
suchungen u'ber des Herren Robert Brown Entdeckunglebender, selbst
im Feuer unzerstorbarer Theilchen in alien Korpern." From "Die
Gesellschaft fur Belorderung der Naturwissenschaften zu Freiburg. ' '
1828.

Compare the pedetic motion with that of a current by slightly in-
clining the tube of the microscope. The small particles will continue

ioo INTERPRE TA TION OF APPEARANCES . [CH. Ill

their independent leaping movements while they are carried along by
the current. The pedetic motion makes it difficult to obtain good
photographs of milk globules and other small particles. The difficulty
may be overcome by mixing the milk with a very weak solution of
gelatin and allowing it to cool (see Ch. .IX).

§ 151. Demonstration of Pedesis with the Polarizing Micro-
scope (Ch. VI). — The following demonstration shows conclusively
that the pedetic motion is real and not illusive. (Ranvier, p. 173.)

Open the abdomen of a dead frog (an alcoholic or formalin
specimen is satisfactory). Turn the viscera to one side and observe
the small, whitish masses at the emergence of the spinal nerves. With
fine forceps remove one of these and place it on the middle of a clean
slide. Add a drop of water, or of water containing a little gum arabic.
Rub the white mass around in the drop of liquid and soon the liquid
will have a milky appearance. Remove the white mass, place a cover-
glass on the milky liquid and seal the cover by painting a ring of
castor oil all around it, half the ring being on the slide and half
on the cover-glass. This is to avoid the production of currents by
evaporation.

Put the preparation under the microscope and examine with, first
a low then a high power (3 mm. or ^ in.). In the field wilt be seen
multitudes of crystals of carbonate of lime ; the larger crystals are
motionless but the smallest ones exhibit marked pedetic movement.

Use the micro-polariscope, light with great care and exclude all
adventitious light from the microscope by shading the object (§ 109) and
also by shading the eye. Focus sharply and observe the pedetic motion
of the small particles, then cross the polarizer and analyzer, that is,
turn one or the other until the field is dark. Part of the large motion-
less crystals will shine continuously and a part will remain dark, but
small crystals between the large ones will shine for an instant, then
disappear, only to appear again the next instant. This demonstration
is believed to furnish absolute proof that the pedetic movement is real
and not illusory.

§ 152. Muscae Volitantes. — These specks or filaments in the
eyes due to minute shreds or opacities of the vitreous sometimes appear
as part of the object as they are projected into the field of vision. They
may be seen by looking into the well lighted microscope when there is
no object under the microscope. They may also be seen by looking
at brightly illuminated snow or other white surface. By studying
them carefully it will be seen that they are somewhat movable and float

CH. III INTERPRETA TION OF APPEARANCES 101

across the field of vision, and thus do not remain in one position as do
the objects under observation. Furthermore, one may, by taking a
little pains, familiarize himself with the special forms in his own eyes
so that the more conspicuous at least may be instantly recognized.

§ 153. In addition to the above experiments it is very strongly
recommended that the student follow the advice of Beale, p. 248, and
examine first with a low then with a higher power, mounted dry,. then
in water, lighted with reflected light, then with transmitted light, the
following : Potato, wheat, rice, and corn starch, easily obtained by
scraping the potato and the grains mentioned ; bread crumbs ; portions
of feather. Portions of feather accidentally present in histological
preparations have been mistaken for lymphatic vessels (Beale, 288).
Fibers of cotton, linen and silk. Textile fibers accidentally present
have been considered nerve fibers, etc. Human and animal hairs.
Study with especial care hairs from various parts of the body of the
animals used for dissection in the laboratory where you work. These
are liable to be present in histological preparations, and unless their
character is understood there is chance for much confusion and erro-
neous interpretation. The scales of butterflies and moths, especially
the common clothes moth. The dust swept from carpeted and wood
floors. Tea leaves and coffee grounds. Dust found in living rooms
and places not frequently dusted. In the last will be found a regular
museum of objects.

For figures (photo-micrographs, etc.) of the various forms of starch,
see Bulletin No. 13 of the Chemical Division of the U. S. Department
af Agriculture. For Hair and Wool, see Bulletin of the National Asso-
ciation of Wool Growers, 1875, P- 47°> Proc. Amer. Micr. Soc., 1884,
pp. 65-68. Herzfeld, translated by Salter. — The technical testing of
yarns and textile fabrics, London, 1898.

For different appearances due to the illuminator, see Nelson, in
Jour. Roy, Micr. Soc., 1891, pp. 90-105 ; and for the illusory appear-
ances due to diffraction phenomena, see Carpenter-Dallinger, p. 434.
Mercer. Trans. Amer. Micr. Soc., pp. 321-396.

If it is necessary to see all sides of an ordinary gross object, and
to observe it with varying illumination and under various conditions of
temperature, moisture, etc., in order to obtain a fairly accurate and
satisfactory knowledge of it, so much the more is it necessary not to be
satisfied in microscopical observation until every means of investigation
and verification has been called into service, and then of the image
that falls upon the retina, only such details will be noted as the brain
behind the eye is ready to appreciate.

102

INTERPRETATION OF APPEARANCES

[CH. Ill

To summarize this chapter and leave with the beginning student
the result of the experience of many eminent workers :

1. Get all the information possible with the unaided eye. See
the whole object and all sides of it, so far as possible.

2. Examine the preparation with a simple microscope in the same
thorough way for additional detail.

3. Use a low power of the compound microscope.

4. Use a higher power.

5. Use the highest power available and applicable. In this way
one sees the object as a whole and progressively more and more details.
Then as the object is viewed from two or more aspects, something like
a correct notion may be gained of its form and structure.

THE MICROSCOPE IN SECTION

1. Positive ocular.

2. Draw-tube.

3. Main tube or body.

4-5. Society screws in the
^draw-tube and body.

6. Objective in position.

7. Stage.

8. Spring for holding

slides.

9. Sub-stage condenser.
10. Iris diaphragm.

11. Plane and concave mir-

ror.

12. Horse-shoe base.

13. Rack and pinion for

condenser.

14. Flexible pillar.

15. Spiral spring of fine ad-

justment.

16. Fine adjustment

17. Coarse adjustment.

CHAPTER IV

MAGNIFICATION AND MICROMETRY

APPARATUS AND MATERIAL FOR THIS CHAPTER

Simple and compound microscope (§ 156, 158); Steel scale or rule divided to
millimeters and iths ; Block for magnifier and compound microscope (§ 156, 160);
Dividers (§ 156, 160); Stage micrometer (g 159); Wollaston camera lucida (§ 160);
Ocular screw-micrometers (Figs. 106-107); Micrometer ocular (Figs. 104-105).
Abbe camera lucida (Fig. 101). Necturus red blood corpuscles (§ 168).

§ 154. The Magnification, Amplification or Magnifying Power
of a simple or compound microscope is the ratio between the real and
the apparent size of the object examined. The apparent size is ob-
tained by measuring the virtual image (Figs. 21, 38). The object for
determining magnification must be of known length and is designated
a micrometer (§ 159). In practice a virtual image is measured by the
aid of some form of camera lucida (Figs. 97, 101), or by double vision
(§156). As the length of the object is known, the magnification is
easily determined by dividing the apparent size of the image by the
actual size of the object. For example, if .the virtual image measures
40 mm. and the object magnified, 2 mm., the amplification must be
40 -r- 2 = 20, that is, the apparent size is 20 fold greater than the real
size.

Magnification is expressed in diameters or times linear, that is, but
one dimension is considered. In giving the scale at which a micro-
scopical or histological drawing is made, the word magnification is fre-
quently indicated by the sign of multiplication thus : X 450, upon a
drawing would mean that the figure or drawing is 450 times as large
as the object.

§ 155. Magnification of Real Images.— In this case the mag-
nification is the ratio between the size of the real image and the size of
the object, and the size of the real image can be measured directly. By
recalling the work on the function of an objective (§ 53), it will be
remembered that it forms a real image on the ground glass placed on
the top of the tube, and that this real image could be looked at with the

104

MAGNIFICATION AND MICROMETRY

[C//. IV

eye or measured as if it were an actual object. For example, suppose
the object were three millimeters long and its image on the ground glass
measured 15 mm., then the magnification must be, 15 -r- 3 = 5, that is,
the real image is 5 times as long as the object. The real images seen
in photography are mostly smaller than the objects, but the magnifica-
tion is designated in the same way by dividing the size of the real im-
age measured on the ground glass by the size of the object. For
example, if the object is 400 millimeters long and its image on the
ground glass is '25 mm. long, the ratio is 25^-400=-^. That is, the
image is ^g-th as long as the object and is not magnified but reduced.
In marking negatives, as with drawings, the sign of multiplication is
put before the ratio, and in the example the designation would be
XTVth.

MAGNIFICATION OF A SIMPLE MICROSCOPE

§ 156. The Magnification of
a Simple Microscope is the ratio
between the object magnified (Fig.
1 6, A'B'), and the virtual image
(A3B3). To obtain the size of this
virtual image place the tripod mag-
nifier near the edge of a support of
such a height that the distance
from the upper surface of the mag-
nifier to the table is 250 millimeters.

FIG. 94. Tripod Magnifier.

As object, place a scale of some kind ruled in millimeters on the
support under the magnifier. Put some white paper on the table at
the base of the support and on the side facing the light.

0

FIG. 95. Ten Centimeter Rule. The upper edge is divided into millimeters,
the lower into centimeters at the left and half centimeters at the right.

Close one eye, and hold the head so that the other will be near the
upper surface of the lens. Focus if necessary to make the image clear

CH. 1 V ] MA GNIFICA TION A ND MICRO ME TRY 105

(§ n). Open the closed eye and the image of the rule will appear as
if on the paper at the base of the support. Hold the head very still,
and with dividers get the distance between any two lines of the image.
This is the so-called method of double vision in which the microscope
image is seen with one eye and the dividers with the other, the two
images appearing to be fused in a single visual field.

§ 157. Measuring the Spread of Dividers. — This should be
done on a steel scale divided to millimeters and -J-ths.

As \ mm. cannot be seen plainly by the unaided eye, place one
arm of the dividers at a centimeter line, and with the tripod magnifier
count the number of spaces on the rule included between the points of
the dividers. The magnifier simply makes it easy to count the spaces
on the rule included between the points of the dividers — it does not, of
course, increase the number of spaces or change their value.

As the distance between any two lines of the image of the scale
gives the size of the virtual image (Fig. 16, A3B3), and as the size of
the object is known, the magnification is determined by dividing the
size of the image by the size of the object. Thus, suppose the distance
between the two lines of the image is measured by the dividers and
found on the steel scale to be 15 millimeters, and the actual size of the
space between the two lines of the object is 2 millimeters, then the
magnification must be 15^-2=7^. That is, the image is 7^ times as
long or wide as the object. In this case the image is said to be
magnified 7^ diameters, or 7^2 times linear.

The magnification of any simple magnifier may be determined
experimentally in the wray described for the tripod.

MAGNIFICATION OF A COMPOUND MICROSCOPE

§ 158. The Magnification of a Compound Microscope is the
ratio between the final or virtual image (Fig. 21, B3A3), and the object
magnified (A B).

The determination of the magnification of a compound microscope
may be made as with a simple microscope (§ 156), but this is very
fatiguing and unsatisfactory.

§ 159. Stage, Object or Objective Micrometer. — For deter-
mining the magnification of a compound microscope and for the
purpose of micrometry, it is necessary to have a finely divided scale
or rule on glass or on metal. Such a finely divided scale is called a
micrometer, and for ordinary work one mounted on a glass slide
(i X 3 in, 25 x 76 mm.) is most convenient.

io6 MAGNIFICATION AND MICROMETRY [Cff. IV

The spaces between the lines should be y1^ and y^ mm. (or if in
inches, y^ and T^TF i*1-) Micrometers are sometimes ruled on the
slide, but more satisfactorily on a cover-glass of known thickness,
preferably o. 15-0. 18 mm. The covers should be perfectly clean before
the ruling, and afterwards simply dusted off with a camel's hair
duster, and then mounted, lines downward over a shellac or other
good cell. (See Ch. VII). If one rubs the lines the edges of the
furrow made by the diamond are liable to be rounded and the sharp-
ness of the micrometer is lost. If the lines are on the slide and un-
covered one cannot use the micrometer with an oil immersion, as the
oil obliterates the lines. Cleaning the slide makes the lines less sharp
as stated. If the lines are very coarse, it is an advantage to fill them
with plumbago. This may be done either with some very fine plum-
bago on the end of a so ft cork, or by using an exceedingly soft lead
pencil. Lines properly filled may be covered with balsam and a cover-
glass as in ordinary balsam mounting (Ch. VII).

§ 1 60. Determination of Magnification. — This is most readily
accomplished by the use of some form of camera lucida (Ch. V), that
of Wollaston being most convenient as it may be used for all powers,
and the determination of the standard distance of 250 millimeters at
which to measure the images is very readily determined (Fig. 97, § 162).

Employ the 16 mm. (^i in.) objective and a 37 mm. (or X 8 ocu-
lar with a stage micrometer as object. For this power the T^ mm.
spaces of the micrometer should be used as object. Focus sharply.

FIG. 96. Diagram of a stage micrometer,
with a ring on the lines to facilitate finding them.

It is somewhat difficult to find the mi-
crometer lines. To avoid this it is well to
have a small ring enclosing some of the

micrometer lines (Fig. 96). The light must also be carefully regu-
lated. If too much light is used, i. e., too large an aperture, the lines
will be drowned in the light. In focusing with the high powers be
very careful. Remember the micrometers are expensive, and one can-
not afford to break them. As suggested in § 74, focus on the edge of
the cement ring enclosing the lines, then in focusing down to find
the lines, move the preparation very slightly, back and forth.

After the lines are sharply focused, and the slide clamped in posi-
tion make the tube of the microscope horizontal, by bending the flexible
pillar, being careful not to bring any strain upon the fine adjustment
(frontispiece).

CH.

MAGNIFICATION AND MICROMETRY

107

FIG. 97.

FIG. 97. Wollaston^s Camera Lu-
cida, showing the rays from the micro-
scope and from the drawing surface,
also the position of the pupil of the eye.

Axis, Axis. Axial rays from
the microscope and from the drawing
surface (Ch. V).

Camera Lucida. A section of the
quadrangular prism showing the
course of the rays in the prism from
the microscope to the eye. As the rays
are twice reflected, they have the same
relation on entering the eye that they
would have by looking directly into the
ocular.

A. B. The lateral rays from the
microscope and their projection upon
the drawing surface.

C. D Rays .from the drawing
surface to ths eye.

A. D. A' D' . Overlapping portions of the two fields, where both the micro-
scopic image and the drawing surf ace, pencil, etc., can be seen. It is represented
by the shaded part of the overlapping circles at the right.

Ocular. The ocular of the microscope.

P. The drawing pencil. Its point is shown in the overlapping fields.

Put a Wollaston camera lucida (Fig. 97 and Ch. V) in position,
and turn the ocular around if necessary so that the broad flat surface
may face directly upward, as shown in Fig. 97. Elevate the micro-
scope by putting a block under the base, so that the perpendicular dis-
tance from the upper surface of the camera lucida to the table is 250
mm. (§ 162). Place some white paper on the work-table beneath the
camera lucida.

Close one eye, and hold the head so that the other may be very
close to the camera lucida. Look directly down. The image will ap-
pear to be on the table. It may be necessary to readjust the focus
after the camera lucida is in position. If there is difficulty in seeing
dividers and image consult Ch. V. Measure the image with dividers
and obtain the power exactly as above (§156-157).

Thus : Suppose two of the yV^h mm., spaces were taken as object,
and the image is measured by the dividers, and the spread of the
dividers is found on the steel rule to be 9-f millimeters. If now the
object is y^ths of a millimeter and the magnified image is 9f milli-
meters, the magnification (which is the ratio between size of object

io8

MAGNIFICATION AND MICROMETRY

[CH. IV

and image) must be g| -r- fV = 47. That is, the magnification is 47
diameters, or 47 times linear. If the fractional numbers in the above
example trouble the student, both may be reduced to the same denom-
ination, thus : If the size of the image is found to be 9-f mm. this
number may be reduced to tenths mm., so it will be of the same

Image

FIG. 99.

FIGS, 98-99. Figures showing that the size of object and image very directly
as their distance from the center of the lens. In Fig. gg one can also see why it is
necessary to focus down, i. e., bring the object and objective nearer together when
the tube is lengthened. See also fig. 58.

denomination as the object. In 9 mm. there are 90 tenths, and in f
there are 4 tenths, then the whole length of the image is 90 + 4 = 94
tenths of a millimeter. The object is 2 tenths of a millimeter, then

CH. I V ] MA GN1FICA TION AND MIC ROME TRY 109

there must have been a magnification of 94 -r- 2 = 47 diameters in
order to produce an image 94 tenths of a millimeter long.

Put the 25 mm. (i in., C, or X 12) ocular in place of one of 37
mm. focus, and then put the camera lucida in position. Measure the
size of the image with dividers and a rule as before. The power will
be considerably greater than when the low ocular was used. This is
because the virtual image (Fig. 21, B3A3) seen with the high ocular is
larger than the one seen with the low one. The real image (Fig. 21,
A1!*1) remains nearly the same, and would be just the same if positive,
par-focal oculars (§ 37, 72, note) were used.

Lengthen the tube of the microscope 50-60 mm. by pulling out
the draw-tube. Remove the camera lucida, and focus, then replace
the camera and obtain the magnification. It will be greater than with
the shorter tube. This is because the real image (Fig. 99) is formed
farther from the objective when the tube is lengthened, and the
objective must be brought nearer the object. The law is : The size
of object and image varies directly as their distance from the center of the
lens. The truth of this statement is illustrated by Figs. 98 and 99.

§ 161. Varying the Magnification of a Compound Micro-
scope.— It will be seen from the above experiments (§ 160) that in-
dependently of the distance at which the microscopic image is
measured (§ 162), there are three ways of varying the power of a
compound microscope. These are named below in the order of
desirability.

1 i ) By using a higher or lower objective.

(2) By using a higher or lower ocular.

(3) By lengthening or shortening the tube of the microscope (Fig.
99).*

§ 162. Standard Distance of 250 Millimeters at which the
Virtual Image is Measured. — For obtaining the magnification of
both the simple and the compound microscope the directions were to
measure the virtual image at a distance of 250 millimeters. This is not

* Amplifier. — In addition to the methods of varying the magnification given
in \ 161, the magnification is sometimes increased by the use of an amplifier, that
is a diverging lens or combination placed between the objective and ocular and
serving to give the image-forming rays from the objective an increased divergence.
An effective form of this accessory was made by Tolles, who made it as a small
achromatic concavo-convex lens to be screwed into the lower end of the draw-tube
(frontispiece) and thus but a short distance above the objective. The divergence
given to the rays increases the size of the real image about two-fold.

no

MAGNIFICATION AND MICROMETRY

[CH. IV

FIG. 100. Figure showing
the position of the microscope,
the camera lucida, the eye, and
the difference in size of the im-
age depending upon the dis-
tance at which it is projected
from the eye. (a) The size at
25 cm.; (b] at 35 cm., (\ 162).

that the image could not be seen and measured at any other distance, but
because some standard must be selected, and this is the most common
one. The necessity for the adoption of some common standard will be
seen at a glance in Fig. 100, where is represented graphically the fact

FIG. 101. Sectional view
of the Abbe Camera Lucida
to show that in measuring
the standard distance of 250
millimeters, one must meas-
ure along the axis from the
point P, at the left of the
prism, to the mirror, and
from the mirror to the
drawing surface. For a
full explanation of this
camera lucida, see next
chapter.

FIG. 101.

that the size of the virtual image depends directly on the distance at
which it is projected, and this size is directly proportional to the ver-
tical distance from the apex of the triangle, of which it forms a base.
The distance of 250 millimeters has been chosen on the supposition
that it is the distance of most distinct vision for the normal human eye.

CH, IV\

MAGNIFICATION AND MICROMETRY

III

Demonstrate the difference in magnification due to the distance at
which the image is projected, by raising the microscope so that the
distance will be 350 millimeters, then lowering to 150 millimeters.

In preparing drawings it is often of great convenience to make
them at a distance somewhat less or somewhat greater than the stand-
ard. In such a case the magnification must be determined for the
special distance. (See the next chapter, § 181.)

For discussion of the magnification of the microscope, see : Beale,
pp. 41, 355 ; Carpenter-Dallinger, p. 288 ; Nageli and Schwendener,
p. 176 ; Ranvier, p. 29; Robin, p. 126 ; Amer. Soc. Micrs. , 1884, p.
183 ; 1889, p. 22 ; Amer. Jour. Arts and Sciences, 1890, p. 50 ; Jour.
Roy. Micr. Soc., 1888, 1889.

OCULAR OCULAR
37 or 50 mm. 25 mm

OBJECTIVE.

TUBE

IN

TUBE

OUT
MM.

TUBE

IN

TUBE

OUT
MM.

OCULAR MICROMETER
VALUATION.
TUBE IN. OUT MM.

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

x .

X

X

X

X

X

X

X

SIMPLE MICROSCOPE. X

FIG. 102.

§ 163. Table of Magnifications and of the Valuations of the
Ocular Micrometer.— The above table should be filled out by each
student. In using it for Micrometry and Drawing it is necessary to keep
clearly in mind the exact conditions under which the determinations were

112 MAGNIFICATION AND MICROMETRY [CH. IV

made, and also the ways in which variations in magnification and the val-
uation of the ocular micrometer may be produced (§ 161. 162, 172, 176).

MICROMETRY

§ 164. Micrometry is the determination of the size of objects by
the aid of a microscope.

MICROMETRY WITH THE SIMPLE MICROSCOPE

§ 165. With a simple microscope (A), the easiest and best way
is to use dividers and then with the simple microscope determine
when the points of the dividers exactly include the object. The spread
of the dividers is then obtained as above (§ 157). This amount will
be the actual size of the object, as the microscope was only used in
helping to see when the divider points exactly enclosed the object, and
then for reading the divisions on the rule in getting the spread of the
dividers.

(B) One may put the object under the simple microscope and
then, as in determining the power (§ 156), measure the image at the
standard distance. If the size of the image so measured is divided
by the magnification of the simple microscope, the quotient will give
the actual size of the object.

Use a fly's wing or some other object of about that size, and try
to determine the width in the two ways described above. If all the
work is accurately done the results will agree.

MICROMETRY WITH THE COMPOUND MICROSCOPE

There are several ways of varying excellence for obtaining the size
of objects with the compound microscope, the method with the ocular
micrometer (§ 175-176) being most accurate.

§ 166. Unit of Measure in Micrometry. — As most of the ob-
jects measured with the compound microscope are smaller than any of
the originally named divisions of the meter, and the common or decimal
fractions necessary to express the size are liable to be unnecessarily
cumbersome, Harting, in his work on the microscope (1859), proposed
the one thousandth of a millimeter (y^^ mm. or o.ooi mm.) or
one millionth of a meter (-nnriinnr or o.oooooi meter) as the unit. He
named this unit micro-millimeter and designated it mmm. In 1869,
Listing (Carl's Repetorium fur Experimental-Physik, Bd, X, p. 5)

CH. IV] MAGN1FICA TION AND MICROMETR Y 113

favored the thousandth of a millimeter as unit and introduced the name
Mikron or micrum. In English it is most often written Micron (plural
micra or microns, pronunciation Mik'r6n or Mik'r6n). By universal con-
sent the sign or abbreviation used to designate it is the Greek //.
Adopting this unit and sign, one would express five thousandths of a
millimeter (T-<jVo or °-°°5 mm.) thus, 5//.*

\ 167. Micrometry by the use of a stage micrometer on which to mount the ob-
ject.— In this method the object is mounted on a micrometer and then put under
the microscope, and the number of spaces covered by the object is read off directly.
It is exactly like putting any large object on a rule and seeing how many spaces of
the rule it covers. The defect in the method is that it is impossible to properly
arrange objects on the micrometer. Unless the objects are circular in outline they
are liable to be oblique in position, and in every case the end or edges of the object
may be in the middle of a space instead of against one of the lines, consequently
the size must be estimated or guessed at rather than really measured.

§ 1 68 . Micrometry by dividing the size of the image by the magnifica-
tion of the microscope. — For example, employ the 3 mm. (^6 in.) objective,
25 mm. (i in.) ocular, and a Necturus' red blood-corpuscle preparation
as object. Obtain the size of the image of the long and short axes
of three corpuscles with the camera lucida and dividers, exactly as in
obtaining the magnification of the microscope (§ 160). Divide the size
of the image in each case by the magnification, and the result will be
the actual size of the blood-corpuscles. Thus, suppose the image of the
long axis of the corpuscle is 18 mm. and the magnification of the micro-
scope 400 diameters (§ 154), then the actual length of this long axis of
the corpuscle is 18 mm.-r- 400=0.045 mm. or 45^ (§ 166).

FIG. 103. Preparation of blood with a
ring around a group of blood corpuscles.

As the same three blood-corpuscles are to be measured in three
ways, it is an advantage to put a delicate ring around a group of three
or more corpuscles, and make a sketch of the whole enclosed group,

*The term micromillimeter, abbreviation mmm., is very cumbersome, and be-
sides is entirely inappropriate since the adoption of the definite meanings for the
prefixes micro and mega, meaning respectively one-millionth and one million
times the unit before which it is placed. A micromillimeter would then mean
one- millionth of a millimeter, not one-thousandth. The term micron has been
adopted by the great microscopical societies, the international commission on
weights and measures, and by original investigators, and is, in the opinion of the
writer, the best term to employ. Jour. Roy. Micr. Soc., 1888, p. 502 ; Nature,
Vol. XXXVII (1888), p. 388.

1 14 MAGNIFICA TION AND M1CROMETRY [£7/. IV

marking on the sketch the corpuscles measured (Figs. 61-66). The
different corpuscles vary considerably in size, so that accurate com-
parison of different methods of measurement can only be made when
the same corpuscles are measured in each of the ways.

§ 169. Micrometry by the use of a Stage Micrometer and a
Camera Lucida. — Employ the same object, objective and ocular as
before. Put the camera lucida in position, and with a. lead pencil
make dots on the paper at the limits of the image of the blood-
corpuscle. Measure the same three that were measured in § 168.

Remove the object, place the stage micrometer under the micro-
scope, focus well, and draw the lines of the stage micrometer so as to
include the dots representing the limits of the part of the image to be
measured. As the value of the spaces on the stage micrometer is
known, the size of the object is determined by the number of spaces
of the micrometer required to include it.

This simply enables one to put the image of a fine rule on the
image of a microscopic object. It is theoretically an excellent method,
and nearly the same as measuring the spread of the dividers with a
simple microscope (§ 157, 176).

OCULAR MICROMETER

§ 170. Ocular Micrometer, Eye-Piece Micrometer. — This,
as the name implies, is a micrometer to be used with the ocular. It is
a micrometer on glass, and the lines are sufficiently coarse to be clearly
seen by the ocular. The lines should be equidistant and about y^th or
2^th mm. apart, and every fifth line should be longer and heavier to
facilitate counting. If the micrometer is ruled in squares (net micro-
meter} it will be very convenient for many purposes.

The ocular micrometer is placed in the ocular, no matter what the
form of the ocular (i. e., whether positive or negative) at the level at
which the real image is formed by the objective, and the image appears
to be immediately upon or under the ocular micrometer, and hence
the number of spaces on the ocular micrometer required to measure
the real image may be read off directly. This, however, is measuring
the size of the real image, and the actual size of the object can only be
determined by determining the ratio between the size of the real image
and the object. In other words, it is necessary to get the valuation of
the ocular micrometer in terms of a stage micrometer.

§ 171. Valuation of the Ocular Micrometer. — This is the
value of the divisions of. the ocular micrometer for the purposes of

CH.

MAGNIFICATION AND MICROMETRY

micrometry, and is entirely relative, depending upon the magnification
of the real image formed by the objective, consequently it changes
with every change in the magnification of the real image, and must be
especially determined for every optical combination (i. e., objective and
ocular), and for every change in the length of the tube of the micro-
scope. That is, it is necessary to determine the ocular micrometer val-
uation for every condition modifying the real image of the microscope

(§ 161).

Any Huygenian ocular (Fig. 30) may, however, be used as a micrometer ocu-
lar by placing the ocular micrometer at the level of the ocular diaphragm, where
the real image is formed. If there is a slit in the side of the ocular , and the
ocular micrometer is mounted in some way it may be introduced through the
opening in the side. When no side opening exists the mounting of the eye-lens
may be unscrewed and the ocular micrometer, if on a cover-glass can be laid on
the upper side of the ocular diaphragm.

FIG. 104. FIG. 105.

FIGS. 104-105. Ocular Micrometer with movable scale. Fig. 104 is a side view
of the octilar while Fig. 105 gives a sectional end view, and shows the ocular mi-
crometer in position . In both the screw which moves the micrometer is shown at
the left. (Bausch & Lomb Opt. Co.)

§ 172. Obtaining the Ocular Micrometer Valuation for an
Ocular Micrometer with Fixed Lines (Figs. 104-105). — Use the
stage micrometer as object. Light the field well and look into the
microscope. The lines of the ocular micrometer should be very sharply
defined. If they are not, raise or lower the eye-lens to make them so ;
that is, focus as with the simple magnifier.

When the lines of the ocular micrometer are distinct, focus the
microscope (§72, 74, 75) for the stage micrometer. The image of the
stage micrometer will appear to be directly under or upon the ocular
micrometer.

Make the lines of the two micrometers parallel by rotating the ocular
or changing the position of the stage micrometer or both if necessary,

Ii6 MAGNIFICATION AND MICROMETRY \_CH. IV

and then make any two lines of the stage micrometer coincide with
any two on the ocular micrometer. To do this it may be necessary to
pull out the draw- tube a greater or less distance. See how many
spaces are included in each of the micrometers.

Divide the value of the included space or spaces on the stage mi-
crometer by the number of divisions on the ocular micrometer required
to include them, and the quotient so obtained will give the valuation of
the ocular micrometer in fractions of the unit of measure of the stage
micrometer. For example, suppose the millimeter is taken as the unit
for the stage micrometer and this unit is divided into spaces of y^th and
y-Q-g-th millimeter. If now, with a given optical combination and tube-
length, it requires 10 spaces on the ocular micrometer to include the
real image of yVth millimeter on the stage micrometer, obviously one
space on the ocular micrometer would include only one-tenth as much,
or yVth mm. -=-10 = y-J^th mm. That is, each space on the ocular mi-
crometer would include y-^oth of a millimeter on the stage micrometer,
or y-Jijth millimeter of the length of any object under the microscope,
the conditions remaining the same. Or, in other words, it would re-
quire 100 spaces on the ocular micrometer to include i millimeter on the
stage micrometer, then as before, i space of the ocular micrometer would
have a valuation of y^-Q-th millimeter for the purposes of micrometry.
The size of any minute object may be determined by multiplying this
valuation of one space by the number of spaces required to include it.
For example, suppose the fly's wing or some part of it covered 8 spaces
on the ocular micrometer, it would be known that the real size of the
part measured is y^th mm. x 8 = y-fo- mm. or 80 ju. (§ 166). See
Mark, Jour. Applied Microscopy, Vol. I, p. 4.

§ 173. Micrometry with the Ocular Micrometer. — Use the 3
mm. (-|- in. ) objective with the preparation of Necturus blood-corpuscles
as object. Make certain that the tube of the microscope is of the same
length as when determining the ocular micrometer valuation. In a
word, be sure that all the conditions are exactly as when the valuation
was determined, then put the preparation under the microscope and
find the same three red corpuscles that were measured in the other
ways (§168-169).

Count the divisions on the ocular micrometer required to enclose
or measure the long and the short axis of each of the three corpuscles,
multiply the number of spaces in both cases by the valuation of the
ocular micrometer for this objective, tube-length and ocular, and the
results will represent the actual length of the axes of the corpuscles
in each case.

CH.

MAGNIFICATION AND MICROMETRY

117

The same corpuscle is, of course, of the same actual size, when
measured in each of the three ways, so that if the methods are correct
and the work carefully enough done, the same results should be ob-
tained by each method. (§ 176).*

FIG. 1 06. Ocular Screw- Micrometer with
compensation ocular X 6. The upper figure
shows a sectional view of the ocular and the
screw for moving the micrometer at the right.
At the left is shown a clamping screw to
fasten the ocular to the upper part of the mi-
croscope tube. Below is a face view, showing
the graduation on the wheel. An ocular
micrometer like this is in general like the
cob-web micrometer and may be used for
measuring objects of varying sizes very accu-
rately. With the ordinary ocular micrometer
very small objects frequently fill but a part of
an interval of the micrometer, but with this
the movable cross lines traverse the object (or
rather its real image} regardless of the minute-
ness of the object. (Zeiss* Catalog}.

\ 174. Obtaining the Valuation of the Screw or Filar Micrometer. — This
micrometer (Fig. 106-107) usually consists of a Ramsden's ocular and cross lines.
As seen in Fig. 107^ there are three lines. The horizontal and one vertical line
are fixed. One vertical line may be moved by the screw back and forth across the
field.

For obtaining the valuation of this ocular micrometer an accurate stage mi-
crometer must be used. Carefully focus the Tiy^th mm. spaces. The lines of the
ocular micrometer should also be sharp. If they are not focus them by moving
the top of the ocular up or down ($ 172). Make the vertical lines of the filar mi-
crometer parallel with the lines of the stage micrometer. Take the precautions
regarding the width of the stage micrometer lines given in $ 176 (see also Fig.
108). Note the position of the graduated wheel and of the teeth of the recording
comb, and then rotate the wheel until the movable line traverses one space on the
stage micrometer. Each tooth of the recording comb indicates a total revolution
of the wheel, and by noting the number of teeth required and the graduations on
the wheel, the revolutions and parts of revolution required to measure the j^ih
mm, of the stage micrometer can be easily noted. Measure in like manner 4 or 5
spaces and get the average. Suppose this average is i#th revolutions or 125
graduations on the wheel, to measure the Tfoth mm. or IQJU (see % 166), then one
of the graduations on the wheel would measure IQJU divided by I25=.o8//. In
using this valuation for actual measurement, the tube of the microscope and the
objective must be exactly as when obtaining the valuation (see \ 175).

Example of Measurement. — Suppose one uses the red blood corpuscles of a dog
or monkey, etc., every condition being as when the valuation was determined, one
notes very accurately how many of the graduations on the wheel are required to
make the movable line traverse the object from edge to edge. Suppose it requires

u8

MAGNIFICATION AND MICROMETRY

[CH.IV

94 of the graduations to measure the diameter, the actual size of the corpuscle
would be 94X.o8 ju = 7.52^.

The advantage of the filar micrometer is that the valuation of one graduation
being so small, even the smallest object to be measured would require several
graduations to measure it. In ocular micrometers with fixed lines, small objects
like bacteria might not fill even one space, therefore estimations, not measure-
ments, must be made. For large objects, like most of the tissue elements, the
ocular micrometers with fixed lines answer very well, for the part which must be
estimated is relatively small and the chance of error is correspondingly small.

FIG. 107. Filar Ocular Micrometer with Field A (Bausch & Lomb, Optical Co.}.
§ 175. Varying the Ocular Micrometer Valuation. — Any
change in the objective, the ocular or the tube-length of the microscope,
that is to say, any change in the size of the real image, produces a cor-
responding change in the ocular micrometer valuation (§ 161, 171,
176).

\ 176. Remarks on Micrometry. — In using adjustable objectives (§24, 103),
the magnification of the objective varies with the position of the adjusting collar,
being greater when the adjustment is closed as for thick cover-glasses than when
open, as for thin ones. This variation in the magnification of the objective pro-
duces a corresponding change in the magnification of the entire microscope, and
the ocular micrometer valuation — therefore it is necessary to determine the mag-
nification and ocular micrometer valuation for each position of the adjusting
collar.

While the principles of micrometry are simple, it is very difficult to get the
exact size of microscopic objects. This is due to the lack of perfection and uni-

*There are three ways of using the ocular micrometer, or of arriving at the size
of the objects measured with it :

(A) By finding the value of a division of the ocular micrometer for each optical
combination and tube-length used, and employing this valuation as a multiplier.
This is the method given in the text, and ;the one most frequently employed.
Thus, suppose with a given optical combination and tube-length it required five
divisions on the ocular micrometer to include the image of r20ths millimeter of the
stage micrometer, then obviously one space on the ocular micrometer would in-
clude ith of T2(jths nvm. or ^th mm.; the size of any unknown object under the

CH. IV\ MA GNIFICA TION AND MICRO ME TRY 119

fortuity of micrometers, and the difficulty of determining the exact limits of the
object to be measured. Hence, all microscopic measurements are only approxi-
mately correct, the error lessening with the increasing perfection of the apparatus
and the skill of the observer.

microscope would be obtained by multiplying the number of divisions on the
ocular micrometer required to include its image by the value of one space, or in
this case, ^th mm. Suppose some object, as the fly's wing, required 15 spaces of
the ocular micrometer to include some part of it, then the actual size of this part
of the wing would be 15 X -h — fths, or 0.6 mm.

(B) By finding the number of divisions on the ocular micrometer required to
include the image of an entire millimeter of the stage micrometer, and using this
number as a divisor. This number is also sometimes called the ocular micrometer
ratio. Taking the same case as in (A), suppose five divisions of the ocular mi-
crometer are required to include the image of T25ths mm., on the stage micrometer,
then evidently it would require 5 -j- T% — 25 divisions on the ocular micrometer to
include a whole millimeter on the stage micrometer, and the number of divisions
of the ocular micrometer required to measure an object divided by 25 would give
the actual size of the object in millimeters or in a fraction of a millimeter. Thus,
suppose it required 15 divisions of the ocular micrometer to include the image of
some part of the fly's wing, the actual size of the part included would be 15 H- 25
— | or 0.6 mm. This method is really exactly like the one in (A), for dividing by
25 is the same as multiplying by ^th.

(C) By having the ocular micrometer ruled in millimeters and divisions of a
millimeter, and then getting the size of the real image in millimeters. In employ-
ing this method a stage micrometer is used as object and the size of the image of
one or more divisions is measured by the ocular micrometer, thus : Suppose the
stage micrometer is ruled in T^th andy^th mm. and the ocular micrometer is ruled
in millimeters and TVth mm. Taking y2ffth mm. on the stage micrometer as object,
as in the other cases, suppose it requires 10 of the y^th mm. spaces or I mm. to
measure the real image, then the real image must be magnified y$-=-y2^ = 5 diame-
ters, that is, the real image is five times as great in length as the object, and the
size of an object may be determined by putting it under the microscope and getting
the size of the real image in millimeters with the ocular micrometer and dividing
it by the magnification of the real image, which in this case is 5 diameters.

Use the fly's wing as object, as in the other cases, and measure the image of
the same part. Suppose that it required 30 of the y1^ mm. divisions = f§ mm. or 3
mm. to include the image of the part measured, then evidently the actual size of
the part measured is 3 mm, -H 5 = f mm., the same result as in the other cases.

In comparing these methods it will be seen that in the first two (A and B ) the
ocular micrometer may be simply ruled with equidistant lines without regard to
the absolute size in millimeters or inches of the spaces. In the last method the
ocular micrometer must have its spaces some known division of a millimeter or
inch. In the first two methods only one standard of measure is required, viz, the
stage micrometer ; in the last method two standards must be used, — a stage mi-
crometer and an ocular micrometer. Of course, the ocular micrometer in the first
two cases must have the lines equidistant as well as in the last case, but ruling
lines equidistant is quite a different matter from getting them an exact division of
a millimeter or of an inch apart.

120

MAGNIFICATION AND MICROMETRY

[CH. IV

A difficulty when one is using high powers is the width of the lines of the
micrometer. If the micrometer is perfectly accurate half the width of each line
belongs to the contiguous spaces, hence one should measure the image of the space
from the centers of the lines bordering the space, or as this is somewhat difficult
in using the ocular micrometer, one may measure from the inside of one border-
ing line and from the outside of the other. If the lines are of equal width this is
as accurate as measuring from the center of the lines. Evidently it would not be
right to measure from either the inside or the outside of both lines (Fig. 108).

It is also necessary in micrometry to use an objective of sufficient power to
enable one to see all the details of an object with great distinctness. The necessity
of using sufficient amplification in micrometry has been especially remarked upon
by Richardson, Monthly Micr. Jour., 1874, 1875, ; Rogers, Proc. Amer. Soc. Micro-
scopists, 1882, p. 239; Ewell, North American Pract., 1890, pp. 97, 173.

FIG. 108. The appearance of the coarse

A B stage micrometer and of the fine ocular mi-

crometer lines when using a high objective.

(A}. The method of measuring the
spaces by ptdting the fine ocular mi-
crometer lines opposite the center of the
coarse stage micrometer lines.

( B) . Method of measuring the spaces
of the stage micrometer by putting one
line of the ocular micrometer (o. m.} at the
inside and one at the outside of the coarse
stage micrometer lines (s. m.).

FIG. 108.

As to the limit of accuracy in micrometry, one who has justly earned the right
to speak with authority expresses himself as follows: ^ I assume that 0.2 n is the
limit of precision in microscopic measures beyond which it is impossible to go with
certainty." W. A. Rogers Proc. Amer. Soc. Micrs., 1883, p. 198.

In comparing the methods of micrometry with the compound microscope given
above ($ 167, 168, 169, 175), the one given in \ 167 is impracticable, that given in
\ 168 is open to the objection that two standards are required, — the stage microme-
ter, and the steel rule ; it is open to the further objection that several different
operations are necessary, each operation adding to the probability of error. Theoret-
ically the method given in \ 169 is good, but it is open to the very serious objection
in practice that it requires so many operations which are especially liable to intro-
duce errors. The method that experience has found most safe and expeditious,
and applicable to all objects, is the method with the ocular micrometer. If the
valuation of the ocular micrometer has been accurately determined, then the only
difficulty is in deciding on the exact limits of the objects to be measured and so
arranging the ocular micrometer that these limits are inclosed by some divisions of
the micrometer. Where the object is not exactly included by whole spaces on the
ocular micrometer, the chance of error comes in, in estimating just how far into a
space the object reaches on the side not in contact with one of the micrometer

[_CH.

MA GNIFICA TION AND MICRO ME TR Y

121

lines. If the ocular micrometer has some quite narrow spaces, and others consid-
erably larger, one can nearly always manage to exactly include the object by some
two lines. The ocular screw micrometers (Fig. 106-107) obviates this entirely as
the cross hair or lines traverse the object or its real image, and whether this
distance be great or small it can be read off on the graduated wheel, and no
estimation or guess work is necessary.

For those especially interested in micrometry, as in its relation to medical
jurisprudence, the following references are recommended. These articles consider
the problem in a scientific as well as a practical spirit : The papers of Prof. Wm.
A. Rogers on micrometers and micrometry, in the Amer. Quar. Micr. Jour., Vol.
!> PP- 97 > 2°8 ; Proceedings Amer. Soc. Microscopists, 1882, 1883, 1887. Dr. M.
D. Ewell, Proc. Amer. Soc. Micrs., 1890 ; The Microscope, 1889, pp. 43-45 ; North
Amer. Pract., 1890, pp. 97, 173. Dr. J. J. Woodward, Amer. Jour, of the Med.
Sci., 1875. M. C. White, Article "Blood-stains," Ref. Hand-Book Med. Sciences,
1885. Medico-Legal Journal, Vol. XII. For the change in magnification due to
a change in the adjustment of adjustable objectives, see Jour. Roy. Micr. Soc.,
iSSo, p. 702 ; Amer. Monthly Micr. Jour., 1880, p. 67. Carpenter-Dallinger, p. 270.

If one consults the medico-legal journals ; the microscopical journals, the
Index Medicus, and the Index Catalog of the Library of the Surgeon General's
Office, under Micrometry, Blood, and Jurisprudence, he can get on track of the
main work which has been and is being done.

f in.

Dry objectives of 16 mm. (f z«. ), 4. mm. (i in.) and homogeneous immersion
objective 0/2 mm. (TV in. ) in their mountings. (Bausch & Lomb Opt. Co.).

CHAPTER V

DRAWING WITH THE MICROSCOPE

APPARATUS AND MATERIAL FOR THIS CHAPTER

Microscope, Abbe and Wollaston's camera lucidas, drawing board, thumb
tacks, pencils, paper, and microscope screen, (Fig. 59), microscopic preparations.

DRAWING MICROSCOPIC OBJECTS

§ 177. Microscopic objects may be drawn free-hand directly from
the microscope, but in this way a picture giving only the general ap-
pearance and relations of parts is obtained. For pictures which shall
have all the parts of the object in true proportions and relations, it is
necessary to obtain an exact outline of the image of the object, and to
locate in this outline all the principal details of structure. It is then
possible to complete the picture free-hand from the appearance of the
object under the microscope. The appliance used in obtaining out-
lines, etc., of the microscope image is known as a camera lucida.

§ 178. Camera Lucida. — This "is an optical apparatus for en-
abling one to see objects in greatly different situations, as if in one
field of vision, and with the same eye. In other words it is an optical
device for superimposing or combining two fields of view in one eye.

As applied to the microscope, it causes the magnified virtual im-
age of the object under the microscope to appear as if projected upon
the table or drawing board, where it is visible with the drawing paper,
pencil, dividers, etc., by the same eye, and in the same field of vision.
The microscopic image appears like a picture on the drawing paper
(see note to § 181). This is accomplished in two distinct ways :

(A) By a camera lucida reflecting the rays from the microscope so
that their direction when they reach the eye coincides with that of the
rays from the drawing paper, pencil, etc. In some of the camera
lucidas from this group (Wollaston's, Fig. 112), the rays are reflected
twice, and the image appears as when looking directly into the micro-
scope. In others the rays are reflected but once, and the image has
the inversion produced by a plane mirror. For drawing purposes this

CH. V]

DRA WING WITH THE MICROSCOPE

123

inversion is a great objection, as it is necessary to similarly invert all
the details added free-hand.

(B) By a camera lucida reflecting the rays of light from the draw-
ing paper, etc. , so that their direction when they reach the eye coin-
cides with the direction of the rays from the microscope (Fig. 58, 109).
In all of the camera lucidas of this group, the rays from the paper are
twice reflected and no inversion appears.

FIG. no.

FIG. in.

FIG. 109.

FIG. 109. Abbe Camera Lucida with
the mirror at 45°, the drawing surface
horizontal, and the microscope vertical.

Axis, Axis. Axial ray from the mi-
croscope and from the drawing surface.
A, B. Marginal rays of the field on the
drawing surface, a b. Sectional view of
the silvered surf ace on the upper of the tri-
angular prisms composing the cubical
prism ( P) . The silvered surface is shown

as incomplete in the center, thus giving passage to the rays from the microscope

Foot. Foot or base of the microscope.

G. Smoked glass seen in section. It is placed between the mirror and the
prism to reduce the light from the drawing surface.

Mirror. The mirror of the camera lucida. A quadrant (Q) has been added
to indicate the angle of inclination of the mirror, which in this case is 45°.

Ocular. Ocular of the microscope over which the prism of the camera lucida
is placed.

P, P. Drawing pencil and the cubical prism over the ocular.

FIG. no. Geometrical figure showing the angles made by the axial ray with
the drawing surface and the mirror.

A, B. The drawing surface.

FIG. in. Ocular showing eye-point, E. P. It is at this point both horizontally
and vertically that the hole in the silvered surface should be placed (| 182).

124 DRAWING WITH THE MICROSCOPE [CH. V

The better forms of camera lucidas (Wollaston's, Grunow's, Abbe's,
etc.), may be used for drawing both with low and with high powers.
Some require the microscope to be inclined (Fig. TOO) while others are
designed to be used on the microscope in a vertical position. As in
biological work, it is often necessary to have the microscope vertical,
the form for a vertical microscope is to be preferred ; but see Figs. 115-

116.

§ 179. Avoidance of Distortion. — In order that the pictiire

drawn by the aid of a camera lucida may not be distorted, it is necessary
that the axial ray from the image on the drawing surface shall be at right
angles to the drawing surface (Figs. 112, 114.)

$ 180. Wollaston's Camera Lucida. — This is a quadrangular prism of glass
put in the path of the rays from the microscope, and it serves to change the
direction of the axial ray 90 degrees. In using it the microscope is made horizon-
tal, and the rays from the microscope enter one-half of the pupil while rays from
the drawing surface enter the other half of the pupil. As seen in the figure (Fig.
112), the fields partly overlap, and where they do so overlap, pencil or dividers
and microscopic image can be seen together.

In drawing or using the dividers with the Wollaston camera lucida it is neces-
sary to have the field of the microscope and the drawing surface about equally
lighted. If the drawing surface is too brilliantly lighted the pencil or dividers
may be seen very clearly, but the microscopic image will be obscure. On the
other hand, if the field of the microscope has too much light the microscopic
image will be very definite, but the pencil or dividers will not be visible. It is
necessary, as with the Abbe camera lucida (£ 182), to have the Wollaston prism
properly arranged with reference to the axis of the microscope and the eye-point.
If it is not, one will be unable to see the image well, and may be entirely unable
to see the pencil and the image at the same time. Again, as rays from the micro-
scope and from the drawing surface must enter independent parts of the pupil of
the same eye, one must hold the eye so that the pupil is partly over the camera
lucida and partly over the drawing surface. One can tell the proper position by
trial. This is not a very satisfactory camera to draw7 with, but it is a very good
form to measure the vertical distance of 250 rnm. at which the drawing surface
should be placed when determining magnification (\ 162).

§181. * Abbe Camera Lucida. — This consists of a cube of glass
cut into two triangular prisms and silvered on the cut surface of the

*For some persons the image and drawing surface, pencil, etc., do not appear
on the drawing board as stated above, but under the microscope, according to the
general principle that "objects appear in space where they could be touched
along a perpendicular to the retinal surface stimulated," — that is in the line of
rays entering the eye. This is always the case with the Wollaston camera lucida.
The explanation of the apparent location of the image, etc., on the drawing board
with the Abbe camera lucida is that the attention is concentrated upon the draw-
ing surface rather than upon the object under the microscope (Dr. W. B.
Pillsbury).

CH. F]

DRA WING WITH THE MICROSCOPE

125

upper one. A small oval hole is then cut out of the center of the sil-
vered surface and the two prisms are cemented together in the form of
the original cube with a perforated 45 degree mirror within it (Fig.
109, ab). The upper surface of the cube is covered by a perforated
metal plate. This cube is placed over the ocular in such a way that
the light from the microscope passes through the hole in the silvered
face and thence directly to the eye. Light from the drawing surface
is reflected by a mirror to the silvered surface of the prism and reflect-
ed by this surface to the eye in
company with the rays from the
microscope, so that the two fields
appear as one, and the image is
seen as if on the drawing surface
(Figs. 109, 114). It is designed
for use with a vertical micro-
scope, but see § 184.

FIG. 112. Wollaston's Camera
Lucida, showing the rays from the
microscope and from, the drawing sur-
face, and the position of the pupil of
the eye.

For full explanation see Fig. 97.

§ 182. Arrangement of the Camera Lucida Prism. — In plac-
ing this camera lucida over the ocular for drawing or the determination
of magnification, the center of the hole in the silvered surface is placed
in the optic axis of the microscope. This is done by properly arrang-
ing the centering screws that clamp the camera to the microscope tube
or ocular. The perforation in the silvered surface must also be at the
level of the eye-point. In other words the prism must be so arranged
vertically and horizontally that the hole in the silvered surface will be
in the axis of the microscope and coincident with the eye-point of
the ocular. If it is above or below, or to one side of the eye-point,
part or all of the field of the microscope will be cutoff. As stated
above, the centering screws are for the proper horizontal arrangement
of the prism. The prism is set at the right height by the makers for
the eye-point of a medium ocular. If one desires to use an ocular
with the eye-point farther away or nearer, as in using high or low
oculars the position of the eye-point may be determined as directd in

126 DRAWING WITH THE MICROSCOPE \_CH. V

§ 59 and the prism loosened and raised or lowered to the proper level ;
but in doing this one should avoid setting the prism obliquely to the
mirror.

In the latest and best forms of this camera lucida special arrange-
ments have been made for raising or lowering the prism so that it may
be used with equal satisfaction on oculars with the eye-point at differ-
ent levels, and the prism is hinged to turn aside without disturbing
the mirror.

One can determine when the
camera is in a proper position by
looking into the microscope through
it. If the field of the microscope
appears as a circle and of about the
same size as without the camera
lucida, then the prism is in a proper
FIG. 113. One of the latest and position. If one side of the field is
best forms of the Abbe Camera Lucida d fc ^ h rf . Qne ^

(Bausch & Lomb Optical Co.).

of the center ; if the field is consid-
erably smaller than when the prism is turned off the ocular, it indicates
that it is not at the correct level, i. e., it is above or below the eye-
point.

§ 183. Arrangement of the Mirror and the Drawing Surface.
—The Abbe camera lucida was designed for use with a vertical micro-
scope (Fig. 109). On a vertical microscope if the mirror is set at an
angle of 45°, the axial ray will be at right angles with the table top or a
drawing board which is horizontal, and a drawing made under these
conditions will be in true proportion and not distorted. The stage of
most microscopes, however, extends out so far at the sides that with
a 45° mirror the image appears in part on the stage of the microscope.
In order to avoid this the mirror may be depressed to some point below
45°, say at 40° or 35° (Fig. 1 14). But as the axial ray from the mirror
to the prism must still be reflected horizontally, it follows that the axial
ray will no longer form an angle of 90 degrees with the drawing sur-
face, but a greater angle. If the mirror is depressed to 35°, then the
axial ray must take an angle of 110° with a horizontal drawing surface
(see the geometrical figure Fig. 114, A). To make the angle 90° again,
so that there shall be no distortion, the drawing board must be raised
toward the microscope 20°. The general rule is to raise the draw-
ing board twice as many degrees toward the microscope as the
mirror is depressed below 45°. Practically the field for drawing

CH. F] DRAWING WITH THE MICROSCOPE

B

I27

FIG. 114. C

Abbe Camera Lucida in position to avoid distortion.

FIG. 114. — The Abbe Camera Lucida with the mirror at 35°.

Axis, Axis. Axial ray from the microscope and from the drawing surface.

A. B. Drawing surface raised toward the microscope 20°.

Foot. The foot or base of the microscope.

Mirror with quadrant (Q). The mirror is seen to be at an angle of 35°.

Ocular. Ocular of the Microscope.

P. P. Drawing pencil and the cubical prism over the ocular.

W. Wedge to support the drawing board.

A. Geometrical figure of the preceding, showing the angles made by the
axial ray with the mirror and the necessary elevation of the drawing board to
avoid distortion. From the equality of opposite angles, the angle of the axial ray
reflected at jj° must make an angle of 110° with a horizontal drawing board. The
board must then be elevated toward the microscope 20° in order that the axial ray
may be perpendicular to it, and thus fulfill the requirements necessary to avoid
distortion ($ 779, 183}.

B. Upper view of the prism of the camera lucid a. A considerable portion of
the face of the prism is covered, and the opening in the silvered surface appears
oval.

C. Quadrant to be attached to the mirror of the Abbe Camera Lucida to in-
dicate the angle of the mirror. As the angle is nearly always 45° , 40°, or 55°,
only those angles are shown.

128 DRAWING WITH THE MICROSCOPE \CH. V

can always be made free of the stage of the microscope, at 45°, at 40°,
or at 35°. In the first case (45° mirror) the drawing surface should
be horizontal, in the second case (40° mirror) the drawing surface
should be elevated 10°, and in the third case (35° mirror) the drawing
board should be elevated 20° toward the microscope. Furthermore it
is necessary in using an elevated drawing board to have the mirror bar
project directly laterally so that the edges of the mirror will be in
planes parallel with the edges of the drawing board, otherwise there
will be front to back distortion, although the elevation of the drawing
board would avoid right to left distortion. If one has a micrometer
ruled in squares (net micrometer} the distortion produced by not having
the axial ray at right angles with the drawing surface may be very
strikingly shown. For example, set the mirror at 35° and use a hori-
zontal drawing board. With a pencil make dots at the corners of
some of the squares, and then with a straight edge connect the dots.
The figures will be considerably longer from right to left than from
front to back. Circles in the object would appear as ellipses in the
drawings, the major axis being from right to left.

The angle of the mirror may be determined with a protractor, but
that is troublesome. It is much more satisfactory to have a quadrant
attached to the mirror and an indicator on the projecting arm of the
mirror. If the quadrant is graduated throughout its entire extent, or
preferably at three points, 45°, 40° and 35°, one can set the mirror at a
known angle in a moment, then the drawing board can be hinged and
the elevation of 10° and 20° determined with a protractor. The draw-
ing board is very conveniently held up by a broad wedge. By marking
the position of the wedge for 10° and 20° the protractor need be used
but once, then the wedge may be put into position at any time for the
proper elevation.

§ 184. Abbe Camera and Inclined Microscope. — It is very
fatiguing to draw continuously with a vertical microscope, and many
mounted objects admit of an inclination of the microscope, when one
can sit and work in a more comfortable position. The Abbe camera is
as perfectly adapted to use with an inclined as with a vertical micro-
scope. All that is requisite is to be sure that the fundamental law is
observed regarding the axial ray of the image and the drawing surface,
viz. , that they should be at right angles. This is very easily accom-
plished as follows : The drawing board is raised toward the microscope
twice as many degrees as the mirror is depressed below 45° (§ 183),
then it is raised exactly as many degrees as the microscope is inclined,

CH. V\ DRAWING WITH THE MICROSCOPE 129

and in the same direction, that is, so the end of the drawing board
shall be in a plane parallel with the stage of the microscope. The
mirror must have its edges in planes parallel with the edges of the
drawing board also (Figs. 115, 116.)

FIG. 115. Arrangement of jo"
the drawing board for using the
microscope in an inclined position
with the Abbe camera lucida (de-
signed by Mrs. S. P. Gage, 1887.)

§ 185. Drawing with the Abbe Camera Lucida. — (A) The
light from the microscope and from the drawing surface should be of
nearly equal intensity, so that the image and the drawing pencil can be
seen with about equal distinctness. This may be accomplished with
very low powers (16 mm. and lower objectives) by covering the mirror
of the microscope with white paper when transparent objects are to be
drawn. For high powers it is best to use a substage condenser. Often
the light may be balanced by using a larger or smaller opening in the
diaphragm. One can tell which field is excessively illuminated, for
it is the one in which objects are most distinctly seen. If it is the
microscopic, then the image of the microscopic object is very distinct
and the pencil is invisible or very indistinct. If the drawing surface is
too brilliantly lighted the pencil can be seen clearly, but the micro-
scopic image will be obscure.

When opaque objects, that is objects which must be lighted with
reflected light (§ 63), like dark colored insects, etc., are to be drawn
the light must usually be concentrated upon the object in some way.
The microscope may be placed in a very strong light and the drawing
board shaded or the light may be concentrated upon the object by
means of a concave mirror or a bull's eye condenser (Fig. 53).

If the drawing surface is too brilliantly illuminated, it may be
shaded by placing a book or a ground glass screen between it and the
window, also by putting one or more smoked glasses in the path of the
ra}7s from the mirror (Fig. 109 G). If the light in the microscope is
too intense, it may be lessened by using white paper over the mirror,
or by a ground glass screen between the microscope mirror and the
source of light (Piersol, Amer. M. M. Jour., 1888, p. 103). It is also
an excellent plan to blacken the end of the drawing pencil with carbon

130

DRAWING WITH THE MICROSCOPE

\CH. V

ink. Sometimes it is easier to draw on a black surface, using a white
pencil or style. The carbon paper used in manifolding letters, etc.,
may be used, or ordinary black paper may be lightly rubbed on one
side with a moderately soft lead pencil. Place the black paper over
white paper and trace the outlines with a pointed style of ivory or
bone. A corresponding dark line will appear on the white paper
beneath. (Jour. Roy. Micr. Soc., 1883, p. 423).

FIG. 1 1 6. Bernhard's Drawing Board for the Abbe Camera Lucida. This
drawing board is adjustable vertically, and the board may be inclined to prevent
distortion. It is also arranged for use with an inclined microscope, having the
base board hinged. Microscope and drawing surface are then inclined together.
(Zeit. wiss. Mikroskopie, vol. vi (1894, p. 208). (Zeiss Catalog}.

(A) It is desirable to have the drawing paper fastened with thumb
tacks, or in some other way. (B) The lines made while using the
camera lucida should be very light, as they are liable to be irregular.
(C) Only outlines are drawn and parts located with a camera lucida.
Details are put in free-hand. (D) It is sometimes desirable to draw
the outline of an object with a moderate power and add the details with
a higher power. If this is done it should always be clearly stated. It
is advisable to do this only with objects in which the same structure is
many times duplicated, as a nerve or a muscle. In such an object all

CH. F] DRAWING WITH THE MICROSCOPE 131

the different structures could be shown, and by omitting some of the
fibers the others could be made plainer without an undesirable enlarge-
ment of the entire figure.

(E) If a drawing of a given size is desired and it cannot be ob-
tained by any combination of oculars, objectives and lengths of the
tube of the microscope, the distance between the camera lucida and
the table may be increased or diminished until the image is of the
desired size. This distance is easily changed by the use of a book or
a block, but more conveniently if one has a drawing board with adjust-
able drawing surface like that shown in Fig. 1 16. The image of a few
spaces of the micrometer will give scale of enlargement, or the power
may be determined for the special case (§ 186-187).

(F) It is of the greatest advantage, as suggested by Heinsius (Zeit.
w. Mikr., 1889, P- 367) i to have the camera lucida hinged so that the
prism may be turned off the ocular for a moment's glance at the prepa-
ration, and then returned in place without the necessity of loosening
screws and readjusting the camera. This form is now made by several
opticians, and the quadrant is added by some. (Fig. 114.) -Any skilled
mechanic can add the quadrant.

§ 186. Magnification of the Microscope and size of Draw-
ings with the Abbe Camera Lucida. — In determining the standard
distance of 250 millimeters at which to measure the image in getting
the magnification of the microscope, it is necessary to measure from
the point marked P on the prism (Fig. 109) to the axis of the mirror
and then vertically to the drawing board.

In getting the scale to which a drawing is enlarged the best way
is to remove the preparation and put in its place a stage micrometer,
and to trace a few (5 or 10) of its lines upon one corner of the drawing.
The value of the spaces of the micrometer being given, thus :

,^th mm.

FIG. 117. Showing the method of indicating the scale at which a drawing
was made.

The enlargement of the figure can then be accurately determined
at any time by measuring with a steel scale the length of the image of
the micrometer spaces and dividing it by their known size.

Thus, suppose the 5 spaces of the scale of enlargement given with
a drawing were found to measure 25 millimeters and the spaces on the

132 DRAWING WITH THE MICROSCOPE [CH. V

micrometer were y1 th millimeter, then the enlargement would be
25 -3- yf^ = 500. That is, the image was drawn at a magnification of
500 diameters.

If the micrometer scale is added to every drawing, there is no
need of troubling one's self about the exact distance at which the
drawing is made, convenience may settle that, as the special magnifi-
cation in each case may be determined from the scale accompanying
the picture. It should be remembered, however, that the conditions
when the scale is drawn must be exactly as when the drawing was
made.

§ 187. Drawing at Slight Magnification. — Some objects are of
considerable size and for drawings should be enlarged but a few diame-
ters,— 5 to 20. By using sufficiently low objectives and different ocu-
lars a great range may be obtained. Frequently, however, the range
must be still further increased. For a moderate increase in size the
drawing surface may be put farther off, or, as one more commonly
needs less rather than greater magnification, the drawing surface may
be brought nearer the mirror of the camera lucida by piling books or
other objects on the drawing board. If one takes the precaution to
draw a scale on the figure under the same conditions, its enlargement
can be readily determined (§ 186).

A very satisfactory way to draw at low magnifications is to use a
simple microscope and arrange a camera lucida over it as over the ocular.
In this way one may get drawings at almost any low magnification.

If one has many
large objects to draw at a
low magnification, then
some form of embryo-
graph is very conven-
ient. (Jour. Roy. Micr.,
Soc., 1899, P- 223.) The
writer has made use of
a photographic camera
and different photo-
graphic objectives for the
purpose. The object is

FIG. 1 1 8. Camera lucida for drawing objects natural size. (H. Bausch
Jour. Applied Microscopy, vol. Hi (/£<%>, p. 801).

illuminated as if for a photograph and in place of the ground glass a
plain glass is used and on this some tracing paper is stretched. Noth-

CH. F]

DRA WING WITH THE MICROSCOPE

133

ing is then easier than to trace the outlines of the object. See also Ch.
VIII.

REFERENCES

Beale, 31, 355 ; Behrens, Kossel and Schiefferdecker, 77 ; Carpenter-Dallinger,
278 ; Van Heurck, 91 ; American Naturalist, 1886, p. 1071, 1887, pp. 1040-1043;
Amer. Monthly Micr. Jour., 1888, p. 103, 1890, p. 94 ; Jour. Roy. Micr. Soc., iSSi,
p. 819, 1882, p. 402, 1883, pp. 283, 560, 1884, p. 115, 1886, p. 516, 1888, pp. 113, 809,
798; Zeit. wiss. Mikroskopie, 1884, pp. 1-21, 1889, p. 367, 1893, pp. 289-295.
Here is described an excellent apparatus made by Winkel. Consult also the latest
catalogs of the opticians.

1O CENTIMETER RULE

The upper edge is in millimeters, the lower in centimeters, and half centimeters.

THE METRIC SYSTEM

UNITS. The most commonly used divisions and multiples.

.„ (Centimeter (c.m.), i-iooth Meter ; Millimeter (m.m.), i-ioooth Meter: Micron
iR< (,x ). i-ioooth Millimeter ; the Micron is the unit in Micrometry & 166).

LENGTH.

( Kilometer, 1000 Meters ; used in measuring roads and other long distances.

THE GRAM FOR \ Milligram (m.g.), i-ioooth Gram.
WEIGHT. . . > Kilogram, 1000 Grams, used for ordinary masses, like groceries, etc.

THE LITER FOR i Cubic Centimeter (c.c. ), i-ioooth Liter. This is more common than the correct
CAPACITY. . \ form, Milliliter.

Divisions of the Units are indicated by the Latin prefixes : deci, i-ioth ; centi, i-iooth ; Milli,
i-ioooth ; Micro, i-i,ooo,oooth of any unit.

Multiples are designated by Greek prefixes : deka, 10 times ; hecto, 100 times ; kilo^ 1000 times
myria, 10,000 times ; Mega, 1,000,000 times any unit.

CHAPTER VI

MICRO-SPECTROSCOPE AND POLARISCOPE,

MICRO-CHEMISTRY, MICRO-MATALLOGRAPHY,

TEXTILE FIBERS

APPARATUS AND MATERIAL REQUIRED FOR THIS CHAPTER

Compound microscope ; Micro-spectroscope (g 188) ; Watch-glasses and small
vials, slides and covers ($ 207) ; Various substances for examination (as blood and
ammonium sulphide, permanganate of potash, chlorophyll, some colored fruit,
etc., (§ 208-217); Micro-polarizer (§ 218); Selenite plate (§ 227); Various doubly
refracting objects, as crystals, textile fibers, starch, section of bone ; Various
chemicals, metals, etc.

MICRO-SPECTROSCOPE

\ 188. A Micro-Spectroscope, Spectroscopic or Spectral Ocular, is a direct
vision spectroscope in connection with a microscope ocular. The one devised by
Abbe and made by Zeiss consists of a direct vision spectroscope prism of the Amici
pattern, and of considerable dispersion, placed over the ocular of the microscope.
This direct vision or Amici prism consists of a single triangular prism of heavy
flint glass in the' middle and one of crown glass on each side, the edge of the
crown glass prisms pointing toward the base of the flint glass prism, i. e., the edge
of the crown and flint glass prisms point in opposite directions. The flint glass
prism serves to give the dispersion or separation into colors, while the crown glass
prisms serve to make the emergent rays approximately parallel with the incident
rays, so that one looks directly into the prism along the axis of the microscope.

The Amici prism is in a special tube which is hinged to the ocular and held in
position by a spring. It may be swung free of the ocular. In connection with
the ocular is the slit mechanism and a prism for reflecting horizontal rays verti-
cally for the purpose of obtaining a comparison spectrum ($ 201). Finally near
the top is a lateral tube with mirror for the purpose of projecting an Angstrom
scale of wave lengths upon the spectrum ($ 202, Figs. 119-120.)

\ 189. Apparent Reversal of the Position of the Colors in a Direct Vision
Spectroscope. — In accordance with the statements in § 188 the dispersion or sepa-
ration into colors is given by the flint glass prism or prisms and in accordance
with the general law that the waves of shortest length, blue, etc., will be bent
most, the colors have the position indicated in the top of Fig. 123, also above
Fig. 119. But if one looks into the direct vision spectroscope or holds the eye
close to the single prism (Fig. 124), the colors will appear reversed as if the red
were more bent. The explanation of this is shown in Fig. 124, where it can be

CH. VI] MICRO-SPECTROSCOPE AND POLARISCOPE

135

readily seen that if the eye is placed at E, close to the prism, the different colored
rays appear in the direction from which they reach the eye and consequently
are crossed in being projected into the field of vision and the real position is in-
verted. The same is true in looking into the micro-spectroscope. The actual

Abbe's Micro-spectroscope.

FIG. 120.

Slit Mechanism separately.
(Plan view, Full size.}

FIG. 119.

Longitudinal Section of
the whole instrument.
( yz Full size. )

"The eye lens is adjustable so as to accurately focus on the slit situated between
the lenses. The mechanism for contracting and expanding the slit is actuated by
the screw F and causes the laminae to move symmetrically {Merz^s movement).
The slit may be made sufficiently wide so as to include the whole visual field. The
screw H serves to limit the length of the slit so as to completely fill the latter with
the image of the object under investigation when the comparison prism is inserted.
The comparison prism is provided with a lateral frame and clips to hold the object
and the illuminating mirror. All these parts are encased in a drum on the ocular.

Above the eye-piece is placed an Amid prism of great dispersion which may be
turned aside about the pivot K, so as to allow of the adjustment of the object. The
prism is retained in its axial position by the spring catch L A scale is projected
on the spectrum by means of a scale tube and mirror attached to the prism casing.
The divisions of the scale indicate in decimals of a micron the wave length of the
respective section of the spectrum. The screw P serves to adjust the scale relative
to the spectrum.

The instrument is inserted in the tube in place of the ordinary eye-piece and is
clamped to the former by means of the screw M in such a position that the mirrors
A and O, which respectively serve to illuminate the comparison prism and the
scale of wave lengths are simultaneously illuminated.'1'1 (Cut loaned by Wm.
Krafft, N. Y. )

136

MICRO-SPECTROSCOPE AND POLARISCOPE \_CH. VI

position of the different colors may be determined by placing some ground glass
or some of the lens-paper near the prism and observing with the eye at the
distance of distinct vision.*

j?o'Ur

$(uctr

FIG. 121. Various Spedrums. — All except that of sodium were obtained by
diffused day-light with the slit of such a width as gave the most distinct Fraunhofer
lines.

It frequently occurs^that with a substance giving several absorption bands (e.
g., chlorophyll] the density or thickness of the solution must be varied to show all
the different bands clearly.

Solar Spectrum. — With diffused day-light and a narrow slit the spectrum is not
visible much beyond the fixed line B. In order to extend the visible spectrum in
the red to the line A, one should use direct sunlight and a piece of ruby glass in
place of the watch-glass in Fig. 123.

Sodium Spectrum. — The line spectrum (§/?/) of sodium obtained by lighting
the microscope with an alcohol flame in which some salt of sodium is glowing.
With the micro-spectroscope the sodium line seen in the solar spectrum and with
the incandescent sodium appears single, except under very favorable circumstances.
(| 102). By using a comparison spectrum of day-light with the sodium spectrum
the light and dark D-lines will be seen to be continuous as here shown.

Permanganate of Potash. — This spectrum is characterized by the presence of
five absorption bands in the middle of the spectrum and is best shown by using a ^
per cent, solution of permanganate in water in a watch-glass as in Fig. 123.

Met-hemoglobin. — The absorption spectrum of met-hemoglobin is characterized
by a considerable darkening of the blue end of the spectrum and of four absorption
bands, one in the red near the line C and two between D and E nearly in the place
of the two bands of oxy-hemoglobin ; finally there is a somewhat faint, wide, band
near- F. Such a met-hemoglobin spectrum is best obtained by making a solution of
blood in water of such a concentration that the two oxy-hemoglobin bands run
together (§ 211], and then adding three or four drops of a ^ per cent, aqueous
solution of permanganate of potash or a few drops of hydrogen dioxid (H2O2 ).
Soon the bright red will change to a brownish color, when it may be examined.

*The author wishes to acknowledge the aid rendered by Professor E. L.
Nichols in giving the explanation offered in this section.

CH. VI] MICRO-SPECTROSCOPE AND POLARISCOPE 137

VARIOUS KINDS OF SPECTRA

By a spectrum is meant the colored bands appearing when the light traverses
a dispersing prism or a diffraction grating, or is affected in any way to separate
the different wave lengths of light into groups. When daylight or some good
artificial light is thus dispersed one gets the appearance so familiar in the
rainbow.

\ 190. Continuous Spectrum. — In case a good artificial light as the electric
light is used the various rainbow or spectral colors merge gradually into one
another in passing from end to end of the spectrum. There are no breaks or gaps.

§ 191. Line Spectrum. — If a gas is made incandescent, the spectrum it pro-
duces consists, not of the various rainbow colors, but of sharp, narrow, bright lines,
the color depending on the substance. All the rest of the spectrum is dark.
These line spectra are very strikingly shown by various metals heated till they are
in the form of incandescent vapor.

\ 192. Absorption Spectrum. — By this is meant a spectrum in which there are
dark lines or bands in the spectrum. The most striking and interesting of the
absorption spectra is the Solar Spectrum , or spectrum of sunlight. If this is exam-
ined carefully it will be found to be crossed by dark lines, the appearance being as
if one were to draw pen marks across a continuous spectrum at various levels,
sometimes apparently between the colors and sometimes in the midst of a color.
These dark lines are the so-called Fraunhofer Lines. Some of the principal ones
have been lettered with Roman capitals, A, B, C, D, E, F, G, H, commencing at
the red end. The meaning of these lines was for a long time enigmatical, but it
is now known that they correspond with the bright lines of a line spectrum (§ 191).
For example, if sodium is put in the flame of a spirit lamp it will vaporize and
become luminous. If this light is examined there will be seen one or two bright
yellow bands corresponding in position with D of the solar spectrum (Fig. 121).
If now the spirit-lamp flame, colored by the incandescent sodium, is placed in the
path of the electric light, and it is examined as before, there will be a continuous
spectrum, except for dark lines in place of the bright sodium lines. That is, the
comparatively cool yellow light of the spirit lamp cuts off or absorbs the intensely
hot yellow light of the electric light ; and although the spirit flame sends a yellow
light to the spectroscope it is so faint in comparison with the electric light that the
sodium lines appear dark. It is believed that in the sun's atmosphere there are
incandescent metal vapors (sodium, iron, etc.), but that they are so cool in com-
parison with the rays of their wave length in the sun that the cooler light of the
incandescent metallic vapors absorb the light of corresponding wave length, and
are, like the spirit lamp-flame, unable to make up the loss, and therefore the pres-
ence of the dark lines.

\ 193. Absorption Spectra from Colored Substances. — While the solar spec-
trum is an absorption spectrum, the term is more commonly applied to the spectra
obtained with light which has passed through or has been reflected from colored
objects which are not self-luminous.

It is the special purpose of the micro-spectroscope to investigate the spectra of
colored objects which are not self-luminous, i. e., blood and other liquids, various
minerals, as monazite, etc. The spectra obtained by examining the light reflected
from these colored bodies or transmitted through them, possess, like the solar

138

MICRO-SPECTROSCOPE AND POLARISCOPE \_CH. VI

spectrum dark lines or bands, but the bands are usually much wider and less
sharply defined. Their number and position depend on the substance or its con-
stitution (Fig. 122), and their width, in part, upon the thickness of the body.
With some colored bodies, no definite bands are present. The spectrum is simply
restricted at one or both ends and various of the other colors are considerably
lessened in intensity. This is true of many colored fruits.

\ 194. Angstrom and Stokes' Law of Absorption Spectra. — The waves of
light absorbed by a body when light is transmitted through some of its substance

FIG. 122. Absorption spectrum of Oxy-hemoglobin or arterial blood (/) and
of Ho moglobin or venous blood (2). (From Gamgee and McMunn}.

A, B, C, D, E, F, G, H. Some of the Prinipal Fraunhofer lines of the solar
spectrum (\ 192}.

.90, .80, .70, .60, .'jo, .40. Wave lengths in microns, as shown in Angstrom's
scale (§ 202). It will be seen that the wave lengths increase toward the red and
decrease toward the violet end of the spectrmn.

Red, Yellow, Orange, etc. Color regions of the spectrum. Indigo should
come between the blue and the violet to complete the seven colors usually given. It
was omitted through inadvertence.

are precisely the waves radiated from it when it becomes self-luminous. For ex-
ample, a piece of glass that is yellow when cool, gives out blue light when it is hot
enough to be self-luminous. Sodium vapor absorbs two bands of yellow light (D
lines); but when light is not sent through it, but itself is luminous and examined
as a source of light its spectrum gives bright sodium lines, all the rest of the
spectrum being dark (Fig. 121).

§ 195. Law of Color. — The light reaching the eye from a colored, solid,
liquid or gaseous body lighted with white light, will be that due to white light less
the light waves that have been absorbed by the colored body. Or in other words,
it will be due to the wave lengths of light that finally reach the eye from the ob-
ject. For example, a thin layer of blood under the microscope will appear
yellowish green, but a thick layer will appear pure red. If now these two layers
are examined with a micro-spectroscope, the thin layer will show all the colors, but
the red end will be slightly, and the blue end considerably restricted, and some of
the colors will appear considerably lessened in intensity. Finally there may ap-
pear two shadow-like bands, or if the layer is thick enough, two well-defined
dark bands in the green (g 210).

If the thick layer is examined in the same way, the spectrum will show only
red with a little orange light, all the rest being absorbed. Thus the spectroscope
shows which colors remain, in part or wholly, and it is the mixture of this remain-
ing or unabsorbed light that gives color to the object.

CH, VI} MICRO-SPECTROSCOPE AND POLARISCOTE 139

\ 196. Complementary Spectra. — While it is believed that Angstrom's law
(I 194) is correct, there are many bodies on which it cannot be tested, as they
change in chemical or molecular constitution before reaching a sufficiently high
temperature to become luminous. There are compounds, however, like those of
didymium, erbium and terbium, which do not change with the heat necessary to
render them luminous, and with them the incandescence and absorption spectra
are mutually complementary, the one presenting bright lines where the other
presents dark ones (Daniell).

ADJUSTING THE MICRO-SPECTROSCOPE

§ 197. The micro-spectroscope, or spectroscopic ocular, is put in
the place of the ordinary ocular in the microscope, and clamped to the
top of the tube by means of a screw for the purpose.

§ 198. Adjustment of the Slit. — In place of the ordinary dia-
phragm with circular opening, the spectral ocular has a diaphragm
composed of two movable knife edges by which a slit-like opening of
greater or less width and length may be obtained at will by the use of
screws for the purpose. To adjust the slit, depress the lever holding
the prism-tube in position over the ocular, and swing the prism aside.
One can then look into the ocular. The lateral screw should be used
and the knife edges approached till they appear about half a millimeter
apart. If now the Amici prism is put back in place and the micro-
scope well lighted, one will see a spectrum by looking into the upper
end of the spectroscope. If the slit is too wide, the colors will overlap
in the middle of the spectrum and be pure only at the red and blue
ends ; and the Fraunhofer or other bands in the spectrum will be
faint or invisible. Dust on the edges of the slit gives the appearance
of longitudinal streaks on the spectrum.

§ 199. Mutual Arrangement of Slit and Prism. — In order
that the spectrum may appear as if made up of colored bands going
directly across the long axis of the spectrum, the slit must be parallel
with the refracting edge of the prism. If the slit and prism are not
thus mutually arranged, the colored bands will appear oblique, and
the whole spectrum may be greatly narrowed. If the colored bands
are oblique, grasp the prism tube and slowly rotate it to the right or to
the left until the various colored bands extend directly across the spec-
trum.

§ 200. Focusing the Slit. — In order that the lines or bands in
the spectrum shall be sharply denned, the eye-lens of the ocular should
be accurately focused on the slit. The eye-lens is movable, and when
the prism is swung aside it is very easy to focus the slit as one focused

140

MICRO-SPECTROSCOPE AND POLARISCOPE \_CH. VI

for the ocular micrometer (§ 172). If one now uses daylight there
will be seen in the spectrum the dark Fraunhofer lines (Fig. 121 E. F.,
etc.).

Coxrx]}.

•• '
*•*!

\\.xi?trt

\

i i '.

i C "i '

/

FIG. 123. FIG. 124. FIG. 125.

FIG. 123. (i). Section of the tube and stage of the microscope with the spectral
ocular or micro-spectroscope in position.

Amid Prism (\ 188). — The direct vision prism of Amid in which the central
shaded pi ism of flint glass gives the dispersion or separation into colors, luhile the

CH. VI] MICRO-SPECTROSCOPE AND POLARISCOPE 141

end prisms of crown glass cause the rays to emerge approximately parallel with
the axis of the microscope. A single ray is represented as entering the prism and
this is divided into three groups (Red, Yellow, Blue}, which emerge from the
prism, the red being least and the blue most bent toward the base of the flint prism
(see Fig. 124).

Hinge. — The hinge on which the prism tube turns when it is swung off the
ocular.

Ocular (\ 1 88) — The ocular in which the slit mechanism takes the place of the
diaphragm (§198). The eye-lens is movable as in a micrometer ocular, so that
the slit may be accurately focused for the different colors (| 200).

S. Screw for setting the scale of wave lengths ( \ 202) .

S' ' . Screw for regulating the width of the slit ( \ 198).

S//. Screw for clamping the micro-spectroscope to the tube of the microscope.

Scale Tube. — The tube near the upper end containing the Angstrom scale and
the lenses for projecting the image upon the upper face of the Amid prism, whence
it is reflected upward to the eye with the different colored rays. At the right is a
special mirror for lighting the scale.

Slit. — The linear opening between the knife edges. Through the slit the light
passes to the prism. It must be arranged parallel with the refracting edge of the
prism, and of such a width that the Fraunhofer or Fixed Lines are very clearly and
sharply defined when the eye-lens is properly Jocused (\ 198-200).

Stage. — The stage of the microscope. This supports a watch-glass with sloping
sides for containing the colored liquid to be examined.

(3) Comparison Prism with tube for colored liquid (C. L.}, and mirror. The
prism reflects horizontal rays vertically, so that when the prism is made to cover
part of the slit two parallel spectra may be seen, one from light sent directly through
the entire microscope and one from the light reflected upward from the comparison
prism.

(4) View of the Slit Mechanism from below. — Slit, the linear space between
the knife edges through which the light passes.

P. Comparison prism beneath the slit and covering part of it at will.

S. S' '. Screws for regulating the length and width of the slit.

FIG. 124. Flint-Glass Prism showing the separation or dispersion of white
light into the three groups of colored rays (Red, Yellow, Blue], the blue rays being
bent the most from the refracting edge (\ 189).

FIG. 125. Sectional View of a Microscope with the Polariscope in position
(8218-227).

Analyzer and Polarizer. — They are represented with corresponding faces par-
allel so that the polarized beam could traverse freely the analyzer. If either Nicol
were rotated 90° they would be crossed and no light would traverse the analyzer
unless some polarizing substance were used as object, (a) Slot in the analyzer
tube so that the analyzer may be raised or lowered to adjust it for difference of
level of the eye- point in different oculars (\ 59, 200).

Pointer and Scale. — The pointer attached to the analyzer and the scale or divided
circle clamped (by the screw S) to the tube of the microscope. The pointer and
scale enable one to determine the exact amount of rotation of the analyzer (\ 220).

Object — The object whose character is to be investigated by polarized light.

142 MICRO-SPECTROSCOPE AND POLAR/SCOPE [CH. VI

To show the necessity of focusing the slit, move the eye-lens down
or up as far as possible, and the Fraunhofer lines cannot be seen.
While looking into the spectroscope move the ocular lens up or down,
and when it is focused the Fraunhofer lines will reappear. As the dif-
ferent colors of the spectrum have different wave lengths, it is neces-
sary to focus the slit for each color if the sharpest possible pictures are
desired.

It will be found that the eye-lens of the ocular must be farther
from the slit for the sharpest focus of the red end than for the sharpest
focus of the lines at the blue end. This is because the wave length of
red is markedly greater than for blue light.

Longitudinal dark lines of the spectrum may be due to irregular-
ity of the edge of the slit or to the presence of dust. They are most
troublesome with a very narrow slit.

§ 201. Comparison or Double Spectrum. — In order to com-
pare the spectra of two different substances it is desirable to be able to
examine their spectra side by side. This is provided for in the better
forms of micro-spectroscopes by a prism just below the slit, so placed
that the light entering it from a mirror at the side of the drum shall be
totally reflected in a vertical direction, and thus parallel with the rays
from the microscope. The two spectra will be side by side with a
narrow dark line separating them. If now the slit is well focused and
daylight be sent through the microscope and into the side to the reflect-
ing or comparison prism, the colored bands and the Fraunhofer dark
lines will appear directly continuous across the two spectra. The
prism for the comparison spectrum is movable and may be thrown en-
tirely out of the field if desired. When it is to be used, it is moved
about half way across the field so that the two spectra shall have
about the same width.

§ 202. Scale of "Wave Lengths. — In the Abbe micro-spectro-
scope the scale is in a separate tube near the top of the prism and at
right angles to the prism-tube. A special mirror serves to light the
scale, which is projected upon the spectrum by a lens in the scale-tube.
This scale is of the Angstrom form, and the wave lengths of any part
of the spectrum may be read off directly, after the scale is once set in
the proper position, that is, when it is set so that any given wave
length on the scale is opposite the part of the spectrum known by pre-
vious investigation to have that particular wave length. The point
most often selected for setting the scale is opposite the sodium line
where the wave length is, according to Angstrom, 0.5892 fji. In ad-

CH. FT] MICRO-SPECTROSCOPE AND POLARISCOPE 143

justing the scale, one may focus very sharply the dark sodium line of
the solar spectrum and set the scale so that the number 0.589 is oppo-
site the sodium or D line, or a method that is frequently used and
serves to illustrate § 191—2, is to sprinkle some salt of sodium (carbon-
ate of sodium is good) in an alcohol lamp flame and to examine this
flame. If this is done in a darkened place with a spectroscope, a
narrow bright band will be seen in the yellow part of the spectrum.
If now ordinary daylight is sent through the comparison prism, the
bright line of the sodium will be seen to be directly continuous with
the dark line at D in the solar spectrum (Fig. 121). By reflecting
light into the scale-tube the image of the scale will appear on the
spectrum, and by a screw just under the scale- tube but within the
prism-tube, the proper point on the scale (0.589 /*) can be brought
opposite the sodium band. All the scale will then give the wave
lengths directly. Sometimes the scale is oblique to the spectrum.
This may be remedied by turning the prism-tube slightly one way or
the other. It may be due to the wrong position of the scale itself. If
so, grasp the milled ring at the distal end of the scale-tube and, while
looking into the spectroscope, rotate the tube until the lines of the scale
are parallel with the Fraunhofer lines. It is necessarj* in adjusting
the scale to be sure that the larger number, o. 70, is at the red end of
the spectrum.

The numbers on the scale should be very clearly defined. If the}*
do not so appear, the scale-tube must be focused by grasping the outer
tube of the scale-tube and moving it toward or from the prism-tube
until the scale is distinct. In focusing the scale, grasp the outer scale-
tube with one hand and the prism-tube with the other, and push or
pull in opposite directions. In this way one will be less liable to injure
the spectroscope.

§ 203. Designation of Wave Length. — Wave lengths of light
are designated by the Greek letter A., followed by the number indicat-
ing the wave length in some fraction of a meter. With the Abbe
micro- spectroscope the micron is taken as the unit as with other micro-
scopical measurements (§ 166). Various units are in use, as the one
hundred thousandth of a millimeter, millionths or ten millionths of a
millimeter. If these smaller units are taken, the wave lengths will be
indicated either as a decimal fraction of a millimeter or as whole num-
bers. Thus, according to Angstrom, the wave length of sodium light
is 5892 ten millionths mm., or 589.2 millionths, or 58.92 one hundred
thousandths, or 0.5892 one thousandth mm., or 0.5892 J/u. The last
would be indicated thus, A. D '= 0.5892 yu.

144 MICRO-SPECTROSCOPE AND POLARISCOPE \CH. VI

§ 204. Lighting for Micro-specftroscope. — For opaque objects
a strong light should be thrown on them either with a concave mirror
or a condensing lens. For transparent objects the amount of the sub-
stance and the depth of color must be considered. As a general rule
it is well to use plenty of light, as that from an Abbe illuminator with
a large opening in the diaphragm, or with the diaphragm entirely
open. For very small objects and thin layers of liquids it may be
better to use less light. One must try both methods in a given case,
and learn by experience.

The direct and the comparison spectra should be about equally
illuminated. One can manage this by putting the object requiring the
greater amount of illumination on the stage of the microscope and
lighting it with the Abbe illuminator. In lighting it is found in gen-
eral that for red or yellow objects, lamp- light gives very satisfactory
results. For the examination of blood and blood crystals the light
from a petroleum lamp is excellent. For objects with much blue or
violet, daylight or artificial light rich in blue light is best.

Furthermore, one should be on his guard against confusing the
ordinary absorption bands with the Fraunhofer lines when daylight is
used. With lamp-light the Fraunhofer lines are absent and, therefore,
not a source of possible confusion.

§ 205. Objectives to Use with the Micro-spectroscope. — If
the material is of considerable bulk, a low objective (16 to 50 mm.) is
to be preferred. This depends on the nature. of the object under ex-
amination, however. In case of individual crystals one should use
sufficient magnification to make the real image of the crystal entirely
fill the width of the slit. The length of the slit may then be regulated
by the screw on the side of the drum, and also by the comparison
prism. If the object does not fill the whole slit the white light enter-
ing the spectroscope with the light from the object might obscure the
absorption bands. For opaque objects illuminating objectives are
useful (§28, 233.)

In using high objectives with the micro-spectroscope one must
very carefully regulate the light ( Ch. II) and sometimes shade the
object.

§ 206. Focusing the Objective. — For focusing the objective the
prism-tube is swung aside, and then the slit made wide by turning the
adjusting screw at the side. If the slit is open one can see objects
when the microscope is focused as with an ordinary ocular. After an
object is focused, it may be put exactly in position to fill the slit of the

CH. VF] MICRO-SPECTROSCOPE AND POLARISCOPE 145

spectroscope, then the knife edges are brought together till the slit is
of the right width ; if the slit is then too long it may be shortened by
using one of the mechanism screws on the side, or if that is not suffi-
cient, by bringing the comparison prism farther over the field. If one
now replaces the Amici prism and looks into the microscope, the
spectrum is liable to have longitudinal shimmering lines. To get rid
of these focus up or down a little so that the microscope will be
slightly out of focus.

§ 207. Amount of Material Necessary for Absorption Spectra
and its Proper Manipulation. — The amount of material necessary to
give an absorption spectrum varies greatly with different substances,
and can be determined only by trial. It a transparent solid is under
investigation it is well to have it in the form a wedge, then succes-
sive thicknesses can be brought under the microscope. If a liquid sub-
stance is being examined, a watch glass with sloping sides forms an
excellent vessel to contain it, then successive thicknesses of the liquid
can be brought into the field as with the wedge-shaped solid. Fre-
quently only a very weak solution is obtainable ; in this case it can be
placed in a homoeopathic vial, or in some glass tubing sealed at the
end, then one can look lengthwise through the liquid and get the
effect of a more concentrated solution. For minute bodies like crystals
or blood corpuscles, one may proceed as described in the previous
section.

MICRO-SPECTROSCOPE — EXPERIMENTS*

§ 208. Put the micro-spectroscope in position, arrange the slit
and the Amici prism so that the spectrum will show the various spec-
tral colors going directly across it (§ 198-199) and carefully focus the
slit. This may be done either by swinging the prism-tube aside and
proceeding as for the ocular micrometer (§ 172), or by moving the
eye- lens of the ocular up and down while looking into the micro-
spectroscope until the dark lines of the solar spectrum are distinct. If
they cannot be made distinct by focusing the slit, then the light is too
feeble or the slit is too wide (§ 198). With the lever move the com-
parison prism across half the field so that the two spectra shall be of
about equal width. For lighting, see § 204.

*If one does not possess a micro-spectroscope, quite satisfactory results may be
obtained by using a microscope with a 16 to 12 mm. objective and a pocket, direct-
vision spectroscope in place of the eye-piece. (Bleile, Trans. Amer. Micr. Soc.
1900, p. 8).

146 MICRO-SPECTROSCOPE AND POLARISCOPE {CH. VI

§ 209. Absorption Spectrum of Permanganate of Potash.—
Make a solution of permanganate of potash in water of such a strength
that a stratum 3 or 4 mm. thick is transparent. Put this solution in a
watch-glass with sloping sides, and put it under the microscope. Use
a 50 mm. or 16 mm. objective, and use the full opening of the illumi-
nator. Light strongly. Look into the spectroscope and slowly move
the watch-glass into the field. Note carefully the appearance with the
thin stratum of liquid at the edge and then as it gradually thickens on
moving the watch-glass still farther along. Count the absorption
bands and note particularly the red and blue ends. Compare carefully
with the comparison spectrum (Figs. 121, 122). For strength of solu-
tion see § 207.

§ 210. Absorption Spectrum of Blood. — Obtain blood from a
recently killed animal, or flame a needle, and after it is cool prick the
finger two or three times in a small area, then wind a handkerchief or
a rubber tube around the base of the finger, and squeeze the finger
with the other hand. Some blood will ooze out of the pricks. Rinse this
off into a watch-glass partly filled with water. Continue to add the blood
until the water is quite red. Place the watch-glass of diluted blood un-
der the microscope in place of the permanganate, using the same object-
ive, etc. Note carefully the spectrum. It would be advantageous to
determine the wave length opposite the center of the dark bands. This
may easily be done by setting the scale properly as described in § 202.
Make another preparation, but use a homeopathic vial instead of a
watch-glass. Cork the vial and lay it down upon the stage of the
microscope. Observe the spectrum. It will be like that in the watch-
glass. Remove the cork and look through the whole length of the vial.
The bands will be much darker, and if the solution is thick enough
only red and a little orange will appear. Re-insert the cork and incline
the vial so that the light traverses a very thin layer, then gradually
elevate the vial and the effect of a thicker and thicker layer may be
seen. Note especially that the two characteristic bands unite and
form one wide band as the stratum of liquid thickens. Compare with
the following :

Add to the vial of diluted blood a drop or two of ammonium sul-
phide, such as is used for a reducing agent in chemical laboratories.
Shake the bottle gently and then allow it to stand for ten or fifteen
minutes. Examine it and the two bands will have been replaced by a
single, less clearly defined band in .about the same position. The
blood will also appear somewhat purple. Shake the vial vigorously

CH. VI} MICRO-SPECTROSCOPE AND POLARISCOPE 147

and the color will change to the bright red of fresh blood. Examine
it again with the spectroscope and the two bands will be visible. After
five or ten minutes another examination will show but a single band.
Incline the bottle so that a thin stratum may be examined. Note
that the stratum of liquid must be considerably thicker to show the
absorption band than was necessary to show the two bands in the first
experiment. Furthermore, while the single band may be made quite
black on thickening the stratum, it will not separate into two bands
with a thinner stratum. In this experiment it is very instructive to
have a second vial of fresh diluted blood, say that from the watch-
glass, before the opening of the comparison prism. The two banded
spectrum will then be in position to be compared with the spectrum of
the blood treated with the ammonium sulphide.

The two banded spectrum is that of oxy-hemoglobin, or arterial blood,
the single banded spectrum of hemoglobin (sometimes called reduced
hemoglobin) or venous blood, that is, the respiratory oxygen is present
in the two banded spectrum but absent from the single banded spectrum.
When the bottle was shaken the hemoglobin took up oxygen from the
air and became oxy-hemoglobin, as occurs in the lungs, but soon the
ammonium sulphide took away the respiratory oxygen, thus reducing
the oxy-hemoglobin to hemoglobin. This may be repeated many
times (Fig. 122).

§ 211. Met-Hemoglobin. — The absorption spectrum of met-
hemoglobin is characterized by a considerable darkening of the blue
end of the spectrum and of four absorption bands, one in the red near
the line C and two between D and E, nearly in the place of the two
bands of oxy-hemoglobin ; finally there is a somewhat faint, wide
band near F. Such a met-hemoglobin spectrum is best obtained by
making a solution of blood in water of such a concentration that the
two oxy-hemoglobin bands run together (§ 210), 'and then adding
three or four drops of a y1^ per cent, aqueous solution of permanganate
of potash. Soon the bright red will change to a brownish color, when
it may be examined (Fig. 121). Instead of the permanganate one
may use hydrogen dioxide (H2O2).

§ 212. Carbon Monoxide Hemoglobin (CO-Hemoglobin). —
To obtain this, kill an animal in illuminating gas, or one may allow
illuminating gas to bubble through some blood already taken from the
body. The gas should bubble through a minute or two. The oxygen
will be displaced by carbon monoxide. This forms quite a stable com-
pound with hemoglobin, and is of a bright cherry-red color. Its

148 MICRO-SPECTROSCOPE AND POLARISCOPE [CH. VI

spectrum is nearly like that of oxy-hemoglobin, but the bands are
farther toward the blue. Add several drops of ammonium sulphide and
allow the blood to stand some time. No reduction will take place,
thus forming a marked contrast to solutions of oxy-hemoglobin. By
the addition of a few drops of glacial acetic acid a dark brownish red
color is produced.

§ 213. Carmine Solution. — Make a solution of carmine by put-
ting y^th gram of carmine in 100 cc. of water and adding 10 drops of
strong ammonia. Put some of this in a watch-glass or in a small vial
and compare the spectrum with that of oxy-hemoglobin or carbon
monoxide hemoglobin. It has two bands in nearly the same position,
thus giving the spectrum a striking similarity to blood. If now several
drops, 15 or 20, of glacial acetic acid are added to the carmine, the
bands remain and the color is not markedly changed, while with
either oxy-hemoglobin or CO-hemoglobin the color would be de-
cidedly changed from the bright red to a dull reddish brown, and
the spectrum, if any could be seen, would be markedly different.
Carmine and O- hemoglobin can be distinguished by the use of ammo-
nium sulphide, the carmine remaining practically unchanged while the
blood shows the single band of hemoglobin (§ 210). The acetic acid
serves to differentiate the CO-hemoglobin as well as the O-hemoglobin.

§ 214. Colored Bodies not giving Distinctly Banded Absorp-
tion Specftra. — Some quite brilliantly colored objects, like the skin of
a red apple, do not give a banded spectrum. Take the skin of a red
apple, mount it on a slide, put on a cover-glass and add a drop of
water at the edge of the cover. Put the preparation under the micro-
scope and observe the spectrum. Although no bands will appear, in
some cases at least, yet the ends of the spectrum will be restricted and
various regions of the spectrum will not be so bright as the comparison
spectrum. Here the red color arises from the mixture of the unab-
sorbed waves, as occurs with other colored objects. In this case,
however, not all the light of a given wave length is absorbed, conse-
quently there are no clearly defined dark bands, the light is simply
less brilliant in certain regions and the red rays so predominate that
they give the prevailing color.

§ 215. Nearly Colorless Bodies with Clearly Marked Ab-
sorption Spectra. — In contradistinction to the brightly colored
objects with no distinct absorption bands are those nearly colorless
bodies and solutions which give as sharply defined absorption bands as
could be desired. The best examples of this are afforded by solutions

CH. VI] MICRO-SPECTROSCOPE AND POLARISCOPE 149

of the rare earths, didymium, etc. These in solutions that give
hardly a trace of color to the eye give absorption bands that almost
rival the Fraunhofer lines in sharpness.

§ 216. Absorption Spectra of Minerals. — As example take some
monazite sand on a slide and either mount it in balsam (see § 256),
or cover and add a drop of water. The examination may be made also
with the dry sand, but it is less satisfactory. Light well with trans-
mitted light, and move the preparation slowly around. Absorption
bands will appear occasionally. Swing the prism tube off the ocular,
open the slit and focus the sand. Get the image of one or more grains
directly in the slit, then narrow and shorten the slit so that no light
can reach the spectroscope that has not traversed the grain of sand.
The spectrum will be satisfactory under such conditions. It is
frequently of great service in determining the character of unknown
mineral sands to compare the spectra with known minerals. If the
absorption bands are identical, it is strong evidence in favor of the
identity of the minerals. For proper lighting see § 204.

§ 217. While the study of absorption spectra gives one a great
deal of accurate information, great caution must be exercised in draw-
ing conclusions as to the identity or even the close relationship of
bodies giving approximately the same absorption spectra. The rule
followed by the best workers is to have a known body as control and
to treat the unknown body and known body with the same reagents,
and to dissolve them in the same medium. If all the reactions are
identical then the presumption is strong that the bodies are ident-
ical or very closely related. For example, while one might be in doubt
between a solution of oxy- or CO-hemoglobin and carmine, the addition
of ammonium sulphide serves to change the double to a single band
in the O-hemoglobin, and glacial acetic acid enables one to distinguish
between the CO-blood and the carmine, although the ammonium sul-
phide would not enable one to make the distinction. Furthermore it
is unsafe to compare objects dissolved in different media. The same
objects as "cyanine and aniline blue dissolved in alcohol give a very
similar spectrum, but in water a totally different one." "Totally dif-
ferent bodies show absorption bands in exactly the same position (solid
nitrate of uranium and permanganate of potash in the blue)." (Mac-
Munn). The rule given by MacMunn is a good one : "The recogni-
tion of a body becomes more certain if its spectrum consists of several
absorption bands, but even the coincidence of these bands with those
of another body is not sufficient to enable us to infer chemical identity;

150 MICRO-SPECTROSCOPE AND POLARISCOPE [CH. VI

what enables us to do so with certainty is the fact : that the two solu-
tions give bands of equal intensities in the same parts of the spectrum
which undergo analogous changes on the addition of the same reagent. ' '

REFERENCES TO THE MICRO-SPECTROSCOPE AND
SPECTRUM ANALYSIS

The micro-spectroscope is playing an ever-increasingly important role in the
spectrum analysis of animal and vegetable pigments, and of colored mineral and
chemical substances, therefore a somewhat extended reference to literature will be
given. Full titles of the books and periodicals will be found in the Bibliography
at the end.

Angstrom, Recherches sur le spectre solaire, etc. Also various papers in
periodicals. See Royal Soc's Cat'l Scientific Papers ; Anthony & Brackett ; Beale,
p. 269 ; Behrens, p. 139 ; Kossel und Schiefferdecker, p. 63 ; Carpenter, p. 323 ;
Browning, How to Work with the Spectroscope, and in Monthly Micr. Jour., II,
p. 65 ; Daniell, Principles of Physics. The general principles of spectrum analysis
are especially well stated in this work, pp. 435-455 ; Davis, p. 342 ; Dippel, p.
277 ; Frey ; Gamgee, p. 91 ; Halliburton ; Hogg, p. 122 ; also in Monthly Micr.
Jour., Vol. II, on colors of flowers; Jour, Roy. Micr. Soc., 1880, 1883, and in
various other vols. ; Kraus ; Lockyer ; M'Kendrick ; MacMunn ; and also in
Philos, Trans. R. S., 1886; various vols. of Jour. Physiol.; Nageli und Schwend-
ener ; Proctor; Ref. Hand-Book Med. Science, Vol. I, p. 577, VI, p. 516, VII, p.
426; Roscoe ; Schellen ; Sorby, in Beale, p. 269; also Proc. R. S., 1874, p. 31,
1867, p. 433 ; see also in the Scientific Review, Vol. V, p. 66, Vol. II, p. 419.
The larger works on Physiology, Chemistry and Physics may also be consulted
with profit.

Vogel, Spectrum analysis ; also in Nature, Vol. xix, p. 495, on absorption spec-
tra. The bibliography in MacMunn is excellent and extended.

For hemochromogen in medico-legal cases see Bleile, Trans. Amer. Micr.
Soc., 1900, p. 9.

MICRO-POLARISCOPB

g 218. The micro-polariscope, or polarizer, is a polariscope used in connection
with a microscope.

The most common and typical form consists of two Nicol prisms, that is, two
somewhat elongated rhombs of Iceland spar cut diagonally and cemented together
with Canada balsam. These Nicol prisms are then mounted in such a way that
the light passes through them lengthwise, and in passing is divided into two rays
of plane polarized light. The one of these rays obeying most nearly the ordinary
law of refraction is called the ordinary ray, the one departing farthest from the
law is called the extra-ordinary ray. These two rays are not only polarized, but
polarized in planes almost exactly at right angles to each other. The Nicol prism
totally reflects the ordinary ray at the cemented surface as it meets that surface at
an angle greater than the critical angle, and only the extraordinary or less refracted
ray is transmitted.

CH. VI~\ MICRO-SPECTROSCOPE AND POLARISCOPE 151

§ 219. Polarizer and Analyzer. — The polarizer is one of the Nicol prisms. It is
placed beneath the object and in this way the object is illuminated with polarized
light. The analyzer is the other Nicol and is placed at some level above the object,
very conveniently above the ocular.

When the corresponding faces of the polarizer and analyzer are parallel i. e.,
when the faces through which the oblique section passes are parallel, light passes
freely through the analyzer to the eye. If these corresponding faces are at right
angles, that is, if the Nicols are crossed, then the light is entirely cut off and the
two transparent prisms become opaque to ordinary light. There are then, in the
complete revolution of the analyzer, two points, at o° and 180°, where the corre-
sponding faces are parallel and where light freely traverses the analyzer. There
are also two crossing points of the Nicols, at 90° and 270°, where the light is extin-
guished. In the intermediate points there is a sort of twilight.

\ 220. Putting the Polarizer and Analyzer in Position. — Swing the diaphragm
carrier of the Abbe illuminator out from under the illuminator, remove the disk
diaphragm or open widely the iris diaphragm and place the analyzer in the dia-
phragm carrier, then swing it back under the illuminator. Remove the ocular,
put the graduated ring on the top of the tube and then replace the ocular and put
the analyzer over the ocular and ring. Arrange the graduated ring so that the indi-
cator shall stand at o° when the field is lightest. This may be done by turning the
tube down so that the objective is near the illuminator, then shading the stage so
that none but polarized light shall enter the microscope. Rotate the analyzer until
the lightest possible point is found, then rotate the graduated ring till the index
stands at o°. The ring may then be clamped to the tube by the side screw for the
purpose. Or, more easily, one may set the index at o°, clamp the ring to the
microscope, then rotate the draw-tube of the microscope till the field is lightest.

I 221. Adjustment of the Analyzer. — The analyzer should be capable of
moving up and down in its mounting, so that it can be adjusted to the eye-point
of the ocular with which it is used. If on looking into the analyzer with parallel
Nicols the edge of the field is not sharp, or if it is colored, the analyzer is not in
a proper position with reference to the eye-point, and should be raised or lowered
till the edge of the field is perfectly sharp and as free from color as the ocular
with the analyzer removed.

§ 222. Objectives to Use with the Polariscope. — Objectives of the lowest pow-
ers may be used, and also all intermediate forms up to a 2 mm. homogeneous im-
mersion. Still higher objectives may be used if desired. In general, however,
the lower powers are somewhat more satisfactory. A good rule to follow in this
case is the general rule in all microscopic work, — use the power that most clearly
and satisfactorily shows the object under investigation.

$ 223. Lighting for Micro- Polariscope Work. — Follow the general directions
given in Chapter II. It is especially necessary to shade the object so that no un-
polarized light can enter the objective, otherwise the field cannot be sufficiently
darkened. No diaphragm is used over the polarizer for most examinations. Direct
sunlight may be used to advantage with some objects, and as a rule the object
would best be very transparent.

\ 224. Mounting Objects for the Polariscope. — So far as possible objects
should be mounted in balsam to render them transparent. In many cases objects
mounted in water do not give satisfactory polariscope appearances. For example,

152 MICRO-SPECTROSCOPE AND POLARISCOPE \_CH. VI

if starch is mounted dry or in water, the appearances are not so striking as in a
balsam mount (Davis, p. 337 ; Suffolk).

\ 225. Purpose of a Micro-Polariscope. — The object of a micro-polariscope is
to determine, in microscopic masses, one or more of the following points : (A)
Whether the body is singly refractive, mono-refringent, or isotropic, that is, opti-
cally homogeneous, as are glass and crystals belonging to the cubical system ; (B)
Whether the object is doubly refractive or anisotropic, uniaxial or biaxial ; (C)
Pleochroism ; (D) The rotation of the plane of polarization, as with solutions of
sugar, etc. ; (E) To aid in petrology and mineralogy ; (F) To aid in the determi-
nation of very minute quantities of crystallizable substances; (G) For the pro-
duction of colors.

For petrological and mineralogical investigations the microscope should possess
a graduated, rotating stage so that the object can be rotated, and the exact angle
of rotation determined. Fig. 126. It is also found of advantage in investigating
object swith polarized light where colors appear, to combine a polariscopic and
spectroscope ( Spectro-Polariscope ) .

MICRO-POLARISCOPE — EXPERIMENTS

§ 226. Arrange the polarizer and analyzer as directed above (§ 220)
and use a 16 mm. objective except when otherwise directed.

(A) Isotropic or Singly Refracting Objects. — Light the mi-
croscope well and cross the Nicols, shade the stage and make the field
as dark as possible (§219). As an isotropic substance, put an ordin-
ary glass slide under the microscope. The field will remain dark. As
an example of a crystal belonging to the cubical system and hence iso-
tropic, make a strong solution of common salt (sodium chloride)
put a drop on a slide and allow it to crystallize, put it under the
microscope, remove the analyzer, focus the crystals and then replace
the analyzer and cross the Nicols. The field and the crystals will re-
main dark.

(B) Anisotropic or Doubly Refracting Objects. — Make a fresh
preparation of carbonate of lime crystals like that described for pedesis
(§ !50» or use a preparation in which the crystals have dried to the
slide, use a 5 or 3 mm. objective, shade the object well, remove the
analyzer and focus the crystals, then replace the analyzer. Cross the
Nicols. In the dark field will be seen multitudes of shining crystals,
and if the preparation is a fresh one in water, part of the smaller
crystals will alternately flash and disappear. By observing carefully,
some of the larger crystals will be found to remain dark with crossed
Nicols, others will shine continuously. If the crystals are in such a
position that the light passes through them parallel with the optic

FIG. 126. Chamofs Microscope
for Micro-Chemical Analysis (Jour-
nal of Applied Microscopy, 1809, p.

This is a modified and simplified
petrographical microscope and has
all the attachments and motions nec-
essary for micro-chemical analysis,
As the objects studied are mostly
liquid or in liquids the microscope
has no joint as it must be used in a
vertical position.

154 MICRO-SPECTROSCOPE AND POLARISCOPE [CH. VI

axis,* the crystals are isotropic like salt crystals and remain dark.
If, however, the light traverses them in any other direction the ray
from the polarizer is divided into two constituents vibrating in planes
at right angles to each other, and one of these will traverse the an-
alyzer, hence such crystals will appear as if self-luminous in a dark-
field. The experiment with these crystals from the frog succeeds well
with a 2 mm. homogeneous immersion.

As a further illustration of anisotropic objects, mount some cotton
fibers in balsam (§ 256), also some of the lens paper (§ 114;. These
furnish excellent examples of vegetable fibers.

Striated muscle fibers are also very well adapted for polarizing
objects.

. As examples of biaxial crystals, allow some borax solution to dry
and crystallize on a slide ; use the crystals as objects. As all doubly
refracting objects restore the light with crossed Nicols, they are some-
times called depolarizing.

(C) Pleochroism. — This is the exhibition of different tints as the
analyzer is rotated. An excellent subject for this will be found in
blood crystals.

§ 227. Production of Colors. — For the production of gorgeous
colors, a plate of selenite giving blue and yellow colors is placed between
the polarizer and the object. If properly mounted, the selenite is very
conveniently placed on the diaphragm carrier of the Abbe illuminator,
just above the polarizer. A thin plate or film of mica also answers
well.

It is not necessary to use selenite or mica for the production of the
most glorious colors in many objects. One of the most beautiful pre-
parations, and one of the most instructive also, may be prepared as
follows : Heat some xylene balsam on a slide until the xylene is nearly
evaporated. Add some crystals of the medicine, sulphonal and warm
till the sulphonal is melted and mixes with the balsam. While the
balsam is still melted put on a cover-glass. If one gets perfect crystals
there will be shown not only the most beautiful colors, but the black
cross with perfection. (Clark).

*The optic axis of doubly refracting crystals is the axis along which the crystal
is not doubly refracting, but isotropic like glass. When there is but one such
axis, the crystal is said to be uniaxial, if there are two such axes the crystal is
said to be bi-axial.

The crystals of carbonate of lime from the frog (see \ 151) are uniaxial crystals.
Borax crystals are bi-axial.

CH. VI~\ MICRO-CHEMISTRY 155

It is very instructive and interesting to examine many organic
and inorganic substances with a micro-polarizer.

REFERENCES TO THE POLARISCOPE AND TO THE USE OF POLARIZED

LIGHT.

Anthony & Brackett, 133 ; Behrens ; Behrens, Kossel und Schiefferdecker ;
Carnoy, 61 ; Carpenter-Dallinger, 317, 1097; Clark; Daniel, 494; Davis; v.
Ebener ; Gamgee ; Halliburton, 36,272 ; Hogg, 133,729 ; Lehmann ; M'Kendrick ;
Nageli undSchwendener, 299 ; Quekett ; Suffolk, 125 ; Valentin ; Physical Review,
I., p. 127. Daniell, Physics for Medical Students. Nichols, Physics.

MICRO-CHEMISTRY

§ 228. During the last decade the microscope has become one of
the necessities of the expert chemist, and the signs of the times
indicate that in every research laboratory of chemistry the microscope
will become as familiar as it now is in research laboratories of biology.
Its proper place in chemistry has been admirably stated by Chamot :

"It is rather remarkable how slow American chemists have been in realizing
the importance of the microscope as an adjunct to every chemical laboratory.
This is, perhaps, largely due to the fact that few of our students in chemistry
become familiar with the construction and manipulation of this instrument, just
as few of them become sufficiently familiar with the spectroscope and its manifold
uses ; and doubtless also because of the prevailing impression that a microscope
is primarily an instrument for the biologist and is of necessity a most expensive
luxury. The fact is, however, that this instrument is now far from being a luxury
to the chemist, and the time is not far distant when it will be conceded to be as
much a necessity in every analytical laboratory as is the balance.

"Nor is the apprenticeship to its use in chemical work long or intricate.

"Micro- chemical analysis should appeal to every chemist because of its neat-
ness, wonderful delicacy, in which it is not excelled even by the spectroscope, and
the expedition with which an analysis can be made. A complete analysis,
intricate though it may be, is a matter of a few minutes rather than of a few hours.

"While there is no good reason to believe, as do some enthusiasts, that this
new system is to displace the old analysis in the wet way, every chemist should,
nevertheless, familiarize himself with the microscope, its accessories, and the
elegant and time-saving methods of micro-analysis, thus enabling him to examine
qualitatively the most minute amounts of material with a rapidity and accuracy
which is truly marvelous ; not to speak of the many substances for which no
other method of identification is known.

"At present the greatest bar to its general use is the absence of any well
denned scheme, and the absolute necessity of being well grounded in general
chemistry. There are no tables which can be followed in a mechanical way by
the student, but on the contrary he is obliged to exercise his knowledge and judg-
ment at every step. For this very reason the introduction of this subject into the
list of those now taught is greatly to be desired. ' '

156 MICRO-CHEMISTRY \.CH. VI

It will be seen by the last paragraph that in chemistry as in
biology, the microscope is only an aid to investigation; it cannot take
the place of thorough training in the fundamentals of the subject
under investigation.

§ 229. The following list of substances is suggested by Dr.
Chamot for beginning practice as the results given are definite and
easily obtained :

SUGGESTIONS FOR STUDY ON CRYSTAL SYSTEMS

"Isometric.

Sodium chlorid, potassium chlorid, potassium iodid. Strontium nitrate.
Barium nitrate. Lead nitrate. Potassium bromid. Sodium bromid.

Alums crystallize in octahedra, cubes or combinations of the two. It is
well to recall that the alums have the general formula, M2(RO4)3.N2RO4.24
H2O, where -M- can be Al, Cr, Mn, Fe, In, Ga, Tl, R ; -N- Na, K, Rb, Cs,
NH4 Ag, or Tl and -R- S or Se. All alums are isomorphous.

Tetragonal.

Potassium copper chlorid. Ammonium copper chlorid. Urea.
Nickel sulfate 6H2O. This salt is dimorphic, crystallizing also in the
monoclinic system. Nickel sulfate yH2O is orthorhombic.

Orthorhombic.

Asparagin. Picric acid. Acetanilid. Resorcin.

Mercuric chlorid. Silver nitrate. Potassium sulfate. Potassium nitrate.
Magnesium sulfate 7H2O. Potassium chromate. Sodium nitrate (also
Hexagonal).

Monoclinic.

Lactose. Napthalene. Potassium ferric oxalate. Sodium ferric oxalate.

Potassium chlorate (sodium chlorate is Isomet. or Tetrag.)

Lead acetate. Copper acetate H2O. Oxalic acid.

Ferrous sulfate, this salt forms normally with 7 H2O and is then Mono-
clinic, but in presence of zinc sulfate becomes Orthorhombic, and in presence
of copper sulfate, triclinic. Sodium Sulfate ioH2O. Borax. Potassium ferri-
cyanid.

Triclinic.

Copper sulfate 5H 2 O. Boric acid. Potassium dichromate.

Hexagonal.

Lead iodid (according to Behrens PbI2 is probably orthorhombic).
Sodium nitrate (also Orthorhombic). Bromoform. lodoform.

% 230. Before performing analytical tests, it is necessary that the student
be familiar with the properties of crystals and also thoroughly familiar with
the appearance of crystals of the chlorids, nitrates, and sulfates of Sodium,
Potassium, and Ammonium ; since some of these salts are sure to appear in
almost every test drop examined.

CH. VI} MICRO-CHEMISTRY 157

Frequently a chemically pure salt cannot be made to yield satisfactory
crystals on the evaporation of its solution, but beautifully formed crystals will
result when in the presence of other compounds. A striking example is found in
Ammonium chlorid. This salt fails to yield other than dendritic masses when
preparations are made from the pure salt, but if formed by metathesis and
especially if in the presence of a difficultly crystallizable salt, well formed isome-
tric crystals (cubes) are seen.

AN EXERCISE FOR PRACTICE

Take a fragment of ammonium chlorid, dissolve in a tiny drop of water on a
slide and try to obtain distinct well formed crystals. Neither slow nor rapid
evaporation nor recrystallization by breathing on the preparation will yield satis-
factory crystals.

Place a small drop of water on a glass slide, add Ferric chlorid until the drop
is distictly yellow. Stir. At the centre of the drop add two or three tiny frag-
ments of Ammonium acetate. The preparation must not be warmed. There is
formed Ferric acetate, Ammonium chlorid and double chlorids of ammonium and
iron. Study the preparation and observe the following points, i. Tendency
toward formation of double salt. 2. That the type crystal of NH4C1 is a cube.
3. Cubes may so grow as to present the appearance of a rectangular prism. 4. In
certain positions cubes have the appearance of a pyramid. 5. In other positions
they exhibit a hexagonal outline, thus simulating a polyhedron of many faces.
6. There is scarcely any tendency in this case toward the formation of the
dendritic masses observed in the first experiment. 7. The crystals often develop
fastest along the diagonal planes so that the regular faces are replaced by
pyramidal depressions. ' '

FIG. 127. Czapski's Ocular Iris-diaphragm with cross
hairs for examining and accurately determining the axial im-
ages of small crystals. The iris diaphragm enables the observer
to make the field as large or small as desired.

A. Longitudinal Section.

B. Transection, showing the cross lines and the iris dia-
phragm with the projecting part at the left, by which the dia-
phragm is opened and closed. (Zeiss* Catalog. )

For directions and hints in micro-chemical work and crystallography, consult
the various volumes of the Journal of the Roy. Micr. Soc.; Zeitschrift fiir physio-
logische Chemie, and other chemical journals ; Wormly ; Kl£ment & Renard ;
Carpenter-Dalliuger ; Hogg ; Behrens, Kossel und Schiefferdecker ; Frey ; Dana',
and other works on mineralogy ; Davis, Behrens, T. H.— Anleitung zur micro-
chemischen Analyse der wichtigsten organischen Verbindungen. Hamburg,
1895-1897. Microchemische Technik, 2d edition, Hamburg, 1900. A manual of
michrochemical analysis with an introductory chapter by J. W. Judd, London.

158 TEXTILE FIBERS \_CH. VI

1894. Bspecial attention is also called to the articles of Dr. E. M. Chamot in the
Journal of Applied Microscopy beginning with vol. ii. p. 502, and continued in
vol. iii. and iv.

TEXTILE FIBERS, FOOD AND PHARMACOLOGICAL PRODUCTS

§ 231. The microscope is coming more and more into use for the
determination of the character of textile fibers, both in the raw state
and after manufacture. As the textile fibers have distinctive char-
acters it is not difficult to determine mixtures in fabrics of various
kinds. The student is advised to study carefully known fibers, as of
cotton, wool, linen, silk, jute etc., so that he is certain of the appear-
ances, and then to determine of what fibers different fabrics are com-
posed. He will be astonished at the amount of "Alabama wool" in
supposedly all wool goods.

For works and articles upon textile fibers see : Herzfeld, J. Trans-
lated by Salter. The technical testing of yarns and textile fabrics
with reference to official specifications. London, 1898. E. A.
Posselt — The structure of fibers, yarns and fabrics. Philadelphia
and London, 1891. Dr. C. Rougher — Des filaments vegetaux em-
ployes dans Tindustrie. Paris, 1873. Wm. P. Wilson and E. Fah-
ring — , The conditioning of wool and other fabrics in the technological
laboratories of the Philadelphia Commercial Museum. Journal of Ap-
plied Microscopy, Vol. II, (1899) pp. 290-292, 457-460. Bulletin of
the National Association of Wool Growers, 1875, p. 470. Proceed-
ings of the Amer. Micr. Soc., 1884, PP- 65-68. Besides these refer-
ences one is liable to find pictures and discussions of various fibers
in general works on the microscope, and in technical and general
cyclopaedias.

§ 232. From the nature of food and pharmacological products
adulterations are in many cases most accurately and easity determined
by microscopic examination. The student will find constant reference
to the microscopical characters of the genuine and spurious substances
in medicines and other pharmacological products in works on pharmacy
or pharmacology ; also in pharmacological journals and in druggists'
reports, e. g. the excellent and well illustrated article by Burt E.
Nelson of the chemical laboratory of the Binghamton State Hospital
on the Microscopical examination of tea, coffee, spices and condiments.
This appeared in Merck's Report, Oct. 15, Dec. 15, 1898. The micro-
scopical Journals also contain occasional articles bearing upon this
subject. See also Food Products in bulletins of the U. S. Dep't Agr.
Mace, E. — Les substances alimentaire, etc., Paris, 1891. Schimper,

CH. VI} MICRO-METALLOGRAPHY 159

A. F. W. Anleitung, etc. Jena, 1900. Hugh Gait, — The Microscopy
of the starches, illustrated by photo-micrographs, London, 1900.

THE MICROSCOPE IN METALLOGRAPHY

§ 233. In the modern investigation of metals and alloys much
light has been thrown upon the structural peculiarities which render
some mixtures satisfactory and others unsatisfactory. There are two
great methods : First, that of studying fractured surfaces without re-
course to any reagents. Second, to polish a metallic surface carefully
with emery or carborundum and finally with rouge or diamantine and
then etch it with some acid for a longer or shorter time. For either
method reflected light must be used. For low powers that obtained at a
good window or by a lamp or a lamp and bulls eye are good. The illum-
inating objectives (§ 28), i. e. objectives in which a prism in the side
of the objective reflects light down through the lenses which act as a
condenser, are preferable for most work and indeed necessary if one uses
high powers. For special microscope see Fig. 126 A.

Elaborate arrangements have been devised for holding the piece
of metal on the stage, but some beeswax, or some clay made plastic
with glycerin answers well. For pictures of the appearances seen
in studying metallic surfaces, see the journals of engineering and
metallurgy, especially the Metallographist, a quarterly publication
devoted to the study of metals with special reference to their physics
and micro-structure, etc. In twenty-five or more of the great metal
manufacturing establishments special laboratories for microscopic
examination and investigation have been established. This is an illus-
tration of what has frequently occurred — great manufacturing interests
have outrun the universities in the appreciation and application of
methods of reasearch. Fortunately, however, laboratories are already
springing up in connection with the universities, and probably within
ten years every great technical school will have its laboratory of
micro-metallography where students will have opportunity to perfect
themselves in the preparation, photography and microscopic study of
the metals and alloys.

Beside the sources of information given above, see Dr. H. Ost und
Dr. Fr. Kolbeck, Lehrbuch der chemischen Technologic mit einem
Schlussabschnitt " Metallurgie. " Hannover, 1901. Behrens, T. H. —
Das mikroskopische Gefiige der Matalle, etc. Hamburg, 1894. For
an excellent bibliography of 188 titles ; see the Metallographist, Vol.
I, 1898, and appended to the special papers in all the volumes. Also
in Iron Age, Jan. 27, 1898. Carpenter- Dallinger, p. 264.

i6o

MICRO-METALLOGRAPHY

\CH. VI

FIG. 126 A. Microscope especially constructed for the study of metals and
alloys. ( The Boston Testing Laboratories').

The stage is rotary, and may be raised or lowered by rack and pinion. Above
the objective is the arrangement for illumination (see Ch. VII}.

CHAPTER VII

SLIDES AND COVER-GLASSES ; MOUNTING ; ISOLATION ;
SECTIONING BY THE COLLODION AND THE PARAF-
FIN METHODS; LABELING AND STORING MICRO-
SCOPICAL PREPARATIONS ; REAGENTS

SLIDES AND COVER-GLASSES

\ 234. Slides, Glass Slides or Slips, Microscopic Slides or Slips. — These are
strips of clear flat glass upon which microscopic specimens are usually mounted for
preservation and ready examination. The size that has been almost universally
adopted for ordinary preparations is 25 x 76 millimeters (1x3 inches). For rock
sections, slides 25 x 45 mm. or 32 x 32 mm. are used ; for serial sections, slides
25 x 76 mm., 50 x 76 mm. or 37 x 87 mm. are used. For special purposes, slides
of the necessary size are employed without regard to any conventional standard.

Whatever size of slide is used, it should be made of clear glass and the edges
should be ground. It is altogether false economy to mount microscopic objects
on slides with unground edges. It is unsafe also as the unground edges are liable
to wound the hands.

For micro-chemical work Dr. Chamot recommends slides of half the length of
those used in ordinary microscopic work. From the rapidity with which they are
destroyed, he thinks the ground edges are unnecessarily expensive. He adds
further: "It is a great misfortune that the colorless glass slips used in America
and so excellent for ordinary microscopic work should be easily attacked by all
liquids ; even water extracts a relatively enormous amount of alkalies and alka-
line earths. The slips of greenish glass, while not as neat or desirable for general
microscopy, seem to be decidedly more resistant, and are therefore preferable."
Transparent celluloid slides are recommended by Behrens for work where hydro-
fluoric acid and its derivatives are to be examined. (Chamot, Jour. Appl. Micr.
vol. iii, p. 793).

\ 235. Cleaning Slides. — For new slides a thorough rinsing in clean water with
subsequent wiping with a soft cloth like glass toweling, or thin cotton cloth like
bleached cheese cloth (bunting or gauze, or absorbent surgical gauze), usually fits
them for ordinary use. If they are not satisfactorily cleaned in this way, soak
them a short time in 50% or 75% alcohol, let them drain for a few moments on a
clean towel or on blotting paper, and then wipe with a soft cloth. In handling
the slides grasp them by their edges to avoid soiling the face of the slide. After
the slides are cleaned they should be stored in a place as free as possible from
dust. For storing, covered glass dishes are best. Use museum jars or glass boxes
(Fig. 150).

162

SLIDES AND COVER-GLASSES

\_CH. VII

FIG. 1 28. Glass slide or slip of the ordinary size for microscopic work ( j x fin.,
j6 x 25 mm.}. ( Cut loaned by the Spencer Lens Company}.

For old slides, if only water, glycerin or glycerin jelly has been used on them,
they may be cleaned with water, or preferably, warm water and then with alcohol
if necessary. Where balsam, or any oily or gummy substance has been used upon
the slides, they may be freed from the balsam, etc., by soaking them for a week
or more in one of the cleaning mixtures for glass. If they are first soaked in
xylene, benzin or turpentine to dissolve the balsam, then soaked in the cleaning
mixture, the time required will be much shortened (\ 242). After all foreign
matter is removed the slides should be thoroughly rinsed in water to remove all
the cleaning mixture. They may then be treated as directed for new slides.

If slides with large covers, as in mounted series, are put into the cleaning
mixture, the swelling of the balsam is liable to break the covers. Dissolving
away the balsam with turpentine, avoids this, and greatly shortens the time neces-
sary for cleaning the old slides and covers.

Another excellent method for balsam mounts is to heat the slides until the
balsam is soft and then remove the cover- glasses. The turpentine cleaning mix-
ture, etc., can then act on the entire surface. It should be said, however, that at
the present price of slides and cover-glasses it costs nearly as much as the slides
and covers are worth to clean those that have been used in balsam mounting.

\ 236. Cover- Glasses or Covering Glasses. — These are circular or quadr-
angular pieces of thin glass used for covering and protecting microscopic objects.
They should be very thin, o.io to 0.25 millimeter (see table, \ 29). It is better
never to use a cover-glass over 0.20 mm. thick, then the preparation may be studied
with a 2 mm. oil immersion as well as with lower objectives. Except for objects
wholly unsuited for high powers, it is a great mistake to use cover-glasses thicker
than the working distance of a homogeneous objective (\ 61). Indeed, if one
wishes to employ high powers, the thicker the sections the thinner should be the
cover-glass (see $ 240).

The cover-glass should always be considerably larger than the object ovei
which it is placed.

\ 237. Cleaning Cover-Glasses. — New cover-glasses should be put into a
glass dish of some kind containing one of the cleaning mixtures ($ 242) and al-
lowed to remain a day or longer. In putting them in, push one in at a time and
be sure that each is entirely immersed, otherwise they adhere very closely and the
cleaning mixture is unable to act freely. Soiled covers should be left a week or
more in the cleaning mixture. An indefinite sojourn in the cleaner does not seem

CH. VII] SLIDES AND COVER-GLASSES 163

to injure the slides or covers. After one day or longer, pour off the cleaning mix-
ture into another glass jar, and rinse the cover-glasses, moving them around with
a gentle rotary motion. Continue the rinsing until all the cleaning mixture is
removed. One may rinse them occasionally, and in the meantime allow a very
gentle stream of water to flow on them, or they may be allowed to stand quietly
and have the water renewed from time to time. When the cleaning mixture is
removed rinse the covers well with distilled water, and then cover them with 50%
to 75% alcohol.

FIGS. 129-130. Figures of square and of circular cover-glasses. See also Fig.
162 for covers on serial sections.

\ 238. Wiping the Cover- Glasses. — When ready to wipe the cover-glasses,
remove several from the alcohol and put them on a soft, dry cloth, or on some of
the lens or filter paper to let them drain. Grasp a cover-glass by its edges, cover
the thumb and index finger of the other hand with a soft, clean cloth or some of the
the lens paper. The bleached cheese cloth ( \ 235 ) is good for wiping covers. Grasp
the cover between the thumb and index and rub the surfaces. In doing this it is
necessary to keep the thumb and index well opposed on directly opposite faces of
the cover so that no strain will come on it, otherwise the cover is liable to be
broken.

When a cover is well wiped, hold it up and look through it toward some dark
object. The cover will be seen partly by transmitted and partly by reflected light,
and any cloudiness will be easily detected. If the cover does not look clear, breathe
on the faces and wipe again. If it is not possible to get a cover clear in this way
it should be put again into the cleaning mixture.

As the covers are wiped put them in a clean glass box or Petri dish. Handle
them always by their edges, or use fine forceps. Do not put the fingers on the
faces of the covers, for that will surely cloud them.

FIG. 131. Glass dish for holding covers
( Whitall, Tatum & Co.}.

\ 239. Cleaning Large Cover-Glasses. — For
serial sections and especially large sections,
large quadrangular covers are used (Fig. 162).
These are to be put one by one into a cleaning
mixture as for the smaller covers and treated in

every way the same. In wiping them one may proceed as for the small covers,
but special care is necessary to avoid breaking them. It is especially desirable
that these large covers should be thin — not over 0.15-0.20 mm. otherwise high ob-
jectives cannot be used in studying the preparations.

$ 240. Measuring the Thickness of Cover-Glasses. — It is of the greatest
advantage to know the exact thickness of the cover-glass on an object ; for, (a)

164

SLIDES AND COVER-GLASSES

VII

In studying the preparation one would not try to use objectives of a shorter work-
ing distance than the thickness of the cover (§61); (b) In using adjustable
objectives with the collar graduated for different thicknesses of cover, the collar
might be set at a favorable point without loss of time ; (c) For unadjustable
objectives the thickness of cover may be selected corresponding to that for which
the objective was corrected (see table, \ 29). Furthermore, if there is a variation
from the standard, one may remedy it, in part at least, by lengthening the tube if
the cover is thinner, and shortening it if the cover is thicker than the standard
(\ 102, Fig. 133.)

In the so called No. i cover-glasses of the dealers in microscopical supplies,
the writer has found covers varying from o. 10 mm. to 0.35 mm. To use cover-
glasses of so wide a variation in thickness without knowing whether one has a
thick or thin one is simply to ignore the fundamental principles by which correct
microscopic images are obtained.

32nds.
1 .0312
3 .0.937
5 .1562
7 .2187
9 .2812
11.3437
13.4062
15.4687

FIG. 132. Micrometer Calipers (Brown and Sharpe}. Pocket Calipers, gradu-
ated in inches or millimeters, and well adapted for measuring cover-glasses.

It is then strongly recommended that every preparation shall be covered with
a cover-glass whose thickness is known, and that this thickness should be indicated
in some way on the preparation.

| 241. Cover-Glass Measurers, Testers or Gauges. For the purpose of
measuring cover-glasses there are three very excellent pieces of apparatus. The
micrometer calipers (Fig. 132) used chiefly in the mechanic arts, are convenient
and from their size easily carried in the pocket. The two cover-glass measurers
specially designed for the purpose are shown in Figs. 133-134. With either of
these the covers may be more rapidly measured than with the calipers.

With all of these measures or gauges one should be certain that the index
stands at zero when at rest. If the index does not stand at zero it should be
adjusted to that point, otherwise the readings will not be correct.

As the covers are measured the different thicknesses should be put into
different boxes and properly labeled. Unless one is striving for the most accurate
possible results, cover-glasses not varying more than 0.06 mm. may be put in the
same box. For example, if one takes 0.15 mm. as a standard, covers varying 0.03
mm. on each side may be put into the same box. In this case the box would con-
tain covers of 0.12, 0.13, 0.14, 0.15, 0.16, 0.17 and o.iS mm.

CH. F/7]

SLIDES AND COVER-GLASSES

FIG. 133. Cover-Glass Measurer (Edward Bausch}.

The cover glass is placed in the notch between the two screws, and the drum is
turned by the milled head at the right till the cover is in contact with the screws.
The thickness is then indicated by the knife edge on the drum and may be read off
directly in o.ooi of a millimeter or inch. In other columns is given the proper
tube-length for various unadjustable objectives (\, i, \, and ^ in. ) (Bausch and
Lomb Optical Company}.

FIG. 134. Zeiss Cover-Glass
Measu rer. With th is the knife edge
jaws are opened by means of a lever
and the cover inserted. The thick-
ness may then be read off on the face
as the pointer indicates the thick-
ness in hundredths millimeter in
the outer circle and in thousandths
inch on the inner circle.

\ 242. Cleaning Mixtures for Glass. — The cleaning mixtures used for clean-
ing slides and cover-glasses are those commonly used in chemical laboratories :
(Dr. G. C. Caldwell's Laboratory Guide in Chemistry).

1 66 MOUNTING AND LABELING [CH. VII

(A) Bichromate of Potash and Sulphuric Acid.

Bichromate of potash (K2Cr2O7) 200 grams

Water, distilled or ordinary - 800 cc.

Sulphuric acid (H2SO4) 1200 cc.

Dissolve the dichromate in the water by the aid of heat, using an agate or
other metal dish, then pour it into a heavy iron kettle lined with sheet lead (Pr.
Trans. Amer. Micr. Soc., 1899, p. 107). Add the sulphuric acid to the dissolved
dichromate in the kettle. The purpose of the lead lined kettle is to avoid break-
age from the great heat developed upon the addition of the sulphuric acid. The
lead is very slightly affected by the acid, iron would be corroded by it.

For making this mixture, ordinary water, commercial dichromate and strong
commercial sulphuric acid may be used. It is not necessary to employ chemically
pure materials.

This is an excellent cleaning mixture and is practically odorless. It is exceed-
ingly corrosive and must be kept in glass vessels. It may be used more than
once, but when the' color changes markedly from that seen in the fresh mixture it
should be thrown away.

( B ) Sulphuric and Nitric Acid Mixture.

Nitric acid (HNO3) 200 cc.

Sulphuric acid (H^SO^) - 300 cc.

The acids should be strong, but they need not be chemically pure. The two
acids are mixed slowly, and kept in a glass stoppered bottle. This is a more cor-
rosive mixture than (A), and has the undesirable feature of giving off stifling
fumes, therefore it must be carefully covered. It may be used several times. It
acts more rapidly than the dichromate mixture, but on account of the fumes is not
so well adapted for general laboratories.

MOUNTING, AND PERMANENT PREPARATION OF MICROSCOPICAL OBJECTS

\ 243. Mounting a Microscopical Object is so arranging it upon some suit-
able support (glass slide) and in some suitable mounting medium that it may be
satisfactorily studied with the microscope.

The cover-glass on a permanent preparation should always be considerably
larger than the object ; and where several objects are put under one cover- glass it is
false economy to crowd them too closely together.

\ 244. Temporary Mounting. — In a great many cases objects do not need to
be preserved ; they are then mounted in any way to enable one best to study
them, and after the study the cover glass is removed, the slide cleaned and made
ready for future use. In the study of living objects, of course only temporary
preparations are possible. With amoebae, white blood corpuscles, and many
other objects both animal and vegetable, the living phenomena can best be studied
by mounting them in the natural medium. That is, for amoebae, in the water in
which they are found ; for the white blood corpuscles, a drop of blood is used and,
as the blood soon coagulates, they are in the serum. Sometimes it is not easy or
convenient to get the natural medium, then some liquid that has been found to
serve in place of the natural medium is used. For many things, water with a

CH.

MOUNTING AND LABELING

167

little common salt (water 100 cc., common salt T%ths gram) is employed. This is
the so-called normal salt or saline solution. For the ciliated cells from frogs and
other amphibia, nothing has been found so good as human spittle. Whatever is
used, the object is put on the middle of the slide and a drop of the mounting
medium added, and then the cover-glass. The cover is best put
on with fine forceps, as shown in Fig. 136. After the cover is in
place, if the preparation is to be studied for some time, it is better
to avoid currents and evaporation by painting a ring of castor oil
around the cover in such a way that part of the ring will be on the
slide and part on the cover (Fig. 165. )

FIG. 135. Needle Holder (Queen & Co.}. By means of the
screw clamp or chuck at one end the needle may be quickly
changed.

FIG. 136. To show the method of putting a cover-glass upon a
microscopic preparation. The cover is grasped by one edge, the
opposite edge is then brought down to the slide, and the cover
gradually lowered upon the object.

FIG. 136.

§ 245. Permanent Mounting. — For making permanent microscopical prepara-
tions, there are three great methods. Special methods of procedure are necessary
to mount objects successfully in each of these ways. The best mounting medium
and the best method of mounting in a given case can only be determined by ex-
periment. In most cases some previous observer has already made the necessary
experiments and furnished the desired information.

The three methods are the following : ( A) Dry or in air (g 246); (B) In some
medium miscible with water, as glycerin or glycerin jelly (| 250); (C) In some
resinous medium like Canada Balsam (§ 255).

§ 246. Mounting Dry or in Air. — The object should be thoroughly dry. If
any moisture remains it is liable to cloud the cover-glass, and the specimen may
deteriorate. As the specimen must be sealed, it is necessary to prepare a cell
slightly deeper than the object is thick. This is to support the cover-glass, and
also to prevent the running in by capillarity of the sealing mixture.

\ 2463. Order of Procedure in Mounting Objects Dry or in Air.

1. A cell of some kind is prepared. It should be slightly deeper than the
object is thick (§ 248).

2. The object is thoroughly dried (dessicated) either in dry air or by the aid
of gentle heat.

3. If practicable the object is mounted on the cover-glass ; if not it is placed
in the bottom of the cell.

4. The slide is warmed till the cement forming the cell wall is somewhat
sticky, or a very thin coat of fresh cement is added ; the cover is warmed and put

1 68 MOUNTING AND LABELING \_CH. VII

on the cell and pressed down all around till a shining ring indicates its adherence

(2 249)-

5. The cover-glass is sealed (\ 249).

6. The slide is labeled (| 308).

7. The preparation is cataloged and safely stored (\ 309, 311).

§ 247. Example of Mounting Dry, or in Air. — Prepare a shallow cell and dry
it (§ 248). Select a clean cover-glass slightly larger than the cell. Pour upon the
cover a drop of 10% solution of salycilic acid in 95% alcohol. Let it dry spon-
taneously. Warm the slide till the cement ring or cell is somewhat sticky, then
warm the cover gently and put it on the cell, crystals down. Press on the cover
all around the edge (§ 246); seal, label and catalog (\ 253, 308, 309). •

A preparation of mammalian red blood corpuscles may be satisfactorily made
by spreading a very thin layer of fresh blood on a cover with the end of a slide.
After it is dry, warm gently to remove the last traces of moisture and mount blood
side down, precisely as for the crystals. One can get the blood as directed for the
Micro-spectroscopic work (g 210).

FIG. 137. Turn-Table for sealing cover-glasses and making shallow mount-
ing cells. ( Queen & Co.)

| 248. Preparation of Mouuting Cells. — (A) Thin Cells. These are most
conveniently made of some of the cements used in microscopy. Shellac is one of
the best and most generally applicable. To prepare a shellac cell place the slide
on a turn-table (Fig. 137) and center it, that is, get the center of the slide over
the center of the turn-table. Select a guide ring on the turn-table which is a little
smaller than the cover-glass to be used, take the brush from the shellac, being
sure that there is not enough cement adhering to it to drop. Whirl the turn-table
and hold the brush lightly on the slide just over the guide ring selected. An
even ring of the cement should result. If it is uneven, the cement is too thick or
too thin, or too much was on the brush. After a ring is thus prepared remove
the slide and allow the cement to dry spontaneously, or heat the slide in some
way. Before the slide is used for mounting, the cement should be so dry when it
is cold that it does not dent when the finger nail is applied to it.

A cell of considerable depth may be made with the shellac by adding succes-
sive layers as the previous one drys.

(B) Deep Cells are sometimes made by building up cement cells, but more
frequently, paper, wax, glass, hard rubber, or some metal is used for the main
part of the cell. Paper rings, block tin or lead rings are easily cut out with gun
punches. These rings are fastened to the slide by using some cement like the
shellac.

CH. VII]

MOUNTING AND LABELING

169

| 249. Sealing the Cover-Glass for Dry Objects Mounted in Cells. — When
an object is mounted in a cell, the slide is warmed until the cement is slightly
sticky or a very thin coat of fresh cement is put on. The cover-glass is warmed
slightly also, both to make it stick to the cell more easily, and to expel any re-
maining moisture from the object. When the cover is put on it is pressed down
all around over the cell until a shining ring appears, showing that there is an in-
timate contact. In doing this use the convex part of the fine forceps or some
other blunt, smooth object ; it is also necessary to avoid pressing on the cover
except immediately over the wall of the cell for fear of breaking the cover. When
the cover is in contact with the wall of cement all around, the slide should be
placed on the turn-table and carefully arranged so that the cover-glass and cell
wall will be concentric with the guide rings of the turn-table. Then the turn-
table is whirled and a ring of fresh cement it painted, half on the cover and half
on the cell wall (Fig. 165.) If the cover-glass is not in contact with the cell wall
at any point and the cell is shallow, there will be great danger of the fresh cement
running into the cell and injuring or spoiling the preparation. When the cover-
glass is properly sealed, the preparation is put in a safe place for the drying of the
cement. It is advisable to add a fresh coat of cement occasionally.

FIG. 138. Centering Card. A card with stops for the slide and circles in the
position occupied by the center of the slide. If the slide is put upon such a card it
is very easy to arrange the object so that it will be approximately in the center oj
the slide. The position of the long cover used for serial sections is also shown
(Fig. 162}. (From the Microscope, December, 1886).

§ 250. Mounting Objects in Media Miscible with Water. — Many objects are
so greatly modified by drying that they must be mounted in some medium other
than air. In some cases water with something in solution is used. Glycerin of
various strengths, and glycerin jelly are also much employed. All these media
keep the object moist and therefore in a condition resembling the natural one.
The object is usually and properly treated with gradually increasing strengths of
glycerin or fixed by some fixing agent before being permanently mounted in
strong glycerin or either of the other media.

170 MOUNTING AND LABELING [CH. VII

In all of these different methods, unless glycerin of increasing strengths has
been used to prepare the tissue, the fixing agent is washed away with water before
the object is finally and permanently mounted in either of the media.

For glycerin jelly no cell is necessary unless the object has a considerable
thickness.

$ 251. Order of Procedure in Mounting Objects in Glycerin.

1. A cell must be prepared on the slide if the object is of considerable thick-
ness (§ 248, 249).

2. A suitably prepared object ( § 250) is placed on the center of a clean slide,
and if no cell is required a centering card is used to facilitate the centering

(Fig. 138)-

3. A drop of pure glycerin is put upon the object, or if a cell is used, enough
to fill the cell.

4. In putting on the cover-glass it is grasped with fine forceps and the under
side breathed on to slightly moisten it so that the glycerin will adhere, then one
edge of the cover is put on the cell or slide and the cover gradually lowered upon
the object (Fig. 136). The cover is then gently pressed down. If a cell is used, a
a fresh coat of cement is added before mounting ($ 249).

FIG. 139. Slide and cover-glass showing method of
anchoring a cover-glass with a glycerin preparation when
no cell is used. A cover-glass so anchored is not liable to
move when the cover is being sealed ($ 253).

FIG. 140. Glass slide with cover-glass, a drop of re-
agent and a bit of absorbent paper to show method of irri-
gation (\262, 263}.

5. The cover-glass is sealed (\ 249).

6. The slide is labeled ( \ 308).

7. The preparation is cataloged and safely stored (§ 309, 311).

$ 252. Order of Procedure in Mounting Objects in Glycerin Jelly.

1. Unless the object is quite thick no cell is necessary with glycerin jelly.

2. A slide is gently warmed and placed on the centering card (Fig. 138) and
a drop of warmed glycerin jelly is put on its center. The suitably prepared object
is then arranged in the center of the slide.

3. A drop of the warm glycerin jelly is then put on the object, or if a cell is
used it is filled with the medium.

4. The cover-glass is grasped with fine forceps, the lower side breathed on
and then gradually lowered upon the object (Fig. 136) and gently pressed down.

5. After mounting, the preparation is left flat in some cool place till the
glycerin jelly sets, then the superfluous amount is scraped and wiped away and
the cover-glass sealed with shellac ($ 253).

6. The slide is labeled (§308).

7. The preparation is cataloged and safely stored (\ 309, 311).

\ 253. Sealing the Cover-Glass when no Cell is used. — (A) For glycerin
mounted specimens. The superfluous glycerin is wiped away as carefully as possi-
ble with a moist cloth, then four minute drops of cement are placed at the edge of
the cover (Fig. 139), and allowed to harden for half an hour or more. These will

CH. VII]

MOUNTING AND LABELING

171

anchor the cover-glass, then the preparation may be put on the turn-table and a
ring of cement put around the edge while whirling the turn-table.

c

FIG. 141. A — Simple form of moist chamber made with a plate and bowl. B,
bowl serving as a bell jar ; P, plate containing the water and over which the bowl is
inverted ; S, slides on which are mounted preparations which are to be kept moist.
These slides are seen endwise and rest upon a bench made by cementing short pieces
of large glass tubing to a strip of glass of the desired length and width.

B — Two covet -glasses (C) made eccentric, so that they may be more easily sepa-
rated by grasping the projecting edge.

C — Slide (S) with projecting cover-glass (C). The projection of the cover en-
ables one to grasp and raise it without danger of moving it on the slide and thus
folding the substance under the cover. (From Proc. Amer. Micr. Soc. , 1891).

(B) For objects in glycerin jelly, Farrants* solution or a resinous medium.
The mounting medium is first allowed to harden, then the superfluous medium is
scraped away as much as possible with a knife, and then removed with a cloth
moistened with water for the glycerin jelly and Farrants' solution or with alcohol,
chloroform or turpentine, etc., if a resinous medium is used. Then the slide is put
on a turn-table and a ring of the shellac cement added. ( C ) Balsam preparations
may be sealed with shellac as soon as they are prepared, but it is better to allow
them to dry for a few days. One should never use a cement for sealing prepara-
tions in balsam or other resinous media if the solvent of the cement is a solvent
also of the balsam, etc. Otherwise the cement will soften the balsam and finally
run in and mix with it, and partly or wholly ruin the preparation. Shellac is
an excellent cement for sealing balsam perparations, as it never runs in. Balsam
preparations are rarely sealed.

| 254. Example of Mounting in Glycerin Jelly. — For this select some stained
and isolated muscular fibres or other suitably prepared objects. (See under isola-
tion § 259). Arrange them on the middle of a slide, using the centering card, and
mount in glycerin jelly as directed in \ 252. Air bubbles are not easily removed
from glycerin jelly preparations, so care should be taken to avoid them.

\ 255. Mounting Objects in Resinous Media. — While the media miscible
with water offer many advantages for mounting animal and vegetable tissues the
preparations so made are liable to deteriorate. In many cases, also, they do not
produce sufficient transparency to enable one to use high enough powers for the
demonstration of minute details.

172

MOUNTING AND LABELING

[CH. VII

By using sufficient care almost any tissue may be mounted in a resinous
medium and retain all its details of structure.

For the successful mounting of an object in a resinous medium it must in
some way be deprived of all water and all liquids not miscible with the resinous
mounting medium. There are two methods of bringing this about : (A) By dry-
ing or desiccation (\ 256), and (B) by successive displacements (g 258).

FIG. 142. Small spirit lamp modified into a balsam
bottle, a glycerin or glycerin -jelly bottle, or a bottle
for homogeneous immersion liquid. For all of these
purposes it should contain a glass rod as shown in the
figure. By adding a small brush, it answers well for
a shellac bottle also (See Fig. 170) .

\ 256. Order of Procedure in Mounting Objects in Resinous Media by
Desiccation :

1. The object suitable for the purpose (fly's wings, etc. ) is thoroughly dried
in dry air or by gentle heat.

2. The object is arranged as desired in the center of a clean slide on the
centering card (Fig. 138).

3. A drop of the mounting medium is put directly upon the object or spread
on a cover-glass.

4. The cover-glass is put on the specimen with fine forceps (Fig. 136), but in
no case does one breathe on the cover as when media-miscible with water are
used.

5. The cover-glass is pressed down gently.

6. The slide is labeled (§308).

7. The preparation is cataloged and safely stored (\ 309, 311).

| 257. Example of Mounting in Balsam by Desiccation. — Find a fresh fly,
or if in winter, procure a dead one from a window sill or a spider's web.
Remove the fly's wings, being especially careful to keep them the dorsal side
up. With a camel's hair brush remove any dirt that may be clinging to them.
Place a clean slide on the centering card, then with fine forceps put the two wings
within one of the guide rings. Leave one dorsal side up, turn the other ventral
side up. Spread some Canada balsam on the face of the cover-glass and with the
fine forceps place the cover upon the wings (Fig. 136). Probably some air-bubbles
will appear in the preparation, but if the slide is put in a warm place these will
soon disappear. Label, catalog, etc., ( $307-31 1).

§ 258. Mounting in Resinous Media by a Series of Displacements. — For ex-
amples of this see the procedure in the paraffin and in the collodion methods

CH. VII] ISOLATION OF ELEMENTS 173

($ 280, 300). The first step in the series is Dehydration, that is, the water is dis-
placed by some liquid which is miscible both with the water and the next liquid
to be used. Strong alcohol (95% or stronger) is usually employed for this. Plenty
of it must be used to displace the last trace of water. The tissue may be soaked
in a dish of the alcohol, or alcohol from a pipette may be poured upon it. Dehy-
dration usually occurs in the thin objects to be mounted in balsam in 5 to 15 min-
utes. If a dish of alcohol is used it must not be used too many times, as it loses
in strength.

The second step is clearing. That is, some liquid which is miscible with the
alcohol and also with the resinous medium is used. This liquid is highly refrac-
tive in most cases, and consequently this step is called clearing and the liquid a
clearer. The clearer displaces the alcohol, and renders the object more or less
translucent. In case the water was not all removed, a cloudiness will appear in
parts or over the whole of the preparation. In this case the preparation must be
returned to alcohol to complete the dehydration.

One can tell when a specimen is properly cleared by holding it over some
dark object. If it is cleared it can be seen only with difficulty, as but little light
is reflected from it. If it is held toward the window, however, it will appear
translucent.

The third and final step is the displacement of the clearer by the resinous
mounting medium.

The specimen is drained of clearer and allowed to stand for a short time till
there appears the first sign of dullness from evaporation of the clearer from the
surface. Then a drop of the resinous medium is put on the object, and finally a
cover-glass is placed over it, or a drop of the mounting medium is spread on the
cover and it is then put on the object.

ISOLATION OF HISTOLOGICAI, ELEMENTS

| 259. For a correct conception of the forms of the cells and fibers of the
various organs of the body, one must see these elements isolated and thus be able
to inspect them from all sides. It frequently occurs also that the isolation is not
quite complete, and one can see in the clearest manner the relations of the cells
or fibers to one another.

The chemical agents or solutions for isolating are, in general, the same as
those used for hardening and fixing. But the solutions are only about one-tenth
as strong as for fixing, and the action is very much shorter, that is, from one or
two hours to as many days. In the weak solution the cell cement or connective
tissue is softened so that the cells and fibers may be separated from one another,
and at the same time the cells are preserved. In fixing and hardening, on the
other hand, the cell cement, like the other parts of the tissue, are made firmer.
In preparing the isolating solutions it is better to dilute the fixing agents with
normal salt solution (§ 331) than merely with water.

\ 260. Isolation by Means of Formaldehyde. — Formaldehyde in normal
salt solution is one of the very best dissociating agents for brain tissue and all the
forms of epithelium. It is prepared as follows : 2 cc. of formal, (that is, a 40%
solution of formaldehyde) are mixed with loco cc. of normal salt solution. This
acts quickly and preserves delicate structures like the cilia of ordinary epithelia,

174

/SOLA TION OF ELEMENTS

\CH. VII

and also of the endymal cells of the brain. It is satisfactory for isolating the
nerve cells of the brain. For the epithelium of the trachea, intestines, etc. , the
action is sufficient in two hours ; good preparations may also be obtained after
two days or more. The action on nerve tissue of the brain and myel or spinal
cord is about as rapid. For the stratified epithelia, like those of the skin, mouth,
etc. , it may require two or three days for the most satisfactory preparations.

ooo

000
000

ooo
ooo

FIG. 143 A. FIG. 143 B. FIG. 144.

FIG. 143. Preparation Vials for Histology and Embryology. This repre-
sents the two vials, natural size, that have been found most useful. They are kept
in blocks with holes of the proper size.

FIG. 144. Block with holes for containing shell vials.

$261. Example of Isolation. — Place a piece of the trachea of a very recently
killed animal, or the roof of a frog's mouth, in the formaldehyde dissociator.
After two hours or more, up to two or three days, excellent preparations of ciliated
cells may be obtained by scraping the trachea or roof of the mouth and mounting
the scrapings on a slide. If one proceeds after two hours, probably most of the
cells will cling together, and in the various clumps will appear cells on end show-
ing the cilia or the bases of the cells, and other clumps will show the cells in pro-
file. By tapping the cover gently with a needle holder or other light object the
cells will be more separated from one another, and many fully isolated cells will
be seen.

\ 262. Staining the Cells. — Almost any stain may be used for the formalin
dissociated cells. For example, one may use eosin. This may be drawn under
the cover of the already mounted preparation (Fig. 140), or a new preparation may
be made and the scrapings mixed with a drop of the eosin before putting on the
cover-glass. It is an advantage to study unstained preparations, otherwise one
may obtain the erroneous opinion that the structure cannot be seen unless it is

CH. VII] ISOLATION OF ELEMENTS 175

stained. The stain makes the structural features somewhat plainer ; it also accen-
tuates some features and does not affect others so markedly.

\ 263. Permanent Preparations of Isolated Cells. — If one desires to make a
permanent preparation of isolated cells it may be done by placing a drop of glycerin
at the edge of the cover and allowing it to diffuse under the cover, or the diffusion
may be hurried by using a piece of blotting paper, as shown in Fig. 140. One
may also make a new preparation and either with or without staining mix the
cells with a drop of glycerin on the slide and then cover, or one may use glycerin
jelly (\ 254. 326).

FIG. 145. Adjustable lens holder with universal joint. This is especially use-
ful f o* gross dissections, and for dissecting the partly isolated elements with needles
(Leitz: Wm. Krafft, N. K).

§ 264. Isolation of Musculer Fibers. — For this the formal dissociator may be
used (\ 260, 324), but the nitric acid method is more successful (\ 330). The fresh
muscle is placed in this in a glass vessel. At the ordinary temperature of a sitting
room ( 20 degrees centigrade) the connective tissue will be so far gelatinized in
from one to three days that it is easy to separate the fascicles and fibers either with
needles or by shaking in a test tube or reagent vial (Fig. 143) with w7ater. It
takes longer for some muscles to dissociate than others, even at the same temper-
ture, so one must try occasionally to see if the action is sufficient. When it is, the
acid is poured off and the muscles washed gently with water to remove the acid.
If one is ready to make the preparations at once they may be isolated and mounted
in water. If it is desired to keep the specimen indefinitely or several days, the
water should be poured off and a half saturated solution of alum added ($ 314).
The alum solution is also advantageous if the specimens are to be stained. The

176 COLLODION SECTIONING \_CH. VII

specimens may be mounted in glycerin, glycerin jelly or balsam. Glycerin jelly
is the most satisfactory, however.

FIG. 146. Pfeiffer'1 s preparation microscope with erecting prism between the
objective and ocular (Leitz ; Wm. Krafft, New York}.

THE PREPARATION OF SECTIONS OF TISSUES AND ORGANS

\ 265. At the present time there are three principal methods of obtaining
thin sections of tissues and organs for microscopic study. These methods are :
The Collodion Method, the Paraffin Method, and the Freezing Method. Each of
these methods has its special application, although the collodion method is per-
haps the most generally applicable, and the freezing method the most restricted,
and is used mostly in pathological work where rapid diagnosis is necessary and
the finest details of structure are not so important. With the paraffin method the
thinnest sections may be made, and in some ways it is the most satisfactory of all.
A good microtome is of great aid in sectioning.

§ 266. The Collodion Method. — In sectioning by this method the tissues are
first hardened properly and then entirely infiltrated with collodion, and the collod-
ion hardened. It is not removed from the tissue, since on account of its transpar-
ency it does no harm.

\ 267. Fixing and Hardening the Tissue. — Any of the approved methods
of hardening and fixing may be employed. A good general method which is
applicable to nearly all of the tissues and organs is that by Picric-Alcohol. P'or
the preparation of the solution see (\ 333). A small piece of tissue or organ not

I

CH.VI1] COLLODION SECTIONING 177

containing more than two to three cubic centimeters is placed in 40 or 50 cc. of
the picric-alcohol and left 6 to 24 hours, when the first picric-alcohol should be
thrown away and fresh added. After one or two days more the picric-
alcohol should be poured off and 67% alcohol added. In a day or two this is re-
placed by 75% or 82% alcohol ; 82% is on the whole most satisfactory, and the
tissue may be left in this till it is ready for dehydration.

\ 268. Dehydration before Infiltration. — When one is ready to imbed for
sectioning, the tissue must first be dehydrated in plenty of 95% or stronger alcohol.
It is better to take only a small piece for this. The smaller the piece the thinner
the sections that may be made. The dehydration will usually be completed in 2 to
24 hours. If the alcohol is changed two or three times the dehydration will be
hastened.

\ 269. Saturating with Ether- Alcohol. (§ 322). — The next step is to remove
the tissue from the alcohol and place it in a vial of ether-alcohol (§ 322) for 2 to
24 hours. The dehydration becomes somewhat more complete by this step, and
the tissue is more perfectly prepared for the reception of the collodion. If the
dehydration is very thorough in the alcohol, this step may be ommitted, however,
but one is surer of success if the ether-alcohol is used.

\ 270. Infiltration with Thin Collodion. — The ether-alcohol is poured off,
and a mixture of thin collodion is added (2 319). Two or three hours will suffice
for objects two or three millimeters in thickness. A stay of one or more days
does no harm. The larger the object the more time is needed.

$ 271. Infiltration with Thick Collodion. — The thin collodion is poured off
and thick collodion (§ 319) added. For very small objects, four or five hours
will suffice to infiltrate, but for larger objects a longer time is necessary. The
tissue does not seem to be injured at all in the thick collodion, and a stay in it
of a day or even a week is more certain to insure a perfect infiltration.

\ 272. Imbedding. — The tissue may be imbedded in a paper box, such as is
used for paraffin imbedding, or in any of the other boxes devised for paraffin. It
is better, if paper is used, to put a small amount of oil on the paper to prevent
the collodion from sticking to it. Vaselin spread over lightly and then re-
moved, so far as possible, with a cloth or with lens paper, gives the right surface.
For small objects it is more convenient to imbed immediately on a holder that
may be clamped into the microtome. Cylinders or blocks of glass, vulcanite,
wood and cork have all been recommended and used. A cork of the proper size
is most convenient, and for many purposes answers well. Some collodion is put
on the end of the cork and a pin put near one edge. The tissue is transferred
from the thick collodion to the cork and leaned against the pin. Drops of the
thick collodion are then poured on the tissue, and by moving the cork properly
the thick viscid mass may be made to surround and envelop the tissue. Drops
of collodion are added at short intervals until the tissue is well surrounded, and
then as soon as a slight film hardens on the surface, the cork bearing the tissue is
inverted in a wide-mouth vial of considerably larger diameter than the cork (Fig.
143). The vial should contain sufficient chlorform to float the cork. The vial is
then tightly corked. In imbedding somewhat larger objects on the end of a cork
or other holder, it is frequently advantageous to wind oiled paper around the
holder or cork, tie it tightly and have the projecting hollow cylinder sufficiently
long to receive the object. The tissue is then put into the cylinder and sufficient

178 COLLODION SECTIONING [Ctf. VII

'collodion added to completely immerse it. As soon as a film has formed over the
exposed end, the cork may be inverted and immersed in chloroform as described
above. For the use of "deck plugs" see \ 274.

§ 273. Hardening and Clarifying the Collodion. — After a few hours the
collodion is hardened by the chloroform. If it acts long enough, and if no water
is present, the imbedding mass is rendered entirely transparent. Whenever the
collodion is hard, whether it is clear or not, the chloroform is poured off and the
castor-xylene* clarifier (g 317) added. In a few hours the imbedded mass will be-
come as transparent as glass and the tissue will seem to have nothing around it.
The tissue may remain for years in the castor-xylene. Sometimes the collodion
remains white and opaque for a considerable time. So far as the writer has been
able to judge, this is due to moisture. If one breathes on the mass too much while
imbedding, or if it is very damp in the room, opacity may result. Sometimes, in
objects of considerable size, this may remain for a week. This is the exception,
however, and if the mass seems sufficiently hard and tough, the cutting may pro-
ceed even if the clarification is incomplete. |

In case the imbedding mass will not clarify after a few days the imbedded
object may be placed in 95% alcohol for a day for dehydration, and then passed
through chloroform and into the clarifier. There is usually no trouble in getting
the mass perfectly clear in this way.

If one is in a great hurry, the collodion may be hardened in 10 or 15 minutes
by heating the bottle containing the chloroform in a water bath. The imbedding
block of hardened collodion may then be transferred to the castor-xylene clarifier
and kept warm. It will soon clear the collodion. One can then cut the sections.

I 274. Cutting the Sections.— For cutting the sections the collodion block is
usually fastened to some form of holder. For small objects cork is fairly good.
Blocks of glass, vitrified fiber, etc., have been used. If one uses wood the "deck
plugs" of the shipwright are satisfactory. They are about the right length when
one plug is made into two holders by sawing in two (Ewing & Ferguson). To
fasten the collodion block to any form of holder, remove it from the castor-xylene,
trim as desired, then dry the end on blotting paper, pour some thick collodion on
the holder and press the collodion block down into the collodion. The evapora-
tion usually fixes it in two or three minutes, when the holder may be clamped in
the jaws of the microtome and the cutting proceed. For collodion sectioning a
long, drawing cut is necessary in order to obtain thin, perfect sections. The ob-
ject is, therefore, put in the jaws of the microtome at the right level, and the
knife arranged so that half or more of the blade of the knife is used in cutting the
section. It is advantageous also to have the object with its long diameter parallel
with the edge of the knife. The surrounding collodion mass should be cut away,
as in sharpening a lead pencil, so that there is not more than a thickness of about

*The hydrocarbon xylene (C8H10) is called xylol in German. In English,
members of the hydrocarbon series have the termination "ene," while members
of the alcohol series terminate in "ol."

t The imbedded object may remain in the castor-xylene clarifier indefinitely
without harm. The collodion grows somewhat tougher by a prolonged stay in it.
After cutting all the sections desired at one time, the imbedded tissue is returned
to the clarifier for future sections.

CH.

COLLODION SECTIONING

179

two millimeters all around the tissue. This is to render the diameter of the end to
be cut as small as possible. The smaller the object the thinner can the sections be
made. With an object two or three millimeters thick and not over five milli-
meters wide, and a good sharp knife, sections 5^ to 6>u can be cut without diffi-
culty. When knife and tissue are properly arranged the tissue and the knife are
flooded with the clarifier. Make the sections with a steady motion of the knife.
Then draw the section up toward the back of the knife with an artist's brush and
make the next section. Arrange the sections in serial order on the knife blade
till enough are cut to fill the area that the cover-glass will cover. For large objects
one can cut thinner sections by a kind of sawing cut.

| 275. Transferring the Sections to the Slide. — If the clarifier has evaporated
so as to leave the sections somewhat dry on the knife, add a small amount. Take
a piece of thin absorbent, close-meshed paper about twice the size of a slide and
place it directly upon the sections. Press the paper down evenly all around and
then pull the paper off the edge of the knife. The sections will adhere to the
paper. Place the paper, sections down, on a slide, taking care that the sections
are in the desired position on the slide. Use some ordinary lens paper or some
absorbent paper, and press it down gently upon the transfer paper. This will ab-
sorb the oil, and then the transfer paper may be lifted, with a rolling motion,
from the slide. The sections will remain on the slide. (See notes p. 180).

§ 276. Fastening the Sections to the Slide. — Drop just enough ether-
alcohol (equal parts of sulphuric ether and 95% alchol) on the sections to moisten
them. This will melt the collodion and fasten the sections to the slide. Allow the
slide to remain in the air till the surface begins to look slightly dull or glazed.

Sometimes, especially when the air is moist, the sections wrinkle badly when
the ether-alcohol is put on to fasten them to the slide. The excessive wrinkling
can be avoided by using one part alcohol and two parts ether instead of using
equal parts of each. Perhaps also it would be advantageous in this case to use
absolute alcohol.

FIG. 147. Reagent bottle with combined cork and pipette ( This
is made by taking a cork of the proper size and making in it a hole
with a cork borer for the glass tube. It is advantageous to have a
string tied tightly around the rubber bulb as shown).

\ 277. Removing the Oil from the Sections. — As soon as the
ether-alcohol has evaporated sufficiently to leave the surface dull,
place the slide in a jar of ordinary commercial benzin. It may be
left here a day or more without injury to the sections, but if
moved around in the jar the oil will be removed in three to five
minutes. From the benzin transfer to a jar of 95% alcohol to
wash away the benzin. One may use alcohol in the beginning,
but it dissolves the oil far less rapidly than the benzin. The slide
may remain in the alcohol half a day or more if one wishes, but a
stay of five minutes or a thorough rinsing of half a minute or so
by moving the slide around in the alcohol will suffice.

Xylene is to be preferred to benzin for removing the oil, but
it is more expensive.

FIG. 147.

l8o COLLODION SECTIONING \_CH. VII

g 278. Staining the Sections with an Alcoholic Stain. — If an alcoholic
stain containing 50% or more alcohol (for example, hydrochloric acid carmine in
70% alcohol) is used, the slide may be removed from the 95% alcohol, drained
somewhat and" then the stain poured upon the sections, or preferably, the slide
immersed in a jar of the stain. The stain is finally washed away with 67% or
stronger alcohol, the sections dehydrated in 95% alcohol, cleared and mounted in
balsam.

\ 279. Staining the Sections with an Aqueous Dye. — In staining with a
watery stain, the slide bearing the sections is transferred from the 95% alcohol
and plunged into a jar of water, and either allowed to remain a few minutes or
moved around in the water a moment. Then it is placed horizontally and some
of the stain placed on the sections with a pipette, or preferably, it is immersed in
a jar of the stain ; in case of immersion the slide should stand vertically or nearly
so, then any particles of dust, etc. , in the stain will settle to the bottom of the
vessel and not settle on the sections. When the sections are stained, usually
within five minutes, they are thoroughly washed with water either by the use of a
pipette or preferably by immersing in a jar of water. They may then be counter-
stained for half a minute with some general dye, like eosin or picric acid, or
mounted with but the one stain. t

*Various forms of paper have been used to handle the collodion sections. It
should 'be moderately strong, fine meshed and not liable to shed lint, and fairly
absorbent. One of the first and most successful papers recommended is "closet or
toilet paper. ' ' Cigarette paper is also excellent. In my own work the heavy white
tissue paper has been found almost perfect for the purpose. Ordinary lens paper
or thin blotting paper for absorbing the oil may be used with it. (| 275).

flf one is a long time in cutting a series of sections, it sometimes occurs that
the xylene evaporates, and while the sections may not look dry, they are practically
in castor oil and not easily transferable. In such a case fresh clarifier or even a
little xylene to thin the oil on the sections may be used. If the oil is too thick it
is viscid and there is difficulty in handling the sections with the paper as they
stick rather firmly to the knife. ($275).

Jin the past the plan for changing sections from 95% alcohol to water, for ex-
ample, has been to run them down gradually, using 75, 50 and 35% alcohol, suc-
cessively. Each percentage may vary, but the principle of a gradual passing from
strong alcohol to water was advocated. On the other hand I have found that the
safest method is to plunge the slide directly into water from the 95% alcohol. The
diffusion currents are almost or quite avoided in this way. There is no time for
the alcohol and water to mix, the alcohol is washed away almost instantly by the
flood of water. So in dehydrating after the use of watery stains, the slide is
plunged quickly into a jar of 95% alcohol. The diffusion currents are avoided in
the same way, for the water is removed by the flood of alcohol. This plan has
been submitted to the severe test of laboratory work, and has proved itself perfectly
satisfactory.

CH.

COLLODION SECTIONING

181

FIG. 149.

FIG. 148.

FIG. 150.

FIG. 148. Waste bowl with rack for supporting slides and a small funnel in
which the slides stand while draining. This outfit is easily made by any tinsmith.
The rack is composed of two brass rods about 4 mm. in diameter. The bent end
pieces are sheet lead. The funnel is made of tin, copper or brass. Either copper
or brass is preferable to tin. A glass dish like that shown in Fig. 160 is be tier than
a bowl, as it can be more readily and thoroughly cleaned. (Cut loaned by Wm.
Wood & Co. )

FIG. 149. Round glass aquarium,
for all the uses described for the bowl.

This glass vessel is better than the bowl
Whitall, Tatum & Co.}

FIG. 150. Glass box or ointment jar with cover. These boxes may be had of
various sizes and can be used advantageously J or water, and for cleaning mixture
for slides and cover glasses ( \ 242). ( Whitall, Tatum & Co. )

FIG. 151. Section lifter. This is of thin, springy, flexible metal placed in a
handle as shown. These are made of various sizes for large or small sections. Such
an instrument is exceedingly helpful in handling loose sections. (Queen & Co.)

182

COLLODION SECTIONING

[CH. VII

FIG. 152. Perforated section lifter. This is easily made by soldering a wire
to some very thin sheet brass or copper, and then perforating this with a coarse
needle or fine awl. Any roughness must be removed by using a fine oil stone.

ORDER OF PROCEDURE IN MAKING MICROSCOPICAL PREPARATIONS BY THE

COLLODION METHOD

\ 280. It will be seen from this table, and sections 267-281, that it requires
about five days to get a microscopical preparation if one commences with the
fresh tissue. Other methods of hardening might require as many months. It is
evident, therefore, that one must exercise foresight in histology or much time
will be wasted.

1. Fixing and hardening the tissue s

(§ 267), 4 days or more.

2. Dehydrating the object to be cut in
95% or stronger alcohol (\ 268), 2-
24 hours.

3. Saturating the tissue in ether-alcohol
($ 269), 2-24 hours.

4. Infiltrating with thin collodion
(\ 270), 2 hours to 2 days.

5. Infiltrating in thick collodion($ 271),
5 hours to several days.

6. Imbedding the tissue (§272), 15 to
20 minutes.

7. Hardening the collodion with chlo-
roform (\ 273), 5-24 hours.

8. Clarifying and further hardening the
collodion with castor-xylene ($ 273),
10-36 hours.

9. Cutting the sections ($ 274), 10 min-
utes to 2 hours.

10. Transferring the sections to a slide
with paper (§ 275), i minute.

11. Fastening the sections to the slide
with ether-alcohol (\ 276), i or 2
minutes.

12. Removing the oil from the sections
with benzin and alcohol (§ 277), 3-5
minutes, or 24 hours.

13. Staining the sections with an alco-
holic dye ($ 278), 2 minutes to 24
hours. .

14. Staining the sections with an aque-
ous dye (g 279), 2-10 minutes.

15. Removing the superfluous dye by
washing in water or alcohol (§ 278-
279), 2-5 minutes.

16. Staining with a general dye (§ 279),
15-30 seconds.

17. Washing with water or alcohol
(§ 278-279), i to 2 minutes.

18. Dehydrating the sections in 95% al-
cohol (§281), 5 min. to 24 hours.

19. Clearing the sections (§281 ), 5 min-
utes to 24 hours.

20. Draining the sections, 1-2 minutes.

21. Mounting in Canada balsam (| 281),
1-2 minutes.

22. I/abeling the preparation (§ 308), 2
minutes.

23. Cataloging the preparation (\ 309),
5-10 minutes.

CH. VI I ~\

PARAFFIN SECTIONING

183

\ 281. Mounting in Balsam. — After the sections are stained they must be
dehydrated and cleared before mounting in balsam. For the dehydration the slide
is plunged into a jar of 95% alcohol. For clearing after the dehydration the slide
is drained of alcohol and put down flat and the clearer poured on, or the whole
slide is immersed in a jar of clearer (§318). Clearing usually is sufficient in a
few minutes ; a stay of an hour or even over night does not injure most sections.

In mounting in balsam the clearer is drained away by standing the slide nearly
vertically on some blotting paper, or by using the waste bowl and standing it up
in the little funnel (Fig. 148). Then the balsam is put on the sections or spread
on the cover-glass and that placed over the sections.

For cataloging and labeling, see \ 307-310.

FIG. 153. Small spirit lamp modified into a bal-
sam bottle, or a glycerin or glycerin-jelly bottle, or a
bottle for homogeneous immersion liquid. For all of
these purposes it should contain a glass rod. See also
Fig. 168.

§ 282. The Collodion Method with Alcohol. — A good method of procedure
for making collodion sections is to do exactly as described, including \ 272, and
then instead of hardening the collodion in chloroform and clarifier, it is hardened
in 82% alcohol for a day or two before sectioning. In sectioning the knife and
tissue are kept wet with 82% alcohol and the sections are dehydrated with 95%
alcohol and then fastened to the slide with ether alone or with ether-alcohol.
The staining and mounting (§ 278-281) are as described. One may preserve the
tissue after imbedding for a long time in the 82% alcohol before sectioning and
sections may be made at any time. While this method appears somewhat simpler,
the results are not so satisfactory as by the oil method given above.

THE PARAFFIN METHOD

§ 283. As with the collodion method, the tissues are first properly fixed and
hardened and then entirely filled with the imbedding mass, but unlike the collo-
dion the mass must be entirely removed before the sections are finally mounted.
The tissue thus imbedded and infiltrated is like a homogeneous mass and sections
may be cut of extreme thinness.

§ 284. Harden perfectly fresh tissue in picric-alcohol (\ 333) from one to
three days. (Any good method for fixing and hardening the elements may be
used. One must observe in each case, however, the special conditions necessary

1 84

PARAFFIN SECTIONING

[_CH. VII

for each method. The time might be longer or shorter than for the picric-alcohol.
(See Lee, the Microtomists' Vade-Mecum.)

If picric-alcohol is used, pour it off after the proper time for fixing has
elapsed, and add 67% alcohol. Leave this on the tissue from one to three days,
and if it becomes very yellow it is well to change it two or three times. After two
or three days pour off the 67% alcohol and add 82%. The tissue should remain
in this one or two days, and it may remain indefinitely.

In case the alcohol becomes much yellowed, it should be changed.

\ 285. Dehydration and Preparation for Imbedding. — From the pieces of
tissue fixed and hardened in any approved manner, cut pieces 5 to 10 millimeters
long and 2 to 3 millimeters in breadth. Place one or two pieces in a shell vial
(Fig. 143.) and add 95% alcohol. Change the alcohol after two or three hours,
and within 6 to 24 hours, depending on the size of the piece to be dehydrated, the
dehydration will be completed. The secret of success is the use of plenty of
alcohol and sufficient time. Absolute alcohol for the second change would act
more promptly and efficiently, but if plenty of 95% is used one will succeed,
unless the day, or the climate in general, is too damp.

FIG. 154. Copper pail with
water bottom for melting para-
ffin. This also serves as a water
bath for large bottles in which
saturated solutions of dichro-
mate and other salts are pre-
pared.

(If one is studying organs, then the whole organ may need to be prepared for
imbedding, but for minute structure small pieces are preferable, as thinner
sections may be made. )

\ 286. Displacing Alcohol and Clearing Tissues with Cedar-wood Oil and
Infiltrating with Paraffin. — (Lee, p. 66. Neelson and Schiefferdecker, Arch, fur
Anat. und Physiol., 1882, p. 206.) When the tissue is dehydrated it is removed
to a vial of cedar-wood oil. When the alcohol used for dehydration is displaced
by the oil, the tissue will look clear and translucent. This requires 2 to 24 hours.

CH. VII}

PARAFFIN SECTIONING

185

It is hastened by warmth. It is then removed from the cedar-wood oil, drained,
and placed in pure, melted paraffin, and this is then put into a paraffin oven and
left from 2 to 24 hours (see Ch. X). It is then imbedded for sectioning.*

Paraffin for infiltrating has usually a somewhat lower melting point than that
for imbedding. Equal parts of paraffin of 43 C. and 54 C., answer well. For
imbedding, the paraffin must be of a melting point which will give good ribbons
in the temperature of the room where the sectioning is to be done. In a room of
19 to 20 C. a mixture of i part 43 C. paraffin with two parts of 54 C. usually
answers well.

FIG. 155. Hot filter for
paraffin, gelatin, balsam, etc.
It is entirely surrounded by a
•water jacket. The water is
heated by placing a burner under
the projecting part H. The
wire basket is to hold the strainer
and allow a free flow of the
filtered substance on all sides.
There is a bail for suspending
the filter, and the filtered sub-
stance runs out through the
narrowed part F.

\ 287. Imbedding in Paraffin. — Make a small paper box, fill it nearly full
of hot paraffin, place the box for a half minute on some cold water to make a
thin solid layer on the bottom, then transfer the tissue to the box and arrange
near one end so that the sections may be cut in the desired plane. Now pi ace the
paper box on as cold water as possible so that the paraffin may cool quickly. It
will be more homogeneous if cooled quickly, and will shrink tightly against the
tissue and avoid air spaces.

In imbedding two main things should be looked after : The paraffin should
be hot so that it will thoroughly fuse with the paraffin in the tissue. The tissue
should be kept from the bottom of the box, either by holding it up in the middle
of the box with warmed forceps or a perforated section lifter while a stratum

*Thickened cedar-wood oil like that for oil immersion objectives is recom-
mended by Lee for clearing. This is very expensive, and for most work unneces-
sary. Any form of cedar-wood oil has been found satisfactory in the writer's
laboratory. The great thing is to have the tissue thoroughly dehydrated before
putting it into the oil.

186

PARAFFIN SECTIONING

[CH. VII

cools on the bottom, or a stratum of the paraffin may be cooled on the bottom
before putting the tissue in the box. Cool quickly after the tissue is in place.

FIG. 156. Paraffin re-
ceptacle P, with water
bath and spout for par-
affin imbedding (i 88^ ) .

\ 288. Cutting the Sections. — After the imbedding mass is well cooled, re-
move the paper box and trim the end containing the tissue in a pyramidal form,
clamp the block of paraffin in the holder of the microtome so that the tissue will be
at the proper level for cutting. If a ribbon microtome is used, heat the holder and
melt the end of the block upon it. Cool and place the holder in its place in the
microtome. Use a very sharp, dry razor for cutting the sections. The sections
are made with a rapid, straight cut as in planing. Do not try to section with a
drawing cut as in collodion sectioning. If the temperature of the room is right
for the paraffin used, the sections will remain flat, and if the opposite sides of the
block are parallel, and one edge strikes the knife squarely, the sections will adhere
and thus make a ribbon. If the room is too cold for the paraffin the sections
will roll. If it is too warm the sections will crumple.

Remember the sections must be very thin, from 3// to is// to show fine struc-
tural details to good advantage.

The secret of making good ribbons of sections is to have the block of paraffin
containing the tissue cut square and properly arranged in the microtome so that
the block strikes the edge of the section knife at right angles with the edge ; and
finally the paraffin must be of a proper melting point for the room in which the
sections are to be cut. Remember that the larger the object the thicker must
be the sections, and the softer the paraffin. Frequently one may modify the tem-
perature if too cold by a Bunsen burner flame near the microtome. If it is too
warm one may go to a basement room.

$ 289. Extending Sections with Warm Water. — Paraffin sections are liable
to have fine wrinkles or folds in them. These folds are very annoying and often
obscure the structure. To get rid of them the sections are extended or stretched
upon warm water. One may put a ribbon of sections on warm water and then cut
the ribbon into pieces and transfer the pieces to slides. Practically, however, the

CH. F/7]

PARAFFIN SECTIONING

187

extension is almost always accomplished on the slide itself. A slide is albumen-
ized (| 290) and the ribbon cut into short pieces and placed on the slide. Distilled
or filtered water is then added with a pipette (Fig. 147) until the sections float.
Then the slide is moved back and forth over an alcohol or gas flame to warm the
water. Care must be taken to avoid melting the paraffin. As the water warms
the paraffin containing the sections will flatten and stretch out. One will be sur-
prised at the amount of extension. It is necessary to take pieces considerably
shorter than the cover-glass to be used or when extended the sections will not all
be covered. After the sections are extended, arrange the ribbons carefully on the
slide as shown in Fig. 162 if one is making serial sections. Arrange in the middle
of the slide if only one or two sections are on each slide (Fig. 138). Let the ex-
cess water drain off. Now let the slide stand several hours for the water to evapor-
ate completely. The time will depend on the temperature and the dryness of the
atmosphere. If there is plenty of time, leave the slides 24 or 48 hours. If one
has a register with hot air intake, the slides may be put in the current of hot air.
They will dry out in half an hour or an hour. Sections which have been left for a
year have given excellent results.

FIG. 157. Trays for slides and for ribbons of sections. The figures show the
construction. It is important to have the bordering f tame with rounded corners so
that the trays miy be easily pulled out of a pile or reinserted. The screw eye shown
in A makes it easy to pull out a single tray. For ribbons of sections a piece of paper
is placed in the tray and the ribbons are placed on it. ( A ) Face view, ( B ) Sec-
tional view of the whole tray, (C) Sectional view of one side to show the construc-
tion more clearly. Trays of this kind are so cheap ($15.00 pet hundred for those
holding 50 to 60 slides), that a laboratory can have a great number. (Trans. Amer.
Micr. Soc., 1899, p. 107).

The slide trays (Fig. 157) are excellent for drying preparations of all kinds.
\ 290. Fastening the Sections to the Slide. — To fasten the sections firmly
to the slide, coat the slide with albumen fixative (\ 312) as follows : Put a minute

i88

PARAFFIN SECTIONING

\CH. VII

drop of the albumen on the center of a slide and with a clean finger spread the
albumen over the slide, wiping off all that is possible. Finally beat or tap the
slide with the end of the finger. This will make a very thin (it cannot be too
thin) and even layer.

The sections are extended and dried as described in $ 289. When the sections
are thoroughly dry they are in optical contact with the slide and have a shining
appearance when looking on the back of the slide. When the sections are dry
coat them with $£% collodion made as follows. Take ^ gram of soluble cotton,
put it into a bottle and add 60 cc. of 95% or absolute alcohol, and 40 cc. of sul-
phuric ether. Coat the sections with a soft camel's hair brush. The collodion
should dry in a minute or less. If one uses too much ether in this fixing collo-
dion, the paraffin will be partly dissolved and will look moist for a long time.
The slight excess of alcohol will obviate any such difficulty.

FIG. 158. A slide holder and
bottle for containing the same (Mix,
Journal of Applied Microscopy, vol.
/, 1808, p. 160.)

FIG. 759. Slide holder with the
bail hinged so that it may be turned
aside in inserting or removing the
slides.

When the collodion is dry place the slide in benzin or xylene to dissolve
the paraffin (see $ 291). If the sections are not extended on water, they may be
put directly on the albumenized sides, pressed down with the finger and coated
with collodion. This is much more rapid, but. does not get rid of the fine folds.
(See Dr. Agnes Claypole Trans. Amer. Micro. Soc. 1894, p. 66, 127.)

\ 291. Removing the Paraffin. — Immerse the slide in a vessel of xylene or
benzin. This will dissolve the paraffin. An hour will usually suffice. One can

CH. VII}

PARAFFIN SECTIONING

189

hasten the solution of the paraffin by moving the slide in the solvent. In this way
it may be dissolved in 5 to 10 minutes, or even less. It will do no harm to leave
the slide in the benzin or xylene over night. Two or three days even might not
do any harm, but it is usually better to proceed at once to the other operations.

FIG. 1 60. Apparatus and regents with which the slide holders are used. With
this apparatus it is easy to prepare specimens in large numbers very expeditiously.
After the sections are fastened to the slide and placed in the holder, the slides need
not be touched during all the operations until they are finally ready to be mounted
in balsam. Each holder contains from 12 to 14 slides. (§277-300). The bottles
for the reagents are glass stoppered specimen or museum bottles. (Mix, Jour. Ap.
Micr. /8o8, p. ///. ) Compare Fig. 148.

§ 292. Removing the Xylene or Benzin — From the xylene or benzin plunge
the slide bearing the sections into a jar of 95% alcohol, and leave it for a few
minutes, or move it around in the alcohol for half a minute or so.

\ 293. Staining the Sections with an Alcoholic Dye. — With an alcoholic
stain like hydrochloric acid carmine, remove the slide from the alcohol, and add
the stain directly after draining the slide, Do not allow the stain to become dry,
for that would injure the tissue. Wash away the stain with 67% alcohol, then
dehydrate with 95% alcohol, clear and mount in balsam as described below.

\ 294. Staining with as Aqueous Dye. — Wash away the 95% alcohol from
the slide bearing the sections by plunging it into a jar of water and moving it
around a moment. Then add the stain to the sections with a pipette, or immerse
the slide in a jar of the stain, and allow the stain to act from 5 to 10 minutes.
Wash thoroughly with water.

\ 295. Staining with a General Dye — Counterstaining. — If it is desired to
give a general stain after the nuclear dye (§ 294), carmine stained preparations
may be tinted with picric-alcohol for half a minute or more ( \ 333), and the hem-
atoxylin stained specimens with eosin ( \ 321 ). It usually takes less than a minute
for this. Wash away the counterstain with water.

190

PARAFFIN SECTIONING

\_CH. VII

\ 296. Counterstaining with Picro-fuchsin. —For a general dye to use with
hematoxylin, eosin is good, but to differentiate the tissues more completely,
especially connective tissue, which is present in practically every section made, it
is better to use Van Gieson's mixture of picric acid and acid fuchsin. (Picric
acid, saturated aqueous solution 75 cc., water 25 cc. i% aqueous solution of acid
fuchsin, 10 cc.) Sections are first strongly stained with hematoxylin, well washed
with water and then stained 3 seconds to 15 minutes in the picro-fuchsin. They
are then washed in distilled water ; or in tap water, to which has been added a drop
or two of glacial acetic acid to 100 cc. of water. They are then dehydrated,
cleared and mounted in acid balsam, that is in balsam which has not been neutral-
ized ($ 315). If glycerin or glycerin jelly is used as amounting medium it should
be slightly acid. Unless the mounting medium is slightly acid, the red of the
acid fuchsin soon fades. In some cases less acid fuchsin should be used, and in
some a greater amount. Acid fuchsin alone without the picric acid is also good
for a counterstain. The picro-fuchsin is a very valuable differential stain and
combined in different proportions with picric acid will give great assistance in al-
most every case. It does not seem to be a permanent stain. (See Freeborn,
Trans. N. Y. Path, Soc., 1893, p. 73. Also studies from the department of pathol-
ogy of the College of Physicians and Surgeons, Columbia University, N. Y.,
1894-1895).

161, Coplin's staining dish. A. The
entire dish ; B. the dish in cross section.
This is made of glass and is a very neat piece
of apparatus. With it ten slides may be
stained at once. ( Whitall, Tatum & Co. )

§ 297. Dehydration of the Stained Sec-
tions.— Place the slide with the stained sec-
tions in a jar of 95% or absolute alcohol and
leave it a few minutes, or wave it around in
the alcohol for half a minute or so.

Remember that the larger and thicker
ihe sections the more time it requires to de-
In a moist atmosphere, 95%
alcohol is not completely satisfactory, and

CROSS-SECTION hvdrate them.

SHOWING SLIDES J

IN POSITION.

one must use a stronger alcohol. When it is dry, 95% answers very well.

| 298. Clearing the Sections. — Drain off the alcohol, and place the slide in a
jar of clearer (§ 318, A or B) or put a drop or two of clearer on the sections. The
clearing is usually accomplished in two or three minutes.

§ 299. Mounting in Balsam. — For this the clearer is drained from the
slide, and wiped away with blotting paper, cloth, etc. The balsam is then put
upon the sections and the cover added, or a cover-glass is spread with the balsam
and then put over the sections. ( If the sections show a whitish appearance and
are opaque they were not sufficiently dehydrated. If natural balsam is used the
sections will clear up in time).

CH. VII}

SERIAL SECTIONS

191

ORDER OF PROCEDURE IN MAKING MICROSCOPICAL PREPARATIONS BY THE

PARAFFIN METHOD

$ 300. It will be seen from this table aiid from sections 283 to 299, that it re-
quires from 2 to 7 days to get a microscopical preparation by the paraffin method
if one starts with a fresh tissue. Depending on the method of fixing and harden-
ing, the time may be much greater. Much time will be lost in waiting unless one
plans ahead in histological work.

1. Fixing and hardening the tissue or 10.

organ (£ 284), 4 days or more.

2. Dehydrating the object to be cut in j n.

95% °r stronger alcohol (| 285), 12.
i to 24 hours containing %% j
eosin. (§301). 13.

3. Displacing the alcohol and clearing

tissues with cedar-wood oil. (See \ 14.
§ 286), 2 to 24 hours.

4. Infiltrating the tissue with paraffin j 15.

in the paraffin oven (| 286), 2 to \

24 hours. 16.

5. Imbedding in paraffin (§ 287), 10

minutes.

6. Cutting the sections (I 288), 10 j 17.

minutes.

7. Extending the sections with warm 18.

water. (See I 289).

8. Fastening the sections to a slide j 19.

(§ 290), 5 minutes to 24 hours.

9. Removing the paraffin (§ 291), 10 20.

minutes to 24 hours.

Removing the xylene or benzin

(3 292).

Washing with water, note, p. 180.
Staining with an aqueons dye

(\ 294), 2 minutes to 24 hours.
Washing away the superfluous

stain with water (§ 294).
Staining with a general dye (§ 295-

296), 10 seconds to 10 minutes.
Washing the sections with water

(§ 295-296).
Dehydrating the stained sections in

95% alcohol (\ 297), 3 minutes to

24 hours.

Clearing the sections ( \ 298) 2 min-
utes to 24 hours.
Mounting in Balsam (§ 299), i to 5

minutes.
Labeling the preparation ( \ 308) , 2

minutes.
Cataloging the preparation ( \ 309),

5 to 10 minutes.

SERIAL SECTIONS : IN TOTO STAINING.

$301. In histological studies it is frequently of the greatest advantage to
have the sections in serial order, then an obscure feature in one section is fre-
quently made clear by the following or preceding sections. While serial sections
are very desirable in histological study, they are absolutely necessary for the solu-
tion of morphological problems presented in complex organs like the brain, in
embryos and in minute animals where gross dissection is impossible.

For all sections whether serial or otherwise, if the sections are to be stained
on the slide it is of great advantage to have the object stained in toto in eosin-
during the dehydration (see 2 of the table above), Very thin sections and minute
parts of embryos then show in the ribbons. The eosin bulk staining does not in
any way interfere with the subsequent staining of the sections.

IQ2 SERIAL SECTIONS [CH. VII

$ 302. Arrangement of Tissues for Sections in Histology. — The}- should be so
arranged that the exact relations of each part to the organ can be readily deter-
mined. For example, an organ like the intestine, a muscle or a nerve, should be
so arranged that exact transections or longisections can be made. Organs like the
liver and other glands, the skin, etc., should be so arranged that sections parallel
with the surface or at right angles to it, ( surface or vertical sections) may be
made. Oblique sections are often very puzzling.

§ 303. Arrangement of Serial Sections. — The numerical order may very con
veniently be like the words on a printed page, i. e., beginning at the upper left
hand corner and extending from left to right.

The position of the various aspects of the sections should be in general such
that when they are under the compound microscope the rights and lefts will corre-
spond with those of the observer.

\ 303 a. Imbedding and Sectioning Embryos and Minute Animals. — Serial
sections of these should be made in the three cardinal sectional planes, viz ;
Transections ; Frontal Sections ; Sagittal Sections.

If models are to be constructed from the sections it may be more conveniently
done if the sections are one of the following thicknesses : 5//. lo/w, i5//, 2ojii, 3O//.
4oju, 5ow, 6o//, 8o//. With an adjustable wax-plate tablet any thickness of sections
may be modeled ($ 437).

(A) Transections, that is sections across the long axis of the embryo or
animal.

Imbed the embryo with the right side down, taking the precautions to have
a layer of paraffin between the embryo and bottom of the box (§ 287).

(1) Mount the block of paraffin containing the embryo so that the tail end
will be next the microtome holder. The head will then be cut first.

(2) Place in the microtome so that the right side of the embryo will meet
the edge of the knife.

(3) Mount as a printed line and the first or cephalic section will be at the
upper left hand corner, and the dorsal aspect of the embryo will be toward the
upper edge of the slide.

Under the microscope the rights and lefts will appear as in the observer's own
body, also the dorsal and ventral aspects so that he can easily locate parts by
comparing them with his own body.

(B) Frontal Sections, that is sections lengthwise of the embryo or animal
and from right to left (dextral and sinistral), so that the embryo is divided into
equal or unequal dorsal and ventral parts.

Imbed the embryo with the right side down in the imbedding box as before.

(1) Mount the paraffin block so that the ventral side of the embryo is next
the microtome holder. The dorsal side will then be cut first.

(2) Let the right side of the embryo meet the edge of the knife.

(3) Mount the first section on the left end of the slide as before and so that
the sections will be crosswise on the slide, the tail toward the upper edge. Under

CH. VI 7 '] SERIAL SECTIONS 193

the compound microscope the head will appear toward the upper edge and the
rights and lefts will be as in the observer's own body.

(4) If the sections are too long to mount crosswise of the slide, cut the
sections apart and mount with the head to the right.

(C) Sagittal Sections, that is sections lengthwise of the embryo or animal
and from the ventral to the dorsal side, thus dividing the body into equal or
unequal right and left parts.

For these sections imbed the embryo with the right side down as before.

1 i ) Put the right side of the embryo next the microtome holder, then the
left side will be cut first.

(2) Let the caudal end meet the knife edge if the embryo is small.

(3) Put the first section in the upper left hand part of the slide as in the
other cases. The sections will be lengthwise of the slide. This will bring the
ventral side up and the head to the right on the slide. Under the microscope the
head will appear at the left and the dorsal side away from the observer.

(4) For large or long embryos place the right side next the microtome holder
as above, but let either dorsal or ventral aspect meet the knife. Cut the sections
apart and mount as in (3).

(D) Axes for Sections. — For transections cut across the longest straight line
from head to tail.

For sagittal sections select the straightest embryo and cut parallel with the
longest axis.

For frontal sections cut parallel with the long axis.

$ 304. For serial sections with collodion imbedded objects it is a great advan-
tage to have the imbedding mass unsymmetrically trimmed, so that if a section is
accidentally turned over it may be easily noticed and rectified.

Furthermore it is imperatively necessary that the object be so imbedded that
the cardinal aspects, dextral and sinistral, dorsal and ventral, cephalic and caudal,
shall be known with certainty.

| 305. Thickness of Cover-Glass and of Serial Sections. — It is a great advan-
tage to use very thin cover-glasses (0.12-0.18 mm.) for serial sections, then the
cover will not prevent the use of high powers. When the ordinary slides (25 X
76 mm., 1X3 inch) are used, cover-glasses 24 X 50 mm. may be advantageously
employed.

The combined thickness of the sections on a slide is easily determined by not-
ing carefully the position of the microtome screw at the first and last sections and
measuring the elevation. With good modern automatic microtomes the successive
sections are almost exactly uniform in thickness, hence it is easy to determine the
combined thickness of the sections on a slide by multiplying the number of sec-
tions by the thickness of each.

\ 306. Labeling Serial Sections. — The label of a slide on which serial sections
are mounted should contain at least the following :

194 SERIAL SECTIONS \_CN. VII

The name of the embryo and the number of the series ; the number of the
slide of that series ; the thickness of the sections, and the number of the first and
last section on the slide ; the date. It is also a convenience to have the informa-
tion repeated in part on the left end and the number of the first section in each
row, as shown in the sample slide of serial sections. (Fig. 211).

LABELING, CATALOGING AND STORING MICROSCOPICAL PREPARATIONS

| 307. Every person possessing a microscopical preparation is interested in
its proper management ; but it is especially to the teacher and investigator that
the labeling, cataloging and storing of microscopical preparations are of import-
ance. "To the investigator, his specimens are the most precious of his possessions,
for they contain the facts which he tries to interpret, and they remain the same
while his knowledge, and hence his power of interpretation, increase. They thus
form the basis of further or more correct knowledge ; but in order to be safe
guides for the student, teacher, or investigator, it seems to the writer that every
preparation should possess two things : viz., a label and a catalog or history. This
catalog should indicate all that is known of a specimen at the time of its prepara-
tion, and all of the processes by which it is treated. It is only by the possession
of such a complete knowledge of the entire history of a preparation that one is
able to judge with certainty of the comparative excellence of methods, and thus
to discard or improve those which are defective. The teacher, as well as the
investigator, should have this information in an accessible form, so that not only
he, but his students can obtain at any time, all necessary information concerning
the preparations which serve him as illustrations and them as examples."

$ 308. Labeling Ordinary Microscopical Preparations. — The label should
possess at least the following information (see \ 306 for serial sections):

The No. of the preparation, its name and date and the thickness of the sec-
tions and of the cover-glass.

0

DATE.

FIG. 163. Example of a label of an ordinary histologic
specimen. (See also Fig. 211 for serial sections}.

/ / /

Fig. 163.

\ 309. Cataloging Preparations. — It is believed from personal experience, and
from the experience of others, that each preparation (each slide or each series)
should be accompanied by a catalog containing at least the information suggested
in the following formula. This formula is very flexible, so that the order may be
changed, and numbers not applicable in a given case maybe omitted. With many
objects, especially embryos and small animals, the time of fixing and hardening
may be months and even years earlier than the time of imbedding. So, too, an
object may be sectioned a long time after it was imbedded, and finally the sections

CH. VII}

LABELING AND CATALOGING

195

may not be mounted at the time they are cut. It would be well in such cases to
give the date of fixing under 2, and under 5, 6 and 8, the dates at which the oper-
ations were performed if they differ from the original date and from one another.
In brief, the more that is known about a preparation the greater its value.

General Formula for Cataloging Mi-
croscopical Preparations :

1. The general name and source.
Thickness of cover glass and of section.

2. The number of the preparation and
the date of obtaining and fixing the
specimen ; the name of the preparator.

3. The special name of the prepara-
tion and the common and scientific
name of the object from which it is de-
rived. Purpose of the preparation.

4. The age and condition of the ob-
ject from which the preparation is de-
rived. Condition of rest or activity ;
fasting or full fed at the time of death.

5. The chemical treatment, — the
method of fixing, hardening, dissociat-
ing, etc., and the time required.

6. The mechanical treatment, — im-
bedded, sectioned, dissected with nee-
dles, etc. Date at which done.

7. The staining agent or agents and
the time required for staining.

8. Dehydrating and clearing agent,
mounting medium, cement used for
sealing.

9. The objectives and other accesso-
ries (micro-spectroscope, polarizer, etc.,)
for studying the preparation.

A Catalog Card Written According to
this Formula :

Muscular Fibers. Cat.

C. 15.
Fibers 20 to 40 n thick.

2. No. 475. (Drr. IX) Oct. i, 1891. S.
H. G. , Preparator.

3. Tendinous and intra-muscular ter-
minations of striated muscular fibres
from the Sartorius of the cat (Felis do-
mestica).

4 . Cat eight months old, healthy and
well nourished. Fasting and quiet for
12 hours.

5. Muscle pinned on cork with vas-
elined pin£ and placed in 20 per cent,
nitric acid immediately after death by
chloroform. Left 36 hours in the acid ;
temperature 20° C. In alum water ()£
sat. aq. sol. ) i day.

6. Fibers separated on the slide with
needles, Oct. 3.

7. Stained 5 minutes with Delafield's
hematoxylin.

8. Dehydrated with 95 % alcohol 5
minutes, cleared 5 minutes with carbol-
turpentine, mounted in xylene balsam ;
sealed with shellac.

9. Use a 1 6 mm . for the general appear-
ance of the fibers, then a 2 or 3 mm. ob-
jective for the details of structure. Try
the micro-polariscope ($ 218).

10. The nuclei or muscle corpuscles are
very large and numerous ; many of the
intra-muscular ends are branched. See
S. P. Gage, Proc. Amer. Micr. Sci., 1890,
p. 132 ; Ref. Hand-book Med., Sci., Vol.
V.,p. 59-

10. Remarks, including references to
original papers, or to good figures and
descriptions in books.

\ 310. General Remarks on Catalogs and Labels. — It is especially desirable
that labels and catalogs shall be written with some imperishable ink. Some form
of water-proof carbon ink is the most available and satisfactory. The water-proof
India ink, or the engrossing carbon ink of Higgins, answers well. As purchased,
the last is too thick for ordinary writing and should be diluted with one-third its
volume of water and a few drops of strong ammonia added.

196 LABELING AND CATALOGING \_CH. VII

If one has a writing diamond it is a good plan to write a label with it on one
end of the slide. It is best to have the paper label also, as it can be more easily
read.

FIG. 164. Writing diamond for writing numbers and labels on glass slides,
cutting cover-glasses, etc. ( Queen and Co. )

The author has found stiff cards, 12^ xy^ cm., like those used for cataloging
books in public libraries, the most desirable form of catalog. A specimen that is
for any cause discarded has its catalog card destroyed. New cards may then be
added in alphabetical order as the preparations are made. In fact a catalog on
cards has all the flexibility and advantages of the slip system of notes (see Wilder
& Gage, p. 45).

Some workers prefer a book catalog. Very excellent book catalogs have been
devised by Ailing and by Ward (Jour. Roy. Micr. Soc., 1887, pp. 173, 348 ; Amer.
Monthly Micr. Jour., 1890, p. 91 ; Amer. Micr. Soc. Proc., 1887, p. 233).

The fourth division has been added as there is coming to be a strong
belief, practically amounting to a certainty, that there is a different structural
appearance in many if not all of the tissue elements depending upon the age of
the animal, upon its condition of rest or fatigue ; and for the cells of the digestive
organs, whether the animal is fasting or full fed. Indeed as physiological histology
is recognized as the only true histology, there will be an effort to determine exact
data concerning the animal from which the tissues are derived. (See Minot, Proc.
Amer. Assoc. Adv. Science, 1890, pp. 271-289 ; Hodge, on nerve cells in rest and
fatigue, Jour. Morph., vol. VII. (1892), pp. 95-168; Jour. Physiol., vol. XVII.,
pp. 129-134; Gage, The processes of life revealed by the microscope ; a plea for
physiological histology, Proc. Amer. Micr. Soc., vol. XVII. (1895), pp. 3-29;
Science, vol. II., Aug. 23, 1895, pp. 209-218.

CABINET FOR MICROSCOPICAL, PREPARATIONS

§ 311. While it is desirable that microscopical preparations should be pro-
perly labeled and cataloged, it is equally important that they should be protected
from injury. During the last few years several forms of cabinets or slide holders
have been devised. Some are very cheap and convenient where one has but a few
slides. For a laboratory or for a private collection where the slides are numerous
the following characters seem to the writer essential :

( i ). The cabinet should allow the slides to lie flat, and exclude dust and light.

(2). Each slide or pair of slides should be in a separate compartment. At
each end of the compartment should be a groove or bevel, so that upon depressing
either end of the slide the other may be easily grasped (Fig. 165). It is also
desirable to have the floor of the compartment grooved so that the slide rests only
on two edges, thus preventing soiling the slide opposite the object.

(3). Each compartment or each space sufficient to contain one slide of the
standard size should be numbered, preferably at each end. If the compartments
are made of sufficient width to receive two slides, then the double slides so fre-
quently used in mounting serial sections may be put into the cabinet in any place
desired.

CH.

LABELING AND CATALOGING

197

FIG. 165. A part of a cabinet drawer seen
from above. In compartment No. 06 is repre-
sented a slide lying flat. The label of the
slide and the number of the compartment are
so placed that the number of the compartment
may be seen through the slide. 7 he sealing
cement is removed at one place to show that in
sealing the cover-glass^ the cement is put
partly on the cover and partly on the slide

(I 249, 253)-

B. — This represents a section of the same
part of the drawer, (a) Slide resting as in
a. No. 06. The preparation is seen to be above
a groove in the floor of the compartment, (b)
One end of the slide is seen to be uplifted by de-
pressing the other into the bevel.

(4). The drawers of the cabinet should be
entirely independent, so that any drawer may
be partly or wholly removed without disturb-
ing any of the others.

(5). On the front of each drawer should be
the number of the drawer in Roman numer-
als, and the number of the first and last com-
partment in the drawer in Arabic numerals
(Fig. 166).

FIG. 1 66. Cabinet for Mi-
croscopical Specimens, show-
ing the method of arrange-
ment and of numbering the
drawers and indicating the
number of the first and last
compartment in each drawer.
It is better to have the slides
on which the drawers rest
somewhat shorter, then the
drawer front may be entire
and not notched as here shown.
(From. Proc. Amer. Micr. Soc.,
1883).

o

70

FIG. 165.

i8g PREPARATION OF REAGENTS [CH. VII

FIG. 167. Slide box for 25 speci-
mens. These are cheap and convenient
and may be stood on end like books,
thus placing the slides in a horizontal
position. Smaller boxes, i. e.for j, 6
and 12 slides are also made, and mail-
ing boxes for a single slide (Bausch &
Lomb Co.).

SOME REAGENTS FOR FIXING, MOUNTING, ETC.

$ 312. Albumen Fixative (Mayer's). — This consists of equal parts of well-
beaten white of egg and glycerin. To each 50 cc. of this I gram of salicylate of
soda is added to prevent putrefactive changes. This must be carefully filtered.
For method of use see $ 290.

\ 313. Alcohol (Ethylic). — Ethyl alcohol is mostly used for histological pur-
poses. (A) absolute alcohol (i. e., alcohol of -^-§-$%} is recommended for many
purposes, but if plenty of 95% alcohol is used it answers every purpose in
histology, in a dry climate or in a warm, dry room. When it is damp dehydration
is greatly facilitated by the use of absolute alcohol.

(B) 82% alcohol made by mixing 5 parts of 95% alcohol with i part of water.

(C) 67% alcohol made by mixing 2 parts of 95% alcohol with i part of water.
Grades of Alcohol. It has been found by careful tests that quite accurate

percentages of alcohol may be obtained by mixing water and alcohol as follows :
Pour alcohol into a graduate until the volume of alcohol corresponds to the de-
sired percentage. Add water until the volume in cubic centimeters corresponds
to the original percentage of the alcohol used. For example, to get 67% from
95% alcohol, pour 67 cc. of 95% into a graduate, and add sufficient water to bring
the volume up to 95 cc. Far 50% alcohol from 75%, put 50 cc. of 75% alcohol in
a graduate, add sufficient water to make the volume 75 cc. From the change in
volume it does not answer to mix given volumes of water and alcohol in these
cases. In the first case, if one mixed 75 cc. of 95% alcohol and 20 cc. of water the
resulting mixture would be over 75% ; but if sufficient water is added to bring the
volume back to the original percentage more than 20 cc. of water is added, that is
enough more to compenate for the shrinkage, and the result is approximately
accurate.

Methyl Alcohol is much cheaper than ethyl, and answers well in most micro-
scopical processes. It has recently been so carefully refined that the disagreeable
odor is very little noticeable.

Synthol. This is said to be a synthetic form of alcohol. It seems to serve the
purpose of alcohol in histology. Absolute synthol is far cheaper for the private
worker than absolute ethyl alcohol.

\ 314. Alum Solution. — For muscle dissociated in nitric acid use a satu-
rated solution (i. e., a solution in which the water holds all the- alum it can. If
one adds an excess so that there will always be some undissolved alum in the ves-
sel he can be sure the solution is saturated after it has stood a few days. An easy
way to get a saturated solution is to take 500 cc. of water and add TOO grams of
alum and heat the water in an agate dish. All the alum will be melted, but on

CH. VII ] PREP A RA TION OF RE A GENTS 1 99

cooling a part will crystallize out, leaving a cold saturated solution). The satu-
rated solution may be used but, if a half saturated solution is employed, it will
answer all the purposes. For a half saturated solution take 100 cc. of water, and
100 cc. of saturated alum water and mix the two. In case preparations are to be
kept some time in alum water, 2% of chloral hydrate should be added to prevent
mold.

\ 315. Balsam, Canada Balsam, Balsam of Fir; Xvlene Balsam. — This is
one of the oldest and most satisfactory of the resinous media used for mounting
microscopical preparations.

The natural balsam is most often used, but within the last ten years the belief
has arisen that it is better to evaporate the balsam and then dissolve it in xylene
or benzole. It certainly dries out more rapidly if so treated. Natural balsam has
the advantage of being able to take up a small amount of water so that if sections
are not quite dehydrated they will clear up after a time. If xylene balsam is used
the dehydration must be almost complete or the preparation will look cloudy.

Filtering Balsam. Balsam is now furnished already filtered through filter
paper. If one wishes to filter it himself a hot filter like that shown in Fig. 155 is
good. If xylene balsam is used it may be made thin and filtered without heat.
For filtering balsam and all resinous and gummy materials, the writer has found a
paper funnel the most satisfactory. It can be used once and then thrown away.
Such a funnel may be easily made by rolling a sheet of thick writing paper
in the form of a cone and cementing the paper where it overlaps, or winding a
string several times around the lower part. Such a funnel is best used in one of
the rings for holding funnels.

FIG. 1 68. Vessel for homogeneous immersion liquid
(thick cedar-wood oil}. This is jilled only a little above the
lower end of the inner tube. The oil will not then run out if
the vessel is tipped over. For applying the oil there is a wire
loop attached to the upper cork. The side cork is for the pur-
pose of emptying the bottle, and also for the escape of the air
when filling it. ( The Spencer Lens Co.; see also Zeit.
wiss. Mikr. , /&?7, p. 348, and Jour. Roy. Micr. Soc. , 1898,
p. 238}.

Natural Balsam. All the samples of balsam tested by the author have been
found slightly acid. This is an advantage for carmine, and acid fuchsin stains or
any other acid stain. Also for preparations injected with carmine or Berlin blue.
In these cases the color would fade or diffuse if the medium were not slightly
acid. For hematoxylin the acid is detrimental. For example, the slight amount
of acid in the balsam will cause the delicate stain in the finest fibers of Weigert
preparations to fade. Also the fuchsin and other stains which are faded by acids.
To neutralize the balsam add some pure sodium carbonate, set the balsam in a
warm place and shake it occassionally. After a month or so the soda will settle
and the clear supernatant balsam will be found very slightly alkaline. Use this
whenever an acid medium would fade the stain in the specimen.

200 PREPARATION OF REAGENTS \CH. VII

\ 316. Cedar- Wood Oil. — This is used for oil immersion objectives. It is
best kept in a bottle like that shown in Fig. 168. This oil is quite thick.

For penetrating tissues and preparing them for infiltration with paraffin, thick
oil is recommended by Lee. The writer has found, however, that any good cedar
wood oil gives excellent results in ordinary histological and embryological work.
That known as Cedar Wood Oil (Florida) js excellent, also that known as cedar
wood oil (true Lebanon). These forms are far less expensive than that used for
immersion objectives. The tissues should be thoroughly dehydrated before put-
ting them into cedar- wood oil, and they should remain until they are transparent
(\ 286).

\ 317. Clarifier, Castor-Xylene Clarifier. — This is composed of castor oil i
part and xylene* 3 parts. (Trans. Amer. Micr. Soc., 1895, p. 361).

\ 318. Clearing Mixture ($281, 298). — (A). One of the most satisfactory and
generally applicable clearers is carbol turpentine, made by mixing carbolic acid
crystals {Acidum carbolicum. A.phenicum crystallizatum] 40 cc. with rectified
oil of turpentine (Oleum terebinthinae rectificatum} 60 cc. If the carbolic acid
does not dissolve in the turpentine add 5 cc. of 95% alcohol, or increase the tur-
pentine, thus : carbolic acid 30 cc., turpentine 70 cc.

(B). Carbol-Xylene Clearer. — Vasale recommends as a clearer, xylene 75 cc.,
carbolic acid (melted crystals) 25 cc. It is used in the same way as the preceding.

| 319. Collodion. — This is a solution of soluble cottonf or other form of pyroxy-
lin in equal parts of sulphuric ether and 95% alcohol. Two solutions are used :

*The hydrocarbon xylene (C8H10) is called xylol in German. In English
members of the hydrocarbon series have the termination "ene" while members of
the alcohol series terminate in "ol."

fThe substance used in preparing collodion goes by various names, soluble cot-
ton or collodion cotton is perhaps best. This is cellulose nitrate, and consists of a
mixture of cellulose tetranitrate C12Hr6(NO3)4O6, and cellulose pentanitrate,
C12H15(NO3)5O5. Besides the names soluble and collodion cotton, it is called gun
cotton and pyroxylin. Pyroxylin is the more general term and includes several
of the cellulose nitrates. Celloidin is a patent preparation of pyroxylin, more ex-
pensive than soluble cotton, but in no way superior to it for imbedding.

Soluble cotton should be kept in the dark to avoid decomposition. After it is
in solution this decomposition is not so liable to occur. The decomposition of the
dry cotton gives rise to nitrous acid, and hence it is best to keep it in a box loosely
covered so that the nitrous acid may escape.

Cellulose nitrate is explosive under concussion and when heated to 150° centi-
grade. In the air, the loose soluble cotton burns without explosion. It is said not
to injure the hand if held upon it during ignition and that it does not fire gun- .
powder if burned upon it. So far as known to the writer, no accident has ever
occurred from the use of soluble cotton for microscopical purposes. I wish to ex-
press my thanks to Professor W. R. Orndorff, organic chemist in Cornell Univer-
sity, for the above information. Proc. Amer. Micr. Soc., vol. XVII (1895), pp.
361-370.

CH. F/7] PREPARATION OF REAGENTS 201

(A). 6% to 8% or thick collodion. It is made by mixing 50 cc. of sulphuric
ether and 50 cc. of 95% alcohol and adding 6 or 8 grams of soluble cotton. If this
is shaken repeatedly the solution will be complete in a day or two.

(B). 1/4% or thin collodion. To prepare this \y2 grams of soluble cotton are
added to 100 cc. of ether-alcohol (§ 322).

$ 320. ^% collodion or cementing collodion. To prepare it ^ths of a gram
of soluble cotton is added to alcohol, 60 cc. , ether, 40 cc.

The excess of alcohol prevents the mixture from softening the paraffin ($ 290).

§ 321. Eosin. — This is used mostly as a contrast stain with hematoxylin, which
is an almost purely nuclear stain. It serves to stain the cell-body, ground sub-
stance, etc., which would be too transparent and invisible with hematoxylin alone.
If eosin is used alone it gives a decided color to the tissue and thus aids in its
study ($ 144). Eosin is used in alcoholic and in aqueous solutions. A very satis-
factory stain is made as follows : 50 cc. of water and 50 cc. of 95% alcohol are
mixed and i-ioth of a gram of dry eosin added.

The eosin is used after the hematoxylin in most cases (\ 280), and, as it is in
alcoholic solution, it may be washed off with 95% alcohol if the object is to be
mounted in balsam. If it is to be mounted in glycerin or glycerin jelly, the excess
of eosin should be washed away with distilled water.

\ 322. Ether, Ether-Alcohol. — Sulphuric ether is meant when ether is men-
tioned in this book. For the ether-alcohol mentioned in £ 269, 319, etc., a mixture
of equal volumes of sulphuric ether and 95% alcohol is meant.

\ 323. Farrants' Solution. — Take 25 grams of clean, dry, gum arabic ; 25 cc.
of a saturated aqueous solution of arsenious acid ; 25 cc. of glycerin. The gum
arabic is soaked for several days in the arsenic water, then the glycerin is added
and carefully mixed with the dissolved or softened gum arabic.

This medium retains air bubbles with great tenacity. It is much easier to
avoid than to get rid of them in mounting.

\ 324. Formaldehyde Dissociator. — This is composed of 2 cc. of a 40% solu-
tion of formaldehyde in 1000 cc. of water, to which 6 grams of common table salt
(sodium chlorid) have been added. Formaldehyde as bought in the market is a
40% solution in water, and is called formol, formalin, formalose and formal, the
last name being the preferable one. For its use in isolating cells see $ 260. (Micr.
Bulletin and Sci. News, vol. XII. (1895), pp. 4-5).

\ 325. Glycerin. — (A). One should procure pure glycerin for a mounting
medium. It needs no preparation, except in some cases it should be filtered
through filter paper or absorbent cotton to remove dust, etc.

For preparing objects for final mounting, glycerin 50 cc., water 50 cc. , forms
a good mixture. For many purposes the final mounting in glycerin is made in an
acid medium, viz., Glycerin 99 cc., Glacial acetic or formic acid, i cc.

By extreme care in mounting and by occasionally adding a fresh coat to the
sealing of the cover-glass, glycerin preparations last a long time. They are liable
to be disappointing, however. In mounting in glycerin care should be taken
to avoid air-bubbles, as they are difficult to get rid of. A specimen need not be
discarded, however, unless the air-bubbles are large and numerous.

\ 326. Glycerin Jelly. — Soak 25 grams of the best dry gelatin in cold water in
a small agate- ware dish. Allow the water to remain until the gelatin is softened.
It usually takes about half an hour. When the gelatin is softened, as may be

202 PREPARATION OF REAGENTS \_CH.V1I

readily determined by taking a little in the fingers, pour off the superfluous water
and drain well to get rid of all the water that has not been imbibed by the gelatin.
Warm the softened gelatin over a water bath and it will melt in the water it has
absorbed. Add to the melted gelatin about- 5 cc. of egg albumen, white of egg ;
stir it well and then heat the gelatin in the water bath for about half an hour. Do
not heat above 75° or 80° C., for if the gelatin is heated too hot it will be trans-
formed into meta-gelatin and will not set when cold. The heat will coagulate the
albumen and form a kind of floculent precipitate which seems to gather all fine
particles of dust, etc., leaving the gelatin perfectly clear. After the gelatin is
clarified it should be filtered through a hot flannel filter and mixed with an equal
volume of glycerin and 5 grams of chloral hydrate and shaken thoroughly. If it
is allowed to remain in a warm place (i. £,, in a place where the gelatin remains
melted) the air-bubbles will rise and disappear.

In case the glycerin jelly remains fluid or semi-fluid at the ordinary temper-
ature (i8°-2o° C), the gelatin has either been transformed into meta-gelatin by too
high a temperature or it contains too much water. The amount of water may be
lessened by heating at a moderate temperature over a waterbath in an open vessel.
This is a very excellent mounting medium. Air-bubbles should be avoided in
mounting as they do not disappear.

\ 327. Chloral Hematoxylin. — Hematoxylin is one of the most useful stains
employed in histology. An excellent solution for ordinary section staining
may be made as follows : Distilled water 200 cc., and potash alum i\ grams, are
boiled together for 5 minutes, in an agate-ware or glass vessel, and sufficient boiled
water added to bring the water back to 200 cc. After the mixture is cool, 4 grams
of chloral hydrate, and T2oths grams of hematoxylin crystals, previously dissolved
in 20 cc. of 95% alcohol, are added. The boiling seems to destroy any fungi pres-
ent in the alum or water, and the chloral prevents the development of any that
may get in afterward, and this solution therefore is quite permanent.

At first the color will be rather faint, but after a week or two it will become a
a deep purple. The deepening of the color is more rapid if the bottle is left un-
corked in the light and is shaken occasionally. It may be prepared for work
at once by the addition of a small amount of hydrogen dioxid (H2O2).

If the stain is too concentrated it may be diluted with freshly distilled water
or with a mixture of water, alum and chloral. If the stain is not sufficiently con-
centrated, more hematoxylin may be added. With hematoxylin of the strength
given in the formula, sections are usually sufficiently stained in from one to five
minutes.

As may be inferred from what was said above, the boiling is to destroy any
living ferments present in the water or alum, and the chloral hydrate is to prevent
the development of germs which accidentally reach the solution after it is made.

No precaution is necessary in using this stain for sections, except that appli-
cable to all hematoxylin solutions, viz : It must be filtered occasionally and after
staining the surplus stain must be very thoroughly washed away with water ;
otherwise black granules or needles will appear in or upon the sections. If granules
appear in the preparations in spite of the washing, it will be well to boil the solu-
tion three to five minutes and filter through paper or absorbent cotton. The addi-
tion of one or two per cent, of chloral after the boiling is also advantageous.
This stain has not been tried for dyeing in bulk. Other substances than chloral

Cff. F/7] PREPARATION OF REAGENTS 203

were tried, but not with so good success. (Proc. Amer. Micr. Soc., 1892, pp.
125-127).

\ 328. Hematein. This is used iiistead of hematoxylin, as it is believed to
give more satisfactory results. Prepare as follows : Prepare a 5% solution of pot-
ash alum in distilled water and boil or leave in a steam steralizer an hour or two.
While this alum solution is warm add i per cent, of hematein dissolved in a small
quantity of alcohol. After the fluid has cooled add 2 grams of chloral for each
loo cc. of solution. (Freeborn, Jour. Ap. Micr., 1900, p. 1056. )

$ 329. Liquid Gelatin. — Gelatin or clear glue, 75 to 100 grams. Commercial
acetic acid (No. 8) 100 cc. , water 100 cc., or glacial acetic acid 40 cc. and water
160 cc., 95% alcohol 100 cc., glycerin 15 to 30 cc. Crush the glue and put it into
a bottle with the acid, and set in a warm place, and shake occasionally. After
three or more days add the other ingredients. This solution is excellent for
fastening paper to glass, wood or paper. The brush must be mounted in a quill
or wooden handle. For labels, it is best to use linen paper of moderate thickness.
This should be coated with the liquid gelatin and allowed to dry. The labels may
be cut of any desired size and attached by simply moistening them, as in using
postage stamps.

Very excellent blank labels are now furnished by dealers in microscopical
supplies, so that it is unnecessary to prepare them one's self, except for special
purposes Those like that shown in Fig. 163 may be had for about $4 for 10,000.

I 330. Nitric Acid Dissociator. — This is prepared by mixing So cc. of water
with 20 cc. of strong nitric acid. It is used mostly in dissolving the connective
tissue of muscle and thus making it possible to separate the fibers. Alum water
is used as a restrainer (§314 and 264). (Proc. Amer. Micr. Soc., Vol. XI, ( 1889),
pp. 34-45).

\ 33 r- Normal Salt Solution or Saline Solution. — Pure water from its differ-
ing density from the natural lymph acts injuriously on the tissues. The addition
of a little table salt, however, prevents this deleterious action, or greatly lessens it,
hence the name of normal salt solution. It is a -£$% solution of table salt (sodium
chlorid) in water ; water 1000 cc., salt 6 grams, or water 100 cc., salt T% gram.

§ 332. Paraffin. Paraffin is of various melting points, hence at the ordinary
temperature of a laboratory, that melting at the lowest temperature will be moder-
ately soft, hence soft paraffin, while that melting at a higher temperature will be
hard. For the best results one usually has to mix hard and soft paraffins. The
larger the object to be cut and the thicker the sections the softer should be the
paraffin.

\ 333. Picric-Alcohol. — This is an excellent hardener and fixer for almost all
tissues and organs. It is composed of 500 cc. of water and 500 cc. of 95% alcohol,
to which 2 grams of picric acid have been added. (It is a i% solution of picric
acid in 50% alcohol). It acts quickly, in from one to three days. (£ 267, 284).
(Proc. Amer. Micr. Soc., Vol. XII (1890), pp. 120-122).

\ 334. Shellac Cement. —Shellac cement for sealing preparations and for mak-
ing shallow cells (\ 248) is prepared by adding scale or bleached shellac to 95%
alcohol. The bottle should be filled about half full of the solid shellac then enough
95% alcohol added to fill the bottle nearly full. The bottle is shaken occasionally
and then allowed to stand until a clear stratum of liquid appears on the top. This
clear, supernatant liquid is then filtered through absorbent cotton, using a paper

204 PREPARATION OF REAGENTS \_CH.VII

funnel (| 315), into an open dish or a wide-mouth bottle. To every 100 cc. of
this filtered shellac, 2 cc. of castor oil and 2 cc. of Venetian turpentine are added
to render the shellac less brittle. The filtered shellac will be too thin, and must
be allowed to evaporate till it is of the consistency of thin syrup. It is then put
into a capped bottle, and for use, into a small spirit lamp (Fig, 153). In case the
cement gets too thick add a small amount of 95% alcohol or some thin shellac.
The solution of shellac almost always remains muddy, and in most cases it takes a
very long time for the flocculent substance to settle. One can very quickly obtain a
clear solution as follows : When the shellac has had time to thoroughly dissolve,
i. e. , in a week or two in a warm place, or in less time if the bottle is frequently
shaken, a part of the dissolved shellac is poured into a bottle and about one-fourth
as much gasolin or benzin added and the two well shaken. After twenty-four
hours or so the flocculent, undissolved substance will separate from the shellac so-
lution and rise with the gasolin to the top. The clear solution may then be siphoned
off or drawn off from the bottom if one has an aspirating bottle. (R. Hitchcock,
Amer. Monthly Micr. Jour , July, 1884, p. 131).

If one desires to color the shellac, the addition of a strong alcoholic solution
of some of the coal tar colors is good, but it is liable to dissolve in the mounting
medium when shellac is used for sealing. A small amount of lampblack well
rubbed up in very thin shellac and filtered, is good to darken the shellac.
1

ARRANGING AND MOUNTING MINUTE OBJECTS

§ 335- Minute objects like diatoms or the scales of insects may be arranged in
geometrical figures or in some fanciful way. either for ornament or more satisfac-
tory study. To do this the cover-glass is placed over the guide. This guide for
geometrical figures may be a net-micrometer or a series of concentric circles. In
order that the objects may remain in place, however, they must be fastened to the
cover-glass. As an adhesive substance, mucilage or liquid gelatin ($ 329) thinned
with an equal volume of 50% acetic acid answers well. A very thin coating of this is
spread on the cover with a needle, or in some other wray and allowed to dry. The ob-
jects are then placed on the gelatinized side of the cover and carefully got into posi-
tion with a mechanical finger, made by fastening a cat's whisker in a needle holder.
For most of these objects a simple microscope with stand (Figs. 20, 145, 146) will be
found of great advantage. After the objects are arranged, one breathes very gently
on the cover-glass to soften the mucilage or gelatin. It is then allowed to dry and
if a suitable amount of gelatin has been used, and it has been properly moistened,
the objects will be found firmly anchored. In mounting one may use Canada bal-
sam or mount dry on a cell (\ 247, 255). See Newcomer, Amer. Micr. Soc.'s Proc.,
1886, p. 128 ; see also E. H. Griffith and H. L. Smith, Amer. Jour, of Micros., iv,
102, v, 87 ; Amer. Monthly Micr. Jour., i, 66. 107, 113. Cunningham, The Micro-
scope, viii, 1888, p. 237.

For additional apparatus for this chapter, see Ch. X.

CHAPTER VIII

PHOTOGRAPHING OBJECTS WITH A VERTICAL CAMERA ;
PHOTOGRAPHING LARGE TRANSPARENT OBJECTS;
PHOTOGRAPHING WITH A MICROSCOPE : (A) TRANS-
PARENT OBJECTS; (B) OPAQUE OBJECTS AND THE
SURFACES OF METALS AND ALLOYS*

APPARATUS AND MATERIAL FOR THIS CHAPTER

Vertical camera with photographic objectives (Fig. 169), small vertical camera
with special microscope stand for embryos, etc. (Figs. 183-184); arrangement of
camera for large transparent objects (Fig. 181); photo-micrographic cameras ( Figs.
183-184, 192); photographic objectives for gross and microscopic work (Figs. 170-
171, 176-180); microscope, microscopic objectives and projection oculars (Figs. 185,
189, 193); color screens, lamps, dry plates and the chemicals and apparatus neces-
sary for developing, printing, etc.

\ 336. Nothing would seem more natural than that the camera, armed with a
photographic objective or with a microscopic objective, should be called into the
service of science to delineate with all their complexity of detail, the myriads of
forms studied.

For photographing many objects in nature the camera remains horizontal or
approximately so, but for a great many of those studied in botany, zoology, miner-
alogy and in anatomy the specimens cannot be put in a vertical position necessary
for a horizontal camera. This difficulty has been overcome by using a mirror or a
45-degree prism. These are practically alike and have the defect of producing a
picture with the inversion of a plane mirror.

VERTICAL CAMERA

$ 337- To meet all the difficulties the object may be left in a horizontal posi-
tion and the camera made vertical (fig. 169).

For the last twenty-five years such a camera has been in use in the Anatomical
Department of Cornell University for photographing all kinds of specimens ;

*Papers on this subject were given by the writer at the meeting of the Amer-
ican Association for the Advancement of Science in 1879, and at the meeting of
the Society of Naturalists of the eastern United States in 1883 ; and in Science
Vol. Ill, pp. 443, 444.

206 PHOTO-MICROGRAPHY [CH. VIII

among these, fresh brains, and hardened brains have been photographed without
the slightest injury to them. Furthermore, as many specimens are so delicate
that they will not support their own weight, they may be photographed under
alcohol or water with a vertical camera and the result will be satisfactory as a
photograph and harmless to the specimen.

A great field is also open for obtaining life-like portraits of water animals.
Freshly killed or etherized animals are put into a vessel of water with a contrast-
ing back ground and arranged as desired, then photographed. The fins have
something of their natural appearance and the gills of branchiate salamanders
float out in the water in a natural way. In case the fish tends to float in the water
a little mercury injected into the abdomen or intestine will serve as ballast.

The photographs obtainable in water are almost if not quite as sharp as those
made in air. Even the corrugations on the scales of such fishes as the sucker
(Catostomus teres) show with great clearness. Indeed so good are the results
that excellent half tone plates may be produced from the pictures thus made, also
excellent photogravures. In those cases, as in anatomical preparations, where the
photograph rarely answers the requirements of a scientific figure, still a photo-
graph serves as a most admirable basis for such a figure. The photograph is made
of the desired size and all the parts are in correct proportion and in the correct
relative position. From this photographic picture may be traced all the outlines
upon the drawing paper, and the artist can devote his whole time and energy to
giving the proper expression without the tedious labor of making measurements.

"While the use of photography for outlines as bases for figures diminishes the
labor of artists about one-half it increases that of the preparator ; and herein lies
one of its chief merits. The photographs being exact images of the preparations,
the tendency will be to make them with greater care and delicacy, and the result
will be less imagination and more reality in published scientific figures ; and the
objects prepared with such care will be preserved for future reference."

"In the use of photography for figures several considerations arise : (i) The
avoidance of distortion ; (2) The adjustment of the camera to obtain an image of
the desired size ; (3) Focusing ; (4) Lighting and centering the object.

(i). While the camera delineates rapidly, the image is liable to. distortion.
I believe opticians are agreed, that, in order to obtain correct photographic images,
the objective must be properly made, and the plane of the object must be parallel
to the plane of the ground glass. Furthermore, as most of the objects in natural
history have not plane surfaces, but are situated in several planes at different
levels, the whole object may be made distinct by using in the objective a dia-
phragm with a small opening.

$338. Scale of Sizes and Focusing. — (2). By placing the camera on a long
table, and a scale of some kind against the wall, the exact position of the ground
glass for various sizes may be determined once for all, and these positions
noted in some way.

FIG. 169. Vertical Camera for photographing objects in a horizontal position.
The camera is attached to a double frame connected by bent metal pieces
fastened to the lower and sliding in a groove in the upper frame. The two frames
can then slide over each other without separating. For moving the outer frame a
rack work is put on the lower or inner frame and a pinion with a toothed wheel on
the outer one. This is turned by the wheel shown . To prevent the camera run-

CH.

PHO TO-MICROGRAPHY

207

ning down in the vertical position a pawl is held in place by a spring. This may
be released by a smaller wheel than that serving to move the pinion. This rack and
pinion are fine enough for focusing with the photographic objectives employed.

FIG. 169.

The camera bed is graduated in centimeters so that the exact extent of the
bellows can be determined by inspection.

The support on which the specimen rests is of heavy glass on vertical
rods about 10 centimers long. The background is placed on the table top about 10
cm. below. This arrangement of stipport and background serves to avoid the dense
shadows which make it difficult to determine exactly the limits of the specimen.
To make the apparatus steady the right hand end of the table is heavily weighted.
The table s% have leveling screws in the legs.

208

PHO TO-MICROGRAPHY

\_CH. VIII

FIG. 170.

FIG. 170. Turner-Reich
anastigmat objective for pho-
tography, ( Gundlach Opt.
Co.)

In the camera here fig-
ured, the camera bed is ruled
in centimeters so that the
position of the ground glass
can be determined with ac-
curacy and noted. It takes
but a moment to set the
ground glass or focusing
screen at the right level to
give any desired size. In
practice it is convenient to
have attached to the camera
a table giving the position of
the ground glass for various
sizes, and also the distance of
the objective from the object
in each case. By having this
information it takes but a
moment to set the camera
and to place it so that it
will be approximately i n

FIG. 171. Zeiss anastigmat objective
for photography ', (Bausch & Lomb Opti-
cal Co. )

focus. The final focusing is then accom-
plished by the use of the rack and pinion
movement. It is an advantage to use a
focusing glass and a clear focusing screen
or the transparent part of the ordinary
screen (Fig. 174), for the final focusing.
As many objects have not sharp details FIG. 171.

which one can focus on, it is helpful to

place some printed letters on the part to be .brought out with the greatest sharp-
ness. Of course these are removed before the exposure is made.

§ 339. In lighting the object one should take pains to so arrange it with ref-
erence to the light that the details will show with the greatest clearness. Naturally
for the vertical camera the light will come from the side and not from a skylight,
although good results are obtained with a skylight if one so places the camera that
it does not cast objectionable shadows.

CH. VIII}

PHO TO-MICROGRAPHY

209

FIG. 172. Tripod magnifier as focusing glass. This
is carefully focused on a scratch or pencil mark on the
lower or ground surface of the focusing screen. Then
whenever the object is sharply focused the focal plane will
be at the level of sensitive surface.

As shown in Fig. 169, the object is placed upon a
glass support and the background is quite a distance
below the support. For a dark object the background
should be light, and for a light one dark. Black
velveteen is excellent for a back-ground. The advan- pIG ry2

tage of the glass support is that the shadows in the

background which often make it difficult to tell just where the specimen ends and
the background begins, is wholly done away with, and that too without at all
affecting the proper light and shade of the object itself. (Method of W. E.
Rumsey, Canadian Entomologist 1896, p. 84).

FIG. 173. Focusing Glass. "It is achromatic,
consisting of a double convex crown lens and a nega-
tive meniscus flint lens cemented together." It screws
into the brass tube and is thus adjustable, enabling one
to focus the pencil mark in the clear area of the focus-
ing screen (Fig. 174} with great accuracy. It also
serves to focus the image with ease and accuracy. The
eye must not be too close to the upper end ofthefocus-
ing glass or the field will be restricted. (Gundlach
Opt. Co.)

FIG. 173.

FIG. 174. Ground-glass focusing screen
with central transparent area for exact
focusing with a focusing glass when one
does not possess a clear focusing screen. (/)
The ground surface ; (2) Central part with
oblong cover-glass and Canada balsam on the
ground surface to render it transparent. X.
The central point in the entire focusing
screen. It is made with a black lead pencil
on the ground surface. The focusing glass
is focused on this cross, then when the image
is in focus it will be at the level of the sen-
sitive coating of the plate .

FIG. 175.

CH. VIII] PHOTO-MICROGRAPHY 211

FIG. 175. Vertical Camera and special microscope stand for photographing
embryos and other small specimens in liquids and for photographing large sections.
The camera rests on a low table and the operator can stand on the floor while per-
forming all the operations.

The stage of the microscope is attached to the arm in the place of the tube.
This stage has two stories. The specimen is shown on the upper and the back-
ground on the lower story.

In focusing, the coarse and fine adjustment of the special microscope stand are
used. The large mirror is to illuminate embryo chicks mounted entire, and other
large transparent preparations. Trans. Amer. Micr. 1901.

§ 340. Prints. — If the photographic prints are to be used solely for outlines,
the well-known blue prints so much used in engineering and architecture may be
made. If, however, light and shade and fine details are to be brought out with
great distinctness, either an aristotype, platinotype or a bromide print is preferable.

§ 341. Recording, Storing and Labeling Negatives. — In or-
der to get the greatest benefit from past experience it is necessary to
make the results available by means of a careful record. For this pur-
pose the table (§ 360) has been prepared. If one gives the information
called for in this table, whether the result is successful or not, one can
after a time work with great exactness, for the elements of success and
failure will stand out clearly in the table.

§ 342. Labeling the Negatives. — After the negative is dry the
labeling can be done on the gelatin side with carbon ink. Enough
data should be given to enable the certain identification of the negative
at any future time.

§ 343- Storing Negatives. — This is satisfactorily done by put-
ting each into an envelope and writing a duplicate label on the upper
edge, and then the negatives may be placed in drawers in alphabetical
order as are the catalog cards of books in a library. One can then find
any negative with the same facility that the title of a book can be
found in a card catalog.

PHOTOGRAPHING EMBRYOS

For photographing embryos and many other small specimens it is
more convenient to use a smaller apparatus than the vertical camera
just described. It is necessary also to have a more delicate method of
focusing.

§ 344. Camera for Embryos. — This is a vertical camera for
photographing with the microscope, with the photographic objective in
the end of the camera as for an ordinary camera. This is readily ac-
complished by having a society screw adapter, and also adapters for
the micro-planars or oiher objectives which one desires t6 use. The

212 PHOTO-MICROGRAPHY \_CH. VIII

magnification usually required varies from natural size ( X i ) to five
times natural size ( X 5) up to X 20. As with the large camera the
position of the ground glass for each magnification and for each objec-
tive is determined once for all by using a scale in millimeters. The
various positions are accurately noted, then one can set the camera
almost instantly for the desired magnification. The supporting rod is
divided to half centimeters and therefore the exact position is easily
recorded (Fig. 183).

FIG. 176. Zeiss Micro-Planar for photographing
with low magnification and for projection (see Ch. IX}.
These are made from 20 to wo mm. equivalent focus,
those of 20 and 35 mm. equivalent focus have the standard,
Royal Society Screw, the others have a larger screw in
order that the image may not be restricted. ( Cut loaned
by Bausch & Lomb Optical. Co.}

§ 345- Special Microscope Stand. — For the accurate focusing
necessary for embryos one should possess a special microscope
stand with the stage in two or three stories and attached to the arm in
place of the tube of the microscope. The stage proper is absent.
This arrangement of the stage permits the use of the coarse and fine
adjustment of the microscope to be used for focusing. The position of
the camera on a low table (45 to 50 cm. high; makes it possible for the
operator to stand on the floor while making all the adjustments of the
the embryo and for focusing ; and all the parts are within reach

(Fig. 175).

§ 346. Arranging the Embryos. — As usually prepared the em-
bryos are white and therefore require a dark background. This may
be attained either by placing the embryos in a dark dish or on some
paper blackened with water-proof India ink, or by putting them in a
glass vessel like a Petri dish, and a piece of black velveteen on the
stage below. The specimens will of course be in a liquid, usually alcohol.

FIG. 177. Wide angle anastigmat
rJ^__ objective for photographing at low mag-
' tL nification (Bausch & Lomb Optical Co.}.

If several embryos are to be
taken at once, the embryos are ar-
ranged in rows something as the
words on a line. Arrange them

in even vertical as well as horizontal rows so that when the print
is made it will be easy to cut them apart. When the embryos are ar-

CH. VIII} PHOTO-MICROGRAPHY 213

ranged, one should be certain that the light brings out the details most
desired. For example, if one is photographing an embryo which
shows the branchial pockets well, great pains should be taken to so
arrange the embryo with reference to the light that the proper shading
will be given to bring out the gill pockets most emphatically. One can
learn to do this only by practice. It is advantageous to have an assist-
ant, then while the operator is looking into the camera the assistant
can turn the embryo in various directions until the appearance is most
satisfactory.

§ 347- Focusing and Making the Exposure. — For getting a
general focus, and for the general arrangement the ground glass screen
is used, but for the final focusing it is desirable to use a clear glass
screen and a focusing glass. In this way one can focus as satisfactor-
ily as with an ordinary microscope. In daylight with white embryos
and a dark ground 30 to 40 seconds is usually sufficient exposure. One
must learn this also by trial and it facilitates the obtaining of exact data
to make a record of every negative made, whether the negative is good
or bad. A table is given in § 360 to facilitate the record taking. In a
short time one can learn to make the correct exposure. If the result
is unsatisfactory, try again. The rule adhered to by all first rate
workers is to to stick to it until the result is satisfactory.

§ 348. Records of Embryos. — Each specimen or litter of speci-
mens will have its own label giving date and method of preparation.
It is an advantage to write this label with water-proof carbon ink, then
one can put the label in the dish with the embryos and it will form a
part of the picture arid serve as a record.

After the picture is satisfactorily made it is wise to number the
embryos on the back of the negative with a wax crayon, and later
when the negative is dry number on the front with carbon ink. The
embryos are placed in separate bottles each with a copy of the original
label and the number corresponding with that put on the negative.
This is easily accomplished if the embryos are arranged in definite
rows as advised in § 346.

Finally when the embryo is cut into serial sections and mounted,
a picture of the whole embryo should accompany the series.

§ 349. Size of the Pictures. — For all embryos it is well to
make one picture natural size (x i) and then for the smallest ones a
magnification of at least five times natural size (x 5). Here, as with
the magnification of the microscope, linear magnification is always
meant (§ 154-155).

214

PHO TO-MICROGRAPH Y

\CH. VIII

§ 350. — Objectives. — For making pictures from one to five times
natural size objectives of 60 to 100 mm. focus answer well (Figs. 176—
1 80). Short focus (75 to 100 mm. equivalent focus), wide angle pho-
tographic objectives are also admirable for this work.

FIG. 178. FIG. 179.

FIG. 178. Leitz 64 millimeter ob- FIG. 179. Leitz 42 millimeter ob-

jective for photography and for projec- jective f or photography and for projection
tion ( Wm. Krafft, N. Y. ) ( Wm. Krafft, N. Y.)

FIG. 1 80. Zeiss' Apochromatic Projection Objective of 70
mm. equivalent focus, for photo-micrography. (Zeiss' Catalog.)
This, and another of 35 mm. focus, are designed for making
pictures of moderate magnification. Usually rather large ob-
jects are photographed with them. The object may be illumina-
ted in the ordinary way. They are used without an ocular,
like a photographic objective. The one of jj mm. is screwed
into the tube of the microscope like an ordinary objective, but the
one of 70 mm. here shown, is, by means of a conical adapter,
screwed into the ocular end of the tube, Fig. 189.

For illuminating the object, any suitable light may be used,
but it is recommended that the light be concentrated by means
of a buWs eye or some form of combination like the engraving
glass, and that the condenser be so placed that it focuses the light upon the objective,
not upon the object. The object is then illuminated with a converging cone of light.

§ 351. Record of Negatives. — As indicated in § 341-343 each
negative should have a record, see record blank on p. 219. On the
negative itself should be also written the main facts with carbon ink.
The name and magnification, date and any other details which may be
thought desirable can be put on the envelope containing the negative,
and then the negative stored like a catalog card as described above
(§ 343)-

PHOTOGRAPHING LARGE TRANSPARENT OBJECTS

§ 352. There are many large transparent objects which it is de-
sirable to photograph, e. g., chick embryos mounted whole, large sec-
tions of organs like the brain, etc. These must be photographed at a
low magnification.

CH. VIII}

PHO TO-MICROGRAPH Y

21

FIG. 181.

FIG. 181. Camera and special microscope stand for photographing very large
transparent sections. For this the vertical camera is used (fig. /<5p) with the
camera reversed on the sliding frame . This frame is elevated sufficiently to utilize
the sky as background and illuminant. The special microscope stand is inclined to
the horizontal and placed on the fixed frame supporting the camera ; the specimen

216 PHOTO-MICROGRAPHY [C//. VIII

placed on the stage. For objective one of the objectives shown in Figs. 176 to 180 is
used. The objective is screwed into an adapter in place of the ordinary photographic
objective. The focusing is performed roughly by the rack and pinion, and then
with great exactness with the focusing glass. For manipulating the fine adjust-
ment of the special microscope the well known device of a cord over the head of the
micrometer screw is used. (See also Fig. 775.) ( Trans. Amer. Micr. Soc.} igoi. )

Successful photographs require an even lighting and an objective
which has sufficient field to take in the whole object. The camera
used for embryos (Fig. 175) answers very well for objects of moderate
size. For lighting them the specimen is put on the upper stage, and the
back-ground shown in the figure is removed. Then the large mirror
is used to throw light up through the preparation. If necessary the
specimen can be placed on one of the lower stories to bring it nearer
the mirror. The lighting and focusing should be as perfect as possi-
ble. Lamplight and daylight are both good.

§ 353. Photographing Large Transparent Objects. — For this
the large vertical camera (Fig. 169, 181) is reversed in position on the
supporting frames, and elevated only sufficiently to make a sky back-
ground ; or a 45 degree reflector of white cloth or paper of sufficient
size must be used for a horizontal camera. If one has the earth for
back-ground the light will be dull and uneven and a very long expos-
ure is necessary, and the final results unsatisfactory.

§ 354- Use of the Special Microscope Stand. — In order to
hold the specimen in position and to focus it accurately, it is put on
the stage of the special microscope stand (Fig. 175), which is inclined,
and fastened to the fixed part of the frame supporting the camera. As
the stage of this microscope is moved by the coarse or the fine adjust-
ment, the focusing can be accomplished with the same accuracy as the
microscope itself. For the general arrangement of the specimen and
the rough focusing the ground glass is used, then this is replaced by a
clear-glass focusing screen, and by the aid of a focusing glass the speci-
men is put in perfect focus. As one cannot reach the fine adjustment
while focusing, the well known device of a cord over the head of the
micrometer screw is resorted to. The two ends of the cord should be
weighted with about 50 or a hundred grams to keep the cord taut, then
whichever one is pulled, the micrometer screw will respond at once.
To cut off the light a piece of black velveteen is hung over the end of
the objective. This can be removed without jarring the apparatus.
An exposure of a few seconds (3 to 10 seconds), will suffice for many
preparations, unless a color screen is used. The color screen increases
the time of exposure from three to five times (§ 359).

CH. VIII}

PHO TO-MICROGRAPHY

217

FIG. 182. BuWs eye lens and holder. (Bausch & Lomb Opt. Co.

COLOR-CORRECT PHOTOGRAPHY

From the fact that the different wave lengths of light affect the photographic
plate with different degrees of vigor, the ordinary photographic print of a many
colored object or landscape is not satisfactory. All objects whose light is of short
wave lengths, as blue, etc., will appear too light and those which are red, yellow
and green will be too dark relatively. To obviate this difficulty two methods have
been adopted, and for the most complete success they must be combined.

(A) The use of ortho- or iso- chromatic plates and (B) the use of a color screen
or light filter.

§ 355. Orthochromatic or Isochromatic Plates. — These are plates which have
been rendered much more sensitive than ordinary plates to the long waves of red,
orange, yellow and green, they therefore give a much more natural rendering to
many-colored objects than ordinary plates. As they are sensitive to red, orange,
etc. , one must be very careful in exposing them in the dark room even to the light of
the developing lantern. The more nearly the plate can be kept from all light,
except that acting during the exposure in the camera, the more satisfactory will
be the resulting negative.

These color-correct plates are not very enduring, and must be used while they
are fresh, or only weak, foggy negatives will result.

218 PHOTO-MICROGRAPHY \_CH. VIII

For photographing transparent or translucent objects there is a further diffi-
culty introduced, viz, greater or less transparency. Assuming that all the rays of
the spectrum are equally active, there would still be difficulty because the blue
and violet stains used in microscopy are liable to be more transparent than those
stained with red and orange, consequently a blue stained preparation is liable to
lack in contrast since the light reaching the plate from the object and from the
space around it produce so nearly equal effects on the plate.

On the other hand a red stain gives too much contrast because the light pass-
ing through it has little effect as compared with the light going through the space
surrounding the object. So far in the discussion it is assumed that the objects
are practically transparent and that pure red and blue are used. As a matter of
fact the stain renders the specimen somewhat more opaque so that the specimen
would be darker than the background in either case. This is especially true of
hematoxylin. While hematoxylin is blue or purple, it renders the tissues more or
less opaque, so that with a petroleum lamp and isochromatic plates, freshly stained
hematoxylin specimens are very easy to get good pictures of.

1 356. Color Screens. — As a general statement all color screens which have
proved really useful in photographing transparent or translucent microscopic
specimens cut off most of the blue end of1 the spectrum. Others cut off also the
red end, leaving only the middle, visually brightest part of the spectrum free. As
modern achromatic objectives are corrected for the visual rays, a screen cutting
off the blue end of the spectrum serves to obviate any lack of sharpness due to
the aberration of the blue rays in such objectives. There is the further advan-
tage that with red and yellow objects, the color being in general of the wave
length transmitted by the screen would be in true relative shade or contrast be-
tween background and object, and give good detail in the object. For the blue
object this form of screen is also good for, as it cuts off the blue rays, the effect
will be like photographing a gray object where the light and shade depend on the
transparency of different parts. Thfc denser the part the more opaque it is and
therefore the darker it appears with transmitted light. The background allowing
all the colored light to pass is lighter than the blue object and therefore there will
be good contrast and also good detail in the object.

\ 357 Composition of Color Screens. — The most successful color screens are
solutions held in parallel sided glass vessels. Colored glass, gelatin, collodion,
etc., colored with different chemicals are fairly satisfactory, but not so satisfac-
tory as the solutions.

(1) The most generally used and most generally useful screen is a watery
solution of dichromate of potash (K2Cr2O7). This cuts off the violet, the blue
and the bluish green. The amount of light cut out depends upon the density of the
solution and the thickness of the stratum through which the light passes.

(2) Zettnow' s cupro-chr ornate color screen allows wave lengths from 0.570^
to 0.550/1 to pass, that is yellow light. It is made by dissolving in 250 cc. of water,
ii grams of pure dry cupric nitrate and i gram of chromic acid. The thickness of
the stratum of liquid should be about i centimeter.

(3) Gifford's color screen. This is highly spoken of both for observation
with the microscope (Carpenter-Dallinger) and for use in photography (J. R. M.
S. 1894, p. 164). It is composed of a strong solution of malachit green in water,
glycerin, glycerin jelly, etc. Only a thin stratum such as could be mounted be-

HI

o
• «!

a 1 1

_ X .

f I

511

5-0 0

M

220 PHOTO-MICROGRAPHY \CH. VIII

tween two cover-glasses is needed. By combining a little picric acid with the
solution or by the use of a thin piece of signal green glass only light between the
fixed lines E and F, is allowed to pass. This is not therefore so generally useful
as dichromate.

(4) Bothamley's aurantia color screen is a saturated alcoholic solution of the
aurantia added to collodion of 3 to 4%. The collodion is poured on a large cover-
glass or a glass plate and allowed to dry. Pringle advises several screens of aurantia
of different shades. That is easily managed by adding a greater or less amount of
the solution to the collodion. This is a good screen and easily used.

Petroleum light serves as a yellow color screen, and one can often get excel-
lent results with such a light when daylight or the electric light without a color
screen does not give a good picture. For all photography with the microscope
isochromatic or orthochromatic plates are advised. For many objects no color
screen is needed if one uses a petroleum lamp.

$ 358. Position of the Screen. — It does not make much difference where the
color screen is placed provided no light reaches the object which has not passed
through the screen.

\ 359. Exposure with a Color Screen. — The interposition of a color screen
increases the time of exposure from three to five times. One can learn the time
and whether or not to use a color screen, and the kind of a screen to use only by
experiment. To get the full benefit of these experiments for future work, every
negative should be carefully recorded (\ 360, table). It would also aid one materi-
ally, in the beginning at least, if he were to study the color screen used with the
micro-spectroscope and determine the wave lengths which are allowed to pass
through it (\ 195, 202). If this study were supplemented by a spectroscopic ex-
amination of the object to be photographed, one would learn to choose with great
accuracy the color screen which would give the best results.

PHOTOGRAPHING WITH A MICROSCOPE*

$ 361. The first pictures jnade on white paper and white leather, sensitized
by silver nitrate, were made by the aid of a solar microscope ( 1802). The pictures

^Considerable confusion exists as to the proper nomenclature of photography
with the microscope. In German and French the term micro-photography is very
common, while in English photo-micrography and micro-photography mean dif-
ferent things. Thus : A photo-micrograph is a photograph of a small or microscopic
object usually made with a microscope and of sufficient size for observation with
the unaided eye ; while a micro-photograph is a small or microscopic photograph
of an object, usually a large object, like a man or woman and is designed to be
looked at with a microscope.

Dr. A. C. Mercer, in an article in the Proc. Amer. Micr. Soc., 1886, p. 131, says
that Mr. George Shadbolt made this distinction.. See the Liverpool and Manches-
ter Photographic Journal (now British Journal of Photography} , Aug. 15, 1858, p.
203 ; also Button's Photographic Notes, Vol. Ill, 1858, pp. 205-208. On p. 208 of
the last, Shadbolt's word "Photomicrography" appears. Dr. Mercer puts the
case very neatly as follows : "A photo-micrograph is a macroscopic photograph of
a microscopic object ; a micro-photograph is a microscopic photograph of a macro-
scopic object. See also Medical News, Jan. 27, 1894, p. 108.

CH. VIII} PHOTO-MICROGRAPHY 221

were made by Wedgewood and Davy, and Davy says : "I have found that images
of small objects produced by means of the solar microscope may be copied without
difficulty on prepared paper, "t

Thus among the very first of the experiments in photography the microscope
was called into requisition. And naturally, plants and motionless objects were
photographed in the beginnings of the art when the time of exposure required
was very great.

At the present time photography is used to an almost inconceivable degree in
all the arts and sciences and in pure art. Even astronomy finds it of the greatest
assistance.

It has also accomplished marvels in the production of colored plates for book
illustrations, especially in natural history. For an example see Comstock's Insect
Life, 2d edition.

Although first in the field, Photo-Micrography has been least successful of
the branches of photography. This is due to several causes. In the first place,
microscopic objectives have been naturally constructed to give the clearest image
to the eye, that is the visual image as it is sometimes called, is for microscopic ob-
servation, of prime importance. The actinic or photographic image, on the other
hand, is of prime importance for photography. For the majority of microscopic
objects transmitted light ( £ 64) must be used, not reflected light as in ordinary vis-
ion. Finally, from the shortness of focus and the smallness of the lenses, the
proper illumination of the object is accomplished with some difficulty, and the
fact of the lack of sharpness over the whole field with any but the lower powers,
have combined to make photo-micrography less successful than ordinary macro-
photography. So tireless, however, have been the efforts of those who believed in
the ultimate success of photo-micrography, that no%v the ordinary achromatic ob-
jectives with ortho-chromatic or isochromatic plates and a color screen or petrol-
eum light give good results, while the apochromatic objectives with projection
oculars give excellent results, even in hands not especially skilled. The problem
of illumination has also been solved by the construction of achromatic and apoch-
romatic condensers and by the electric and other powerful lights now available.
There still remains the difficulty of transmitted light and of so preparing the
object that structural details stand out with sufficient clearness to make a picture
which approaches in definiteness the drawing of a skilled artist.

The writer would advise all who wish to undertake photo-micrography seri-
ously, to study samples of the best work that has been produced. Among those
who showed th,e possibilities of photo-micrographs was Col. Woodward of the U.

fin a most interesting paper by A. C. Mercer on "The Indebtedness of Pho-
tography to Microscopy," Photographic Times Almanac, 1887, it is shown that : "To
briefly recapitulate, photography is apparently somewhat indebted to microscopy
for the first fleeting pictures of Wedgewood and Davy [1802], the first methods of
producing permanent paper prints [Reede, 1837-1839], the first offering of prints
for sale, the first plates, engraved after photographs for the purpose of book illus-
tration [Donne & Foucalt, 1845], the photographic use of collodion [Archer & Dia-
mond, 1851], and finally, wholly, indebted for the origin of the gelatino-bromide
process, greatest achievement of them all [Dr. R. L. Maddox, 1871]. See further
for the history of Photo-micrography, Neuhauss, also Bousfield.

222

PHO TO-M1CROGRAPHY

[CM. VIII

S. Army Medical Museum. The photo-micrographs made by him and exhibited
at the Centennial Celebration at Philadelphia in 1876, serve still as models, and no
one could do better than to study them and try to equal them in clearness and
general excellence. According to the writer's observation no photo-micrographs
of histological objects have ever exceeded those made by Woodward, and most of
them are vastly inferior. It is gratifying to state, however, that at the present
time many original papers are partly or wholly illustrated by photo-micrographs,
and no country has produced works with photo-micrographic illustrations superior
to those in "Wilson's Atlas of Fertilization and Karyokinesis" and "Starr's Atlas
of Nerve Cells," issued by the Columbia University Press. —

In passing the writer would like to pay a tribute to Mr. W. H. Walmsley who
has labored in advancing photo-micrography for the last twenty years. His con-
venient apparatus and abundant experience have been placed freely at the com-
mand of every interested worker, ;and many a beginner has been helped over
difficulties by him. His last contribution in "International Clinics," vol. i. ser.
u, 12, is encouraging in the highest degree both for its matter and for the
illustrations.

FIG. 183. Zeiss' Vertical Photo-micro-
graphic Camera. A. Set screw holding the
rod (S ) in any desired position. P, Q. Set
screws, by which the bellows are held in place.
B. Stand with tripod base in which the sup-
porting rod (S) is held. This rod is now
graduated in centimeters and is a ready
means of determining the length of the cam-
era. M. Mirror of the microscope. L. The
sleeve serving to make a light-tight connection
between the camera and microscope. O. The
lower end of the camera. R. The upper end
of the camera where the focusing screen and
plate holder are situated. (From Zeiss"1 Photo-
m icrograph ic Ca ta log ) .

As the difficulties of photo-micrography are so much greater than of ordinary
photography, the advice is almost universal that no one should try to learn photo-
graphy andtphoto-micrography at the same time, but that one should learn the

CH. K//7] PHOTO-MICROGRAPHY 223

processes of photography by making portraits, landscapes, copying drawings, etc.,
and then when the principles are learned one can take up the more difficult subject
of photo-micrography with some hope of success.

The advice of Sternberg is so pertinent and judicious that it is reproduced :
"Those who have had no experience in making photo-micrographs are apt to ex-
pect too much and to underestimate the technical difficulties. Objects which
under the microscope give a beautiful picture, which we desire to reproduce by
photography may be entirely unsuited for the purpose. In photographing with
high powers it is necessary that the objects to be photographed be in a single plane
and not crowded together and overlying each other. For this reason photograph-
ing bacteria in sections presents special difficulties and satisfactory results can only
be obtained when the sections are extremely thin and the bacteria well stained.
Even with the best preparations of this kind much care must be taken in selecting
a field for photography. It must be remembered that the expert microscopist, in
examining a section with high powers, has his finger on the fine adjustment screw
and focuses up and down to bring different planes into view. He is in the habit of
fixing his attention on the part of the field which is in focus and discarding the
rest. But in a photograph the part of the field not in focus appears in a promi-
nent way which mars the beauty of the picture. ' '

APPARATUS FOR PHOTO-MICROGRAPHY

| 362. Camera. — For the best results with the least expenditure of time one of
the cameras especially designed for photo-micrography is desirable but is not by
any means indispensable for doing good work. An ordinary photographic camera,
especially the kind known as a copying camera, will enable one to get good results,
but the trouble is increased, and the difficulties are so great at best, that one would
do well to avoid as many as possible and have as good an outfit as can be afforded
(Figs. 184, 192).

The first thing to do is to test the camera for the coincidence of the plane occu-
pied by the sensitive plate and the ground glass or focusing screen. Cameras even
from the best makers are not always correctly adjusted. By using a straight edge
of some kind, one can measure the distance from the inside or ground side of the
focusing screen to the surface of the frame. This should be done all around to see
if the focusing screen is equally distant at all points from the surface of the
frame. If it is not it should be made so. When the focusing screen has been ex-
amined, an old plate, but one that is perfectly flat, should be put into the plate
holder and the slide pulled out and the distance from the surface of the plate
holder determined exactly as for the focusing screen. If the distance is not the
same the position of the focusing screen must be changed to correspond with that
of the glass in the plate holder, for unless the sensitive surface occupies exactly
the position of the focusing screen the picture will not be sharp, no matter how7
accurately one may focus. Indeed, so necessary is the coincidence of the plane of
the focusing screen and sensitive surface that some photo-micrographers put the
focusing screen in the plate holder, focus the image and then put the sensitive
plate in the holder and make the exposure (Cox). This would be possible with
the older forms of plate holders, but not with the double plate holders mostly used
at the present day.

224 PHOTO-MICROGRAPHY \CH. VIII

\ 3623. Size of Camera. — The majority of photo-micrographs do not exceed 8
centimeters in diameter and are made on plates Sx u, lox 13 or 13 x 18 centimeters
(3^x4^ in., 4x5 in., or 5x7 in.). Most of the vertical cameras are for plates not
exceeding lox 13 centimeters (4x5 in. ) but Zeiss' new form will take plates 21 x 21
centimeters (8^x8%" in.).

I 363. Work Room. — It is almost self-evident that the camera must be in
some place free from vibration. Frequently a basement room where the camera
table may rest directly on the cement floor or on a pier is an excellent situation.
Such a place is almost necessary for the best work with high powers. For those
living in cities, a time must also be chosen when there are no heavy vehicles
moving in the streets. For less difficult work an ordinary room in a quiet part of
the house or laboratory building will suffice.

| 364. Arrangement and Position of the Camera and the Microscope. — For
much of photo-micrography a vertical camera and microscope are to be preferred
(Fig. 184). Excellent arrangements were perfected long ago, especially by the
French. (See Moitessier. )

Vertical photo-micrographic cam-eras are now commonly made, and by some
firms only vertical cameras are produced. They are exceedingly convenient, and
do not require so great a disarrangement of the microscope to make the picture as
do the horizontal ones. Van Heurck advises their use, then whenever a structure
is shown with especial excellence it is photographed immediately. The variation
in size of the picture is obtained by the objective and the projection ocular rather
than by length of bellows (see below Fig. 184). It must not be forgotten, how-
ever, that penetration varies inversely as the square of the power, and only in-
versely as the numerical aperture (§ 34), consequently there is a real advantage in
using a low power of great aperture and a long bellows rather than an objective of
higher power with a short bellows. A horizontal camera is more convenient for use
with the electric light also (Fig. 192).

For convenience and rapidity of work a microscope with mechanical stage is
very desirable. It is also an advantage to have a tube of large diameter so that
the field will not be too greatly restricted (Fig. 189). In some microscopes the
tube is removable almost to the nose-piece to avoid interfering with the size of the
image. The substage condenser should be movable on a rack and pinion. The
microscope should have a flexible pillar for work in a horizontal position. While it
is desirable in all cases to have the best and most convenient apparatus that is
made, it is not by any means necessary for the production of excellent work. A
simple stand with flexible pillar and good fine adjustment will answer.

$ 365. Objectives and Oculars for Photo-Micrography. — The belief is almost
universal that the apochromatic objectives are most satisfactory for photography.
They are employed for this purpose with a special projection ocular. Two very
low powers are used without any ocular (Fig. 180). Some of the best work
that has ever been done, however, was done with achromatic objectives (work of
Woodward and others). One need not desist from undertaking photo-micrography
if he has good achromatic objectives. From a somewhat extended series of ex-
periments with the objectives of many makers the good modern achromatic ob-
jectives were found to give excellent results when used without an ocular. Most
of them also gave good results with projection oculars, although it must be said
that the best results were obtained with the apochromatic objectives and projec-

FIG. 184. — Vertical photo-
micrographic camera, screen and
small table. The table is about
centimeters high and in the
legs are large screw eyes for
leveling screws. The operator
can stand on the floor and per-
form all the necessary opera-
tions, and in adjusting the mi-
croscope can sit on a low stool.
The screen is of zinc and
has two heavy lead feet to hold
it steady. Near the lower left
hand corner of the screen is an
aperture for the light to shine
through upon the mirror. This
opening is closed by a black slide
which is just balanced so that it
stays in any position. In mak-
ing the exposure it is raised
sufficiently to admit the light to
the mirror, but the stage is left
in shadow. This screen shades
the microscope and the face of
the operator. ( Trans.
Micr. Soc. 1901.

226

PHO TO-MICROGRAPHY

[ CH. VIII

tion oculars. It does not seem to require so much skill to get good results with
the apochromatics as with the achromatic objectives. The majority of photo-
micrographers do not use the Huygenian oculars in photography, although excel-
lent results have been obtained with them. An amplifier is sometimes used in
place of an ocular. Considerable experience is necessary in getting the proper
mutual position of objective and amplifier. The introduction of oculars
especially designed for projection, has led to the discarding of ordinary oculars
and of amplifiers. However the projection oculars of Zeiss restrict the field very
greatly, hence the necessity of using the objective alone for large specimens.*

No. a.

FIG. 185. Projection Oculars with section
removed to show the construction. Below are
shown the upper end with graduated circle to
indicate the amount of rotation found necessary
to focus the diaphragm on the screen. No. 2,
No. 4. The numbers indicate the amount the
ocular magnifies the image formed by the ob-
jective as with the compensation oculars. (Zeiss'
Catalog. )

$ 366. Difference of Visual and Actinic
Foci. — Formerly there was much difficulty ex-
perienced in photo-micrographing on account
of the difference in actinic and visual foci.
Modern objectives are less faulty in this respect
and the apochromatics are practically free
from it. Since the introduction of orthochromatic or isochromatic plates and, in
many cases the use of colored screens, but little trouble has arisen from differences
in the foci. This is especially true when mono-chromatic light and even when
petroleum light is used. In case the two foci are so unlike in an objective, it
would be better to discard it for photography altogether, for the estimation of the
proper position of the sensitive plate after focusing is only guess work and the
result is mere chance. If sharp pictures cannot be obtained with an objective
when petroleum light and orthochromatic plates are used the fault may not rest
with the objective but with the plate holder and focusing screen. They should be
very carefully tested to see if there is coincidence in position of the focusing
screen and the sensitive film as described in \ 362.

§367. Apparatus for Lighting.— For low power work (35 mm. and longer
focus) and for large objects, some form of bull's eye condenser is desirable
although fairly good work may be done with diffused light or lamp-light reflected
by a mirror. If a bull's eye is used it should be as nearly achromatic as possible.
The engraving glass shown in Fig. 188 answers well for large objects. For smaller

*A comparative study both with projection oculars, and without an ocular
was made with the achromatic objective 25 mm. (i inch), 18 mm. (finch), 5
mm. (\ to \ inch) and 2 mm. (TV inch) homogeneous immersion of the Bausch &
Lomb Optical Co.; Gundlach Optical Co.; Leitz ; Reichert ; Winkel, Zeiss and the
Spencer Lens Co. Good results were obtained with all of these objectives both
with and without projection oculars.

CH. VIII]

PHO TO-MICROGRAPH Y

227

objects a Steinheil lens combination gives a more brilliant light and one also more
nearly achromatic. For high power work all are agreed that nothing will take the
place of an achromatic condenser. This may be simply an achromatic condenser,
but preferably it should be an apochromatic condenser. Whatever the form of the
condenser it should possess diaphragms so that the aperture of the condenser may
be varied depending upon the aperture of the objective. For a long time objec-
tives have been used as achromatic condensers, and they are very satisfactory,
although less convenient than a special condenser whose aperture is great enough
for the highest powers and capable of being reduced by means of diaphragms to the
capacity of the lower objectives. It should also be capable of accurate centering.

FIG. 186. Arrangement for Artificial Illumination.

1. Lamp with metal chimney, easily made by rolling up some ferrotype plate
and making a slit-like opening in one side. This opening should be covered by an
oblong cover-glass. A glass slide, being of considerable thickness, breaks too easily.
The lamp should have a wick about 40 mm. wide, so that the thickness of the flame,
if taken edgewise, will give an intense light. A wide flame also enables one to get
a larger image of the flame, and thus to illuminate a larger object than as though a
small flame was used.

2. BuWs-eye condenser on a separate stand. The engraving glass shown in
Fig. 188, or the tripod magnifier (Fig. 172} answers fairly . The Steinheil lenses
are still better.

j. Screen showing image of the flame inverted.
The lamp and bull's-eye stand are on blocks with screw-eyes as leveling screws.

$ 368. Objects Suitable for Photo-micrographs. — While almost any large ob-
ject may be photographed well with the ordinary camera and photographic objec-
tive, only a small part of the objects mounted for microscopic study can be photo-
micrographed satisfactorily. Many objects that give beautiful images when look-
ing into the microscope and constantly focusing with the fine adjustment, appear
almost without detail on the screen of the photo-micrographic camera and in the
photo-micrograph.

228

PHO TO-MICROGRAPHY

[ CH. V11I

FIG. 187. Adjustable lens holder. This lens holder will take magnifiers of
various sizes, and from its adjustable mechanism is very convenient for dissecting,
or for holding a Steinheil and other lenses for illumination ( The Bausch & Lomb
Opt. Co.).

FIG. 1 88. Engraving glass to
serve as a condenser and for a dis-
secting lens. {Bausch & Lomb Opt.
Co.]

If one examines a series of photo-micrographs the chances are that the greater
number will be of diatoms, plant sections or preparations of insects. That is, they
are of objects having sharp details and definite outlines, so that contrast and defi-
niteness may be readily obtained. Stained microbes also furnish favorable objects
when mounted as cover-glass preparations.

CH. VIII} PHOTO-MICROGRAPHY 229

Preparations in animal histology must approximate as nearly as possible to the
conditions more easily obtained with vegetable preparations. That is, they must
be made so thin and be so prepared that the cell outlines will have something of
the definiteness of vegetable tissue. It is useless to expect to get a clear photo-
graph of a section in which the details are seen with difficulty when studying it
under the microscope in the ordinary way.

Many sections which are unsatisfactory as wholes, may nevertheless have parts
in which the structural details show with satisfactory clearness. In such a case the
part of the section showing details satisfactorily should be surrounded by a delicate
ring by means of a marker (see Figs. 61-66). If one's preparations have been
carefully studied and the special points in them thus indicated, they will be found
far more valuable both for ordinary demonstration and for photography. The
amount of time saved by marking one's specimens can hardly be overestimated.
The most satisfactory material for making the rings is shellac colored with lamp-
black.

Ten years ago many histologic preparations could not be satisfactorily photo-
graphed. But now with improved section cutters, better staining and mounting
methods, and with the color screens (\ 356) and isochromatic plates (§355) almost
any preparation which shows the' elements clearly when looking into the micro-
scope can be satisfactorily photographed. Good photographs cannot, however, be
obtained from poor preparations.

\ 369. Light. — The strongest available light is sunlight. That has the defect of
not always being available, and of differing greatly in intensity from hour to hour,
day to day and season to season. The sun does not shine in the evening when
many workers find the only opportunity for work. Following the sunlight the
electric light is the most intense of the available lights. Then come magnesium,
acetylene, the lime light, the gas-glow or Wellsbach light, and lastly, petroleum
light. The last is excellent for the majority of low and moderate power work.
And even for 2 mm. homogeneous immersion objectives, the time of exposure is
not excessive for many specimens (40 seconds to 3 minutes). This light is also
cheapest and most available and has the advantage of being somewhat yellow, and
therefore in many cases makes the use of a color screen unnecessary if one uses
isochromatic plates. Acetylene light is excellent and may be used where the arc
light is not available.

A lamp with flat wick about 40 mm. (i^ in.) wide has been found most gen-
erally serviceable. For large objects and low powers the flame may be made
large and the face turned toward the mirror. This will light a large field. For
high powers the edge toward the mirror gives an intense light. The ordinary
glass chimney answers well, especially where a metal screen is used as shown
in Fig. 184.

EXPERIMENTS IN PHOTO-MICROGRAPHY

§ 370. The following experiments are introduced to show prac-
tically just how one would proceed to make photo-micrographs with
various powers, and be reasonably certain of fair success. If one con-
sults prints or the published figures made directly from photo-micro-
graphs it will be seen that, excepting the bacteria, the magnification
ranges mostly between 10 and 150 diameters.

230 PHOTO-MICROGRAPHY [CH. VIII

§ 371. Focusing Screen for Photo-Micrography. — One cannot
expect a picture sharper than the image seen on the focusing screen.
Hence the greatest care must be taken in focusing. The general focus-
ing may be done with the unaided eye on the ground glass, but for the
final focusing a clear screen and a focusing glass must be used. (Figs.
172, 173). See § 347. With the clear focusing screen one cannot at
first see the image without using a focusing glass, but with a little ex-
perience the aerial image may be seen as with the microscope (§ 54).

§ 372. Photo-micrographs of 20 to 50 Diameters. — For pic-
tures under 15 or 20 diameters it is better to use the camera for embryos
with the objective in the end of the camera, and the special microscope
stand for focusing (Fig. 175).

For pictures at 25 to 50 diameters one may use the microscope with
a low objective, 25 to 35 mm. equivalent focus, and no ocular (Fig.
184). The object is placed on the stage of the microscope, and focused
as in ordinary observation. If a vertical microscope is used the light
from the petroleum lamp or other artificial light, is reflected upward by
the mirror. It may take some time to get the whole field lighted
evenly. Refer back to § 95 for directions. In some cases it may be
advisable to discard the condenser and use the mirror only. For some
purposes one will get a better light by placing the bull's eye or other
condenser between the lamp and the mirror to make the rays parallel
or even to make a sharp image of the lamp flame on the mirror. Re-
member also that in many cases it is necessary to have a color screen
between the source of light and the object (§ 356).

For a horizontal camera it is frequently better to swing the mirror
entirely out of the way and allow the light to enter the condenser
directly or after traversing the bull's eye (Figs. 182, 186). If the ob-
ject is small an achromatic combination like a Steinheil magnifier or an
engraving glass is excellent (Fig. 188). When the light is satisfac-
tory as seen through an ordinary ocular, remove the ocular.

(A) Photographing without an Ocular. — After the removal of the
ocular put in the end of the tube a lining of black velvet to avoid re-
flections. Connect the microscope with the camera, making a light-
tight joint and focus the image on the focusing screen. One may make
a light-tight connection by the use of black velveteen or more con-
veniently by the Zeiss' double metal hood which slips over the end of
the tube of the microscope, and into which fits a metal cylinder on the
lower end of the camera (Figs. 184, 189, 183). In the last figure the
connection has been made.

CH. VIII'}

PHO TO-MICROGRAPHY

231

FIG. 189. Zeiss1 special photo-micrographic stand. It has a very large tube,
a slow acting fine adjustment, mechanical stage and all appliances for the most sat-
isfactory work. ( Cut loaned by Eimer and Amend}.

It will be necessary to focus down considerably to make the image
clear. Lengthen or shorten the bellows to make the image of the de-
sired size, then focus with the utmost care. In case the field is too
much restricted on account of the tube of the microscope, remove the
draw-tube. When all is in readiness it is well to wait for three to five
minutes and then to see if the image is still sharply focused. If it has
got out of focus simply by standing, a sharp picture could not be ob-

232 PHOTO-MICROGRAPHY \_CH. VIII

FIGS. 190-191. Fine tint, half-tone reproductions of photo-micrographs of sec-
tions made by Mrs. Gage, to show the possibilities of photo-micrography with pho-
tographic objectives and with low microscopic objectives without a projection ocular.

1. Frontal section of the head of a large red Diemyctylus viridescens (red
newt] at the level of the portae of the brain, magnified 10 diameters. Negative
made with a Gundlach perigraphic objective of about oo mm. equivalent focus.

2. Frontal section of a larval Diemyctylus about 10 millimeters in length.
Negative made with a Winkel objective of 22 millimeters equivalent focus ; no
ocular. Magnified 50 diameters. (Mrs. Susanna Phelps Gage, the Wilder
Quarter Century Book}.

tained. If it does not remain in focus, something is faulty. When
the image remains sharp after focusing make the exposure. From 20
to 60 seconds will usually be sufficient time with medium plates and
the light as described. If a color screen is used it will require 40-300
seconds, i. e., 2 to 5 times as long, for a proper exposure (§ 359).

B. Photographing with a Projection Ocular. — If the object is small
enough to be included in the field of a projection ocular (Fig. 185) use
that for making the negative as follows : Swing the camera around so
that it will leave the microscope free. Use an ordinary ocular, focus
and light the object, then insert a projection ocular in place of the or-
dinary one, and swing the camera back over the microscope. It is not
necessary to use an ordinary ocular for the first focusing, but as its
field is larger it is easier to find the part to be photographed. The
first step is then to focus the diaphragm of the projection ocular
sharply on the focusing screen. Bring the camera up close to the mi-
croscope and then screw out the eye-lens of the ocular a short distance.
Observe the circle of light on the focusing screen to see if its edges are
perfectly sharp. If not, continue to screw out the eye lens until it is.
If it cannot be made sharp by screwing it out reverse the operation.
Unless the edge of the light circle, i. e. , the diaphragm of the ocular,
is sharp, the resulting picture will not be satisfactory.

It should be stated that for the X 2 projection ocular the bellows
of the camera must be extended about 30 or 40 centimeters or the
diaphragm cannot be satisfactorily focused on the screen. The X4
projection ocular can be focused with the bellows much shorter. For
either projection ocular the screen distance can be extended almost
indefinitely.

When the diaphragm is sharply focused on the screen, the micro-
scope is focused as though no ocular were present, that is, first with the
unaided eye then with the focusing glass. The exposure is also made
in the same way, although one must have regard to the greater mag-

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CH. VIII} PHOTO-MICROGRAPHY 233

nification produced by the projection ocular and increase the time ac-
cordingly ; thus when the X 4 ocular is used, the time should be at
least doubled over that when no ocular is employed. The time will be
still further increased if a color screen is used (§ 359).

Zeiss recommends that when the bellows have sufficient length the
lower projection oculars be used, but with a short bellows the higher
ones. It is also sometimes desirable to limit the size of the field by
putting a smaller diaphragm over the eye lens. This also aids in
making the field uniformly sharp.

§373. Determination of the Magnification of the Photo-
Micrograph. — After a successful negative has been made, it is desirable
and important to know the magnification. This is easily determined
by removing the object and putting in its place a stage micrometer.
If the distance between two or more of the lines of the image on
the focusing screen is obtained with dividers and the distance meas-
ured on one of the steel rules, the magnification is found by dividing
the size of the image by the known size of the object (§ 154). If now
the length of the bellows from the tube of the microscope is noted, say
on a record table like that in section 360, one can get a close approxi-
mation to the power at some other time by using the same optical com-
bination and length of bellows.

For obtaining the magnification at which negatives are made it is
a great advantage to have one micrometer in half millimeters ruled
with coarse lines for use with the lower powers, and one in o. i and
o.oi millimeter ruled with fine lines for the higher powers.

§ 374. Photo-Micrographs at a Magnification of 100 to 150
Diameters. — For this, the simple arrangements given in the preceding
section will answer, but the objectives must be of shorter focus, 8 to 3
mm. It is better, however, to use an achromatic condenser instead of
the engraving glass or the Steinheil lens.

§ 375. Lighting for Photo-Micrography with Moderate and
High Powers. — (100 to 2,500 diameters). No matter how good one's
apparatus, successful photo-micrographs cannot be made unless the ob-
ject to be photographed is properly illuminated. The beginner can do
nothing better than to go over with the greatest care the directions for
centering the condenser, for centering the source of illumination, and
the discussion of the proper cone of light and lighting the whole field,
as given on pp. 41-52. Then for each picture the photographer must
take the necessary pains to light the object properly. An achromatic
condenser is almost a necessity (§ 80). Whether a color-screen should

234 PHOTO-MICROGRAPHY \_CH. VIII

be used depends upon judgment and that can be attained only by ex-
perience. In the beginning one may try without a screen, and with
different screens and compare results.

A plan used by many skilled workers is to light the object and the
field around it well and then to place a metal diaphragm of the proper
size in the camera very close to the plate holder. This will insure a
clean, sharp margin to the picture. This metal diaphragm must be
removed while focusing the diaphragm of the projection ocular, as the
diaphragm opening is smaller than the image of the ocular dia-
phragm.

If the young photo-micrographer will be careful to select for his
first trials, objects of which really good photo- micrographs have
already been made, and then persists with each one until fairly good
results are attained, his progress will be far more rapid than as if poor
pictures of many different things were made. He should, of course,
begin with low magnifications.

§ 376. Adjusting the Objective for Cover-Glass.— After the
object is properly lighted, the objective, if adjustable, must be cor-
rected for the thickness of cover. If one knows the exact thickness
of the cover and the objective is marked for different thicknesses, it is
easy to get the adjustment approximately correct mechanically, then
the final corrections depend on the skill and judgment of the worker.
It is to be noted too that if the objective is to be used without a projec-
tion ocular the tube-length is practically extended to the focusing screen
and as the effect of lengthening the tube is the same as thickening the
cover- glass, the adjusting collar must be turned to a higher number
than the actual thickness of the cover calls for (see § 103).

§ 377. Photographing Without an Ocular. — Proceed exactly
as described for the lower power, but if the objective is adjustable make
the proper adjustment for the increased tube-length (§ 103).

§ 378. Photographing with a Projection Ocular. — Proceed as
described in § 372 B, only in this case the objective is not to be adjusted
for the extra length of bellows. If it is corrected for the ordinary
ocular, the projection ocular then projects this correct image upon the
focusing screen.

§ 379. Photo-Micrographs at a Magnification of 500 to 2000
Diameters. — For this the homogeneous immersion objective is em-
ployed, and as it requires a long bellows to get the higher magnifica-
tion with the objective alone, it is best to use the projection oculars.

CH. VIII} PHOTO-MICROGRAPHY 235

For this work the directions given in § 372 B must be followed with
great exactness. The edge of the petroleum lamp flame is sufficient to
fill the field in most cases. With many objects the time required with
good lamp light is not excessive ; viz., 40 seconds to 3 minutes. The
reason of this is that while the illumination diminishes directly as the
square of the magnification, it increases with the increase in numerical
aperture, so that the illuminating power of the homogeneous immersion
is great in spite of the great magnification (§ 34).

For work with high powers a stronger light than the petroleum
lamp is employed by those doing considerable photo-micrography.
Good work may be done, however, with the petroleum lamp.

It may be well to recall the statement made in the beginning, that
the specimen to be photographed must be of especial excellence for all
powers. No one will doubt the truth of the statement who undertakes
to make photo-micrographs at a magnification of 500 to 2000 diameters.

If one has a complete outfit with electric arc light (Fig. 192) the
time required for photographing objects is much reduced, i. e. ranging
from i to 20 seconds even with a color screen. As the light is so in-
tense with the arc light it is necessary to soften it greatly for focusing.
Several thicknesses of ground glass placed between the lamp and the
microscope will answer. These are removed before taking the nega-
tive. It is well also to have a water bath on the optical bench to ab-
sorb the heat rays. This should be in position constantly (see Ch. IX).

§380. Use of Oculars in Photo-Micrography. — There is much
diversity of opinion whether or not the ordinary oculars used for ob-
servation should be used in photographing. Excellent results have
been obtained with them and also without them.

For great magnification Zeiss recommends the use of the compen-
sation oculars with the apochromatics.

The Zeiss projection oculars may be used with achromatic objectives
of large aperture as well as with the apochromatics.

PHOTOGRAPHING OPAQUE OBJECTS AND METALLIC SURFACES
WITH A MICROSCOPE

All of the objects cansidered in the first part of this chapter are opaque and
some of them were to be photographed somewhat larger than natural size. To
meet the needs of modern work, especially with metals and alloys one must be
able to examine and photograph prepared surfaces at magnifications ranging from
five or ten to five hundred or more diameters.

\ 381. Microscope for Opaque Objects. — If one does not need to magnify
more than about 100 diameters, any good microscope will answer. For the higher

236

PHO TO -MICROGRA PHY

\CH. VIII

CH. VIII}

PHO TO-MICROGRA PH Y

237

FiG. 192. Buxton's Photo- Micrograp hie outfit for use with the arc light.
(Jour. Ap. Microscopy, igoi, p. 1367}. (Cut loaned by the Bausch & Lomb. Opt.
Co.) As will be seen from the figure this apparatus is for work in the horizontal
Position. The optical bench containing the microscope, water bath, color screen and
the electric light, swings sidewise sufficiently for the operator to arrange the speci-
men exactly as desired. It then swings back into position and is joined to the
camera. This is in two sections for either a short or long bellows. This seems to
be the most convenient of all the expensive outfits for photo-micrography .

powers it is far more convenient to employ a special microscope for metallography
(micro-metalloscope.) (German, Metallmikroskop ; French, Microscope pour
1' etude des surfaces metalliques et des objets opaque). (Fig. 193.)

FIG. 193. Special microscope of the Boston Testing Laboratories for the study
and photography of metals and alloys (\ 381). (Cut loaned by the Boston Testing
Laboratories. )

238 PHOTO-MICROGRAPHY [CH. VIII

Such a microscope has the following general characters : The stage is mova-
ble up and down with rack and pinion, it is rotary and more or less mechanical by
means of centering screws. With some at least the stage may be removed
entirely. No substage condenser is present, and a mirror is only present for occa-
sional transparent objects. A revolving nose-piece is not so good as the objective
changers.

§ 382. Illumination of Opaque Objects. — (A) for 25 to 100 diameters. The
directions of Mr. Walmsley are excellent (Trans. Amer. Micr. Soc., 1898, p. 191).
"Altogether the best light for the purpose is diffused daylight. Proper lighting is
more easily obtained with a vertical camera. An even illumination avoiding deep
shadows is preferable in most cases and is more easily attained with the object in
a horizontal position. For many objects it is better not to use a bull's eye or any
form of condenser but for others the condenser may be needed, but when the con-
denser is used one must avoid too much glare. The now little used parabolic re-
flector and Lieberkiihn serve well in many cases, but he adds "the majority yield
better results under the most simple forms of illumination," i. e., with the dif-
fused light from the window. This has been the experience of the writer also.

In case diffused daylight is employed the camera should be near a good sized
window, and the object should be somewhat below the window ledge so that the
illumination is partly from above and from the side. (This is easily attained with
the small table and vertical camera shown in Figs. 175, 184). The vertical illum-
inator is advantageous for these powers also. See (B. ).

(B) For 100 to 500 diameters, — For the magnifications above 50 it is desirable
and for those above 100 it is necessary to use some form of "vertical illuminator,"
that is some arrangement by which the light is reflected down through the objec-
tive upon the object, the objective acting as a condenser, and from the object
back through the objective and ocular to the eye of the observer. This is accom-
plished in two ways :

1 i ) By means of a small speculum-metal mirror in the tube of the micro-
scope. This is set at an angle of 45 degrees and the light thrown into the tube
upon it is reflected straight down through the objective upon the object. The
speculum metal being opaque cuts out a part of the light. Instead of a metal
mirror a circular disc of glass is now more frequently used. This allows the
major part of the light reflected from the object, up through the objective to
reach the eye.

( 2 ) By means of a small glass 45 degree prism inserted into the side of the
objective or of a special adapter. The light is from the side of the microscope, and
is reflected by the prism straight down through the objective upon the object as
before.*

*The idea of the vertical illuminator apparently originated with Hamilton L.
Smith. He used the metal reflector. Beck substituted a cover-glass and Powell
and L/ealand a disc of worked glass; i. e. glass that had been carefully polished
and leveled on the two sides. Carpenter-Dallinger, pp. 336-338.

The use of the prism with the objective is due to Tolles (see Jour. Roy. Micr.
Soc., vol. iii, 1880, pp. 526, 574).

In Zeiss' catalog the prism form is figured. In the catalog of Nachet both the
glass disc and the prism forms are figured.

CH. VIII} PHOTO-MICROGRAPHY 239

For both these devices uncovered objects are most successful or if the object
is covered it must be in optical contact with the cover-glass. Naturally good re-
flecting surfaces like the rulings on polished metal bars give most satisfactory
images, hence this method of illumination is especially adapted to micro-metal-
lography. Indeed, without some such adequate method of illumination the study
of metals and alloys with high powers would be impossible. So successful is it
that oil immersion objectives may be used. ( Carpenter- Dallinger, pp. 335-338).
§ 383. Light for the Vertical Illuminator. — For moderate
powers one may place the microscope in front of a window, or one ma}*
use a petroleum or gas lamp. For the higher powers acetylene or
preferably the electric arc light is used. In either case it may be neces-
sary to soften the light somewhat either by a color screen or by some
ground glass. The light should be concentrated upon the exposed end
of the prism or into the hole leading to the glass disc. Both the prism
and the disc should be adjustable for different objectives and different
specimens. The cone of light, especially with the electric arc light,
should be enclosed in a hollow metal or asbestos cone to avoid the glare
in the eyes of the operator, and it riiay be necessary to soften the light
with ground glass before attempting to focus and arrange the speci-
men. This ground glass would in most cases be removed before mak-
ing the exposure (§ 379).

With the electric light and for long exposure or observation a
water bath to absorb the heat rays will be necessary to avoid injuring
the lenses. (See also under projection in the next chapter).

As it is somewhat difficult to adjust the light in a way to give the
best effect, one can see the advantage of the adjustment for raising and
lowering the stage. This will serve for all but the finest focusing, and
thus avoid moving the tube for focusing enough to throw the lighting
out of adjustment. It might be advantageous to have a fine adjust-
ment on the stage also.

\ 384. Mounting of Objects.— For observation only and with low powers,
the objects may be mounted either in a liquid or dry as seems best. There should
be a black background for most objects, then light will reach the eye only from
the object. A light background is sometimes desirable, especially where one cares
only for outlines.

§385. Preparation of Metallic Surfaces.— In the first place a flat face is
obtained by grinding or filing, and then this is polished. For polishing, finer
and finer emery or other polishing powders are used, (rouge or diamantine,
or specially prepared alumnina, etc). The aim is to get rid of the scratches so
that the surface will be smooth and free from lines.

\ 386. Etching. — After the surface is polished it should be etched with some
substance. This etching material will corrode the less resistant material, the
edges of crystals, etc., so that the structure will appear clearly. For etching,

240 PHOTO-MICROGRAPHY [C//. VI 7/

tincture of iodine, nitric acid in various degrees of strength, hydrochloric acid,
etc. , are used or one may use electricity, the metal being immersed in an indiffer-
ent liquid. See numerous articles in the Metallographist for methods and micro-
graphs.

After the etching, the surface should be washed well with water to remove the
etcher. L/e Chatelier recommends that the etched surface when dry be coated
with a very thin coating of collodion to avoid tarnishing. The preparation will
then last for several months untarnished.

§ 387. Mounting the Preparations of Metal. — In order to get a satisfactory
image the flat, polished and etched face should be at right angles to the optic axis.
For preliminary observation one can approximate this by mounting the specimen
on a piece of beeswax. (Behrens). Very elaborate arrangements of the stage
have also been devised ( Reichert) . A simple and effective device is shown in Fig.
193 in which the specimen is held against the under side of the plane face of the
stage attachment. Rubber bands answer well to support the metal, and only one
side need be flat.

\ 388. 'Photographing Opaque Objects. — The general directions given in \ 347
should be followed with the necessary modifications. The time of exposure is
usually considerably greater with opaque objects than with transparent ones.
Very few such objects can be photgraphed in less than 30 seconds, even with day-
light For metallic surfaces and magnifications of 100, 150, 250 to 500, with the
electric arc light as illuminant the time required for favorable objects is i, 2,
4 and 7 seconds ; with the Wellsbach lamp the time is 5, 10, 30 and 60 minutes
(Sauveur).

FIG. 194. Rack for drying negatives
(Rochester Opt. Co}.

FIG. 194.

References to Ch. VIII.

See the works and journals dealing with photography.

For Photo-Micrography see Pringle, Bousfield, Neuhauss, Sternberg, Francotte
and the special catalogs on photo-micrography and projection issued by the great
opticians. The Journal of the Royal Microscopical Society and of the Quekett
Micr. Club ; Zeit. wiss. Mikroskopie ; the Trans Amer. Micr. Soc. ; the Amer.
Monthly Micr. Journal ; the Journal of Applied Microscopy.

For the photography of metallic surfaces, see the various journals of engineer-
ing and metallurgy, but especially Sauveur's journal, the Metallographist, begun
in 1898.

CH. VIII} PHOTO-MICROGRAPHY 241

ENLARGEMENTS ; LANTERN SLIDES ; PHOTOGRAPHING
BACTERIAL CULTURES

g 389. Enlargements. — As a low power objective has greater depth of focus
or penetration than a higher power (\ 34), it is desirable in many cases to make a
negative of an object with considerable depth at a low magnification, and then
to enlarge this picture to the desired size. As a rule negatives will not bear an
enlargement of more than five diameters.

For this work the camera shown in Fig. 181 is excellent, and the special mi-
croscope stand shown in this figure and in Fig. 175 serves to enable one to get a
very exact focus.

One must select an objective for the enlargement with a field of sufficient size
to cover the part of the negative to be enlarged. An objective of 60 to 100 mm.
focus will answer in most cases.

For the illumination the camera can be elevated against the sky, or artificial
light may be used. It is not easy to light so large a surface evenly by artificial
light.

(A) Enlargement on Bromide Paper. — For this the negative is put in place
and by pulling out the bellows the proper amount, one gets the right magnifica-
tion. Focus now as for any other object, using the fine adjustment and focusing
glass.

For great exactness one must put a clear glass in the plate holder and focus
on the surface away from the objective. Then place the bromide paper on this
clear glass and put another over it to hold it flat against the first plate of glass.
The sensitive surface will then be in the exact plane of the focus and the picture
will be sharp.

For the development and subsequent treatment of the paper, follow the
directions of the makers.

(B) Enlargement on a Glass Plate. — One may proceed in enlarging as for
making lantern slides and make a positive on a glass plate. If it is then desired
to get a negative for printing, place this positive on the microscope stand and
make a negative from it as if it were an object. Or one may make a contact im-
pression as is frequently done in lantern slide making. By this method one must
make three separate pictures, (i)the original photo-micrographic negative ; (2)
the enlarged positive from this ; (3) a negative from the enlarged positive. With
this negative one may print as from the original negative.

\ 390. Lantern Slides from Negatives. — In preparing lantern slides from
photo-micrographic or ordinary negatives one may use the contact method, or the
camera. With the camera one can enlarge or reduce to suit the particular case.
The camera and special microscope stand shown in Fig. 181 are admirable for the
purpose. For lantern slide work a photographic objective is used and the cone
for enlargement removed. One may put the objective in the front of the camera
or in the middle segment, making use of the little side door.

\ 391. Photographing Bacterial Cultures in Petri Dishes. — For the successful
photographing of these cultures dark ground illumination is employed on the
principal stated in § 92. That is the preparation is illuminated with rays so
oblique that none can enter the objective. These striking the culture are reflected

242 PHOTO-MICROGRAPHY \_CH.VIII

into the objective. The clear gelatin around the growth or colonies does not
reflect the light and therefore the space between the colonies is dark.

For supporting the Petri dishes a hole is made in a front board for the camera.
This hole is slightly larger than the dish. Over it is then screwed or nailed a
rubber ring slightly smaller than the Petri dish. This will stretch and receive the
dish, and grasp it firmly so that it is in no danger of falling out when put in a verti-
cal position. If the camera has two divisions like the one shown the board with
the Petri dish is put in the front of the camera, and the objective in the middle
division through the side door. Otherwise the board holding the Petri dish must
be on a separate support.

The illumination is accomplished by the use of two electric lamps with conical
shades. (The cheap tin shades with white enamel paint on the inside are good).
The lamps are placed at the sides so that a bright light is thrown on the culture,
but at such an angle that none of it enters the objective directly.

A piece of black velveteen is placed 10 to 20 cm. beyond the culture. This
prevents any light from being reflected through the clear gelatin to the objective.
Unless some such precaution were taken the background would be gray instead of
black.

One may use daylight by putting the culture in a support just outside a win-
dow, leaving the camera in the room. The rays from the sky are so oblique that
they do not enter the objective. One must use a black non-reflecting background
some distance beyond the dish as in using artificial light (Atkinson).

$ 392. Photographing Bacterial Cultures in Test-Tubes. — Here the lighting
is as in the preceding section, but a great difficulty is found in getting good re-
sults from the refraction and reflections of the curved surfaces. To overcome this
one applies the principles discussed in § 144, and the test-tubes are immersed in a
bath of water or water and glycerin. The bath must have plane surfaces. Behind
it is the black velvet screen, and the light is in front as for the Petri dishes. As
suggested by Spitta it is well to employ a bath sufficiently thick in order that
streak cultures may be arranged so that the sloping surface will all be in focus
at once by inclining the test-tube.

See the works on photo-micrography and photography for the details of lan-
tern slide making. See for the Petri dishes and test-tubes, Atkinson, Botanical
Gazette, xviii (1893), p. 333 ; Spitta, Photo-Micrography (1899), p. 26.

CHAPTER IX

CLASS DEMONSTRATIONS IN HISTOLOGY AND
EMBRYOLOGY

APPARATUS AND MATERIAL FOR THIS CHAPTER

Demonstration microscopes, simple and compound (Figs. 195-196); Traveling
microscope (Fig. 197-198); Indicator ocular (Fig. 199-201); Marker for putting
rings around the parts of specimens to be demonstrated (Fig. 61 ); Projection mi-
croscope (Fig. 207); Projection objectives (Fig. 211-212); Episcope ( Fig. 214).

DEMONSTRATION MICROSCOPES AND INDICATORS

§ 393- Simple Microscope. — The simple microscope held in
one hand and the specimen in the other, has always been used for
demonstration, but for class demonstration it is necessary to have mi-
croscope and specimen together or the part to be observed by the class
is frequently missed. Originally blocks of various kinds to hold both
microscope and specimen were devised, but within the last few years
excellent pieces of apparatus have been devised by several opticians for
the purpose. The accompanying figure shows one of the best forms.

FIG. 195. Simple Demonstra-
tion Microscope of Leitz ( Wm.
Krafft, N. Y.) As shown in
the figure this consists of a
handle, a stage and a lens holder
which slides up and down for
focusing. Fot observation the
student holds it up to the light.

FIG. 195.

§ 394. Compound Demonstration Microscope. —This was
originally called a clinical or pocket microscope. It is thus described
by Mayall in his Cantor Lectures on the history of the microscope : "A
small microscope was devised by Tolles for clinical purposes which
seems to me so good in every way that I must ask special attention for

244

CLASS DEMONSTRA T1ONS

[ CH. IX

it. The objective is screwed into a sliding tube, and for roughly focus-
ing the sliding motion suffices ; for fine adjustment, the sheath is made
to turn on. a fine screw thread on a cylindrical tube, which serves alsa
as a socket-carrier for the stage. The compound microscope is here
reduced to the simplest form I have met with to be a really serviceable
instrument for the purpose in view ; and the mechanism is of thor-
oughly substantial character. I commend this model to the notice of
our opticians. ' '

FIG. 196. Demonstration compound
microscope of Leitz. Leitz now furnishes
a fine adjustment in the form of an inter-
mediate piece between the objective and
the tube. This has in it a screw which is
turned by a milled ring. For the object-
ives employed it makes an efficient fine
adjustment and renders it possible for each
person to adjust the microscope slightly
without endangering the loss of 'the field '.

FIG. 196.

Since its introduction by Tolles many opticians have produced ex-
cellent demonstration microscopes of this type, but most of them have
not preserved a special mechanism for fine adjustment. With it one
can demonstrate with an objective of 6 mm. satisfactorily. It has a lock
so that once the specimen is in the right position and the instrument fo-
cused it may be passed around the class. For observation it is only
necessary for each student to point the microscope toward a window or
a lamp.

CH.

CLASS DEMONSTRATIONS

245

FIG. 197.
Traveling microscope set up for

work (Leitz ; from Wm.

FIG. 197.
Krafft, N. Y).

A modification of this clinical microscope was made by Zentmayer
in which the microscope was mounted on a board and a lamp for illum-
inating the object was placed at the right position.

246

CLASS DEMONSTRATIONS

\CH. IX

§ 395. Traveling Microscope. — For many years the French
opticians have produced most excellent traveling microscopes. The
opticians of other countries have also brought out serviceable instru-
ments. In the one here figured Mr. L,eitz has combined in an admirable
way a traveling microscope and a laboratory instrument. For the
needs of the pathologist and sanitary inspector a microscope must pos-
sess compactness and also the qualities which render it usable for nearly
all the purposes required in a laboratory. This instrument is a type
of such apparatus which has grown up with the needs of advancing
knowledge.

FIG. 198.
Krafft, N. Y.).

FIG. 198.
Traveling microscope folded up and in its case (Leitz ; from Wm.

CH.

CLASS DEMONSTRATIONS

247

§ 396. Indicator or Pointer Ocular. — This is an ocular in
which a delicate pointer of some kind is placed at the level where the
real image of the microscope is produced. It is placed at the same
level as the ocular micrometer, and the pointer like the micrometer is
magnified with the real image and appears as a part of the projected
image (§ 170). By rotating the ocular or the pointer any part of the
real image may be pointed out as one uses a pointer on a wall or black-
board diagram. By means of the indicator eye-piece one can be cer-
tain that the student sees the desired object, and is not confused by the
multitude of other things present in the field. The method of its use
is indicated in Fig. 201. This device has been invented many times.
It illustrates well the adage : "necessity is the mother of invention,"
for what teacher has not been in despair many times when trying to
make a student see a definite object and neglect the numerous other
objects in the field. So far as the writer has been able to learn,
Quekett was the first to introduce an indicator ocular with a metal
pointer which was adjustable and could be turned to any part of the
field or wholly out of the field. See Fig. 199, § 126.

FIG. 199. FIG. 200. FIG. 201.

FIG. 199. Indicator ocular with metal pointer like the one devised by
Quekett (Leitz ; catalog}.

FIG. 200. Indicator ocular with an eyelash ( cilium ) on the ocular diaphragm
to serve as a pointer ( P) . This projects about half way across the diaphragm open-
ing. On the opposite side are shown two rays from the microscope to indicate that
the real image is formed at the level of the ocular diaphragm.

FIG. 201. Field of the microscope with a mammalian blood preparation to show
the use of the indicator (P} for pointing out a white blood corpuscle.

248

CLASS DEMONSTATIONS

[Cff. IX

It is not known who adopted the simple device of putting the tip
of a cat's whisker or an eye-lash on the diaphragm of the ocular as
shown in Fig. 200. This may be done with any ocular, positive or
negative. One may use a little mucilage, Canada balsam or any
other cement, and stick the eyelash on the upper face of the diaphragm
so that it projects about half way across the opening. When the eye-
lens is screwed back in place the hair should be in focus. If it is not
screw the eye-lens out a little and look again. If it is not now sharp, the
hair is a little too high and should be depressed a little. If it is less
distinct on screwing out the ocular it is too low and should be elevated.
One can soon get it in exact focus. Of course it may be removed at
any time.

§ 397- — Marking the Position of Objects. — In order that one
may prepare a demonstration easily and certainly in a short time the
specimens to be shown must be marked in someway. A very efficient
and simple method is to put rings of black or colored shellac around
the part to be demonstrated. For this the Marker, Fig. 61-62, is
employed as described on p. 66.

FIG. 202.

FIG. 202. Ring around one of the sections of a series for demonstrating some
organ especially well.

FIG. 203. Figure of a microscopical
preparation with a ring around a small
part to show the position of some structural
feature.

CH. IX a] PROJECTION MICROSCOPE 249

CHAPTER IX A

THE PROJECTION MICROSCOPE

One of the most useful and satisfactory means at the disposal of
the teacher of Microscopic Anatomy and Embryology for class demon-
strations is the Projection Microscope. With it he can show two
hundred students as well as one, the objects which come within the
range of the instrument.

It is far more satisfactory than microscopic demonstrations, for
with the projection microscope the teacher can point out on the screen
exactly the structural features and organs which he wishes to demon-
strate, and he can thus be certain that the students know exactly what
is to be studied. Unless one employs a pointer ocular (Fig. 201).
there is no certainty that the student selects from the multitude of
things in the microscopic field the one which is meant by the teacher.
Like all other means, however, the projection microscope is limited.
With it one can show organs both adult and embryonic, and the gen-
eral morphology. For the accurate demonstration of cells and cell
structure the microscope itself must be used. As a general statement
concerning the use of the projection microscope for demonstration
purposes, it may be said that it is entirely satisfactory for objects and
details which show under the microscope with objectives up to 16 mm.
equivalent focus. For objects and details requiring objectives higher
than 1 6 mm. focus for ordinary microscopic observations, the projection
microscope is unsatisfactory for large classes.

All specimens should be studied under the microscope by each
student personally ; however, it is much easier to recognize and
understand the complex structures and structural relations in an object
after they have been demonstrated by the instructor with the projec-
tion microscope.

§ 398. Projection Microscope. — This is an arrangement of the
microscope such that an image of the object under the microscope is
thrown upon a screen of some kind. The picture on the screen is
looked at precisely as one looks at the pictures thrown on the screen

250 PROJECTION MICROSCOPE \_CH. IX a

by an ordinary magic lantern. Indeed the projection microscope is a
magic lantern with short focus objectives. One of the first uses of the
microscope was to throw the images of various objects on a screen so
they could be seen by several persons at once. The light used was
sunlight, hence those early projection microscopes were called solar or
sun microscopes. If sunlight were available at all times and could be
controlled, it would be universally employed ; but it is not at all times
available and whenever it is, a heliostat is needed to keep the light
fixed in a given position. Sunlight is therefore practically discarded
and the electric light is employed for illumination.

§399. Parts of a Projection Microscope. — These are shown
in Fig. 204 and are as follows :

(1) Some form of projection objective on a proper support.

(2) A stage for supporting the object. This stage should be
free from the objective holder ; it should have a range of diaphragms
so that the largest as well as the smallest objects may be shown. The
stage should be freely movable so that the object may be completely
lighted (see Fig. 209).

(3) There should be an attachable mechanical stage with large
movement (B, D. 1).

(4) A large lamp condenser for illuminating the object. If this
is properly constructed and the various parts of the apparatus are
separately movable as here shown, no special substage condenser is
necessary (see Fig. 209).

(5) An electric arc lamp for lighting.

Fig. 204, A, B, C, D. Half tone reproductions of photographs
of the Projection Apparatus used in the department of Histology and
Embryology, Cornell University. A 10 centimeter scale was photo-
graphed with the apparatus in each case.

The arc lamp and the metal bed-piece are screwed fast to a thick
board so that the whole apparatus may be moved sidewise or elevated
without getting any part out of line.

From the weight of the blocks carrying the microscope, the stage
and the condenser and the width of the bed-piece no clamping screws
are necessary for holding the blocks in place even when the board is
considerably elevated. The grooves and V's are so accurately fitted
however that the various blocks can be easily moved along the bed
piece and the centering is as accurate .in one position as in another.

I i

I

I

D

E
FIG. 205

CH. IX a] PROJECTION MICROSCOPE 251

A. The ordinary Magic Lantern and the Projection Microscope
in position for alternate use (see Fig. 206 for wiring, etc.) i, Ammeter
shown at the left ; 2, ordinary stereopticon for lantern slides ; 3
Projection Microscope. This shows well the metal screen at the
bottom of the tube, for cutting off stray light.

B. Projection Microscope with a 105 mm. objective for very
large objects.

1. Stage with mechanical stage, large diaphragm opening and
section of a cat's brain as object.

2. 105 mm. objective in position for projecting a large object such
as a brain section.

3. Stage, near the condenser (See Fig. 209).

4. Condenser with water bath. Under the condenser is a focus-
ing glass for photography.

5. 90 degree, automatic arc lamp.

6. Diaphragm with circular opening and specimen cooler in
position (See Fig. 208 F, G).

C. Projection Microscope with the stage and microscope in posi-
tion for the use of a low objective.

i. Large square diaphragm for the stage. 2. Microscope; 3,
Stage with the specimen cooler in position ; 4 Condenser and water
bath ; 5 Arc lamp.

D. Projection Microscope with the parts in position for high
powers.

1. Mechanical stage with a specimen in position.

2. Microscope with a high power in position ; 3. Stage with the
specimen cooler in position ; 4. Condenser and water bath.

5. Arc lamp ; 6. Large diaphragm.

The white lines on the bed-piece indicate the position of the con-
denser, which is about 8 centimeters from the mica covering of the
condenser. The other line indicates the position of the microscope for
high power projection.

FIG. 205 A. Zeiss' Micro-Planar for projection. (Cut loaned by
Bausch & Lomb Optical Co.)

The micro-planars are of 20, 35, 50, 75, and 100 mm. equivalent
focus. When used for projection the special iris in each should be
wide open. These are used without a projection ocular.

FIG. 205. B. C. Leitz objectives of 64 mm. (B), and 42 mm. (C)
for micro- projection. (Cut loaned by Wm. Krafft, N, Y.)

252 PROJECTION MICROSCOPE \CH. IX a

These are used without a projection ocular. The iris in each
objective should be wide open for micro-projection.

FIG. 205 D. Projection apparatus showing the metal hood for the
lamp. The microscope, the stage and the water bath have been
removed.

A. Screw for adjusting the upper carbon along its axis.

B. Wheel by which the lamp may be regulated by hand. It is
also used in putting the carbon holders in the proper position for
adding new carbons.

C. Screw for adjusting the lamp side-wise.

D. Screw for raising or lowering the lamp.

In the door of the metal covering is a transparent window made
of a piece of red and a piece of blue glass. This kind of a window
enables one to look at the arc without hurting the eyes.

FIG. 205 B. Half tone reproductions of the crater of the Thomp-
son 90 degree automatic arc lamp. The lower carbon shows only its
tip. In the center of the one at the left is shown a faint shadow.
This is in the depression of the soft core. The image of the crater
was thrown on the screen by a 64 mm. projection lens (B) and this
image was then photographed.

§ 400. Blackening the Apparatus. — The metal parts of the en-
tire apparatus should be dead black to avoid the reflections. Reflec-
tions dazzle the eyes of the operator, and spreading over the room,
injure the brilliancy of the image on the screen. The hint to blacken
the mountings of the objectives was given by Dr. Coplin. It was
found by personal experience that not only the objective mountings
but every metal part about the apparatus should be blackened. If
necessary one can do this himself by using thin shellac mixed with
lampblack. This should be filtered through two or three layers of
gauze to avoid lumps. It can be put on with a soft brush like a camel's
hair duster. If the shellac is too thick or if not enough lampblack is
used the surface will be shiny. It should be a dead black.

§ 401. Radiant for the Projection Microscope. — No light for
projection has been found so generally satisfactory as the electric arc
lamp used with the constant current. The alternating current can be
used, but it is very unsatisfactory. For demonstration purposes a truly
automatic lamp is more convenient than a hand feed lamp. In start-
ing the latter the carbons must be brought in contact and then slightly
separated. With the automatic lamp 'the separation is attended to by

CH. IXa\

PROJECTION MICROSCOPE

253

the lamp mechanism. If the carbons do not separate when an automatic
lamp starts it is due to too small an amount of current. If the current
is too weak the magnet is not strong enough to lift the carbon.

FIG. 206. Diagram showing proper
wiring for a projection apparatus.

i -f- Main wire conducting the cur-
rent to the apparatus ; 2 — , Main wire
conducting the current away from the
apparatus ; j, resistance or rheostat. It
should be adjustable to allow a greater or
less amount of current to pass as occasion
demands,

4. Ammeter to show the amount of
current. This is very desirable, but not
absolutely necessary. If it is not used the
wire would take the course indicated by
the dotted line.

5. Arc lamp. The left or upper
carbon marked with the arrow and plus
sign ( -j- ) is the one in which is fouud the
crater giving the illumination ; 6, Arc
lamp similar to 5.

7. Lever of the double pole, double
throw switch. The conducting wires go
to the binding posts at the base of the
lever. In case the crater does not appear
in the upper carbon, but in the one mark-
ed with the minus sign ( — ), the conduct-
ing wires i and 2 are interchanged and
must be reversed ( § See 406).

% 402. Wiring, Rheostat and Ammeter for a Projection Mi-
croscope.— Every person who uses a projection apparatus should
know the simple but fundamental principles involved in its proper
installation and running. Fig. 206 illustrates the arrangement of all
the parts. The arrows indicate the direction of the current and the
plus sign indicates the positive and the minus sign the negative
terminal. It will be seen that all the parts, z". e. the resistance or
rheostat (3) the ammeter (4) the lamp (5 or 6), are placed in series
along one wire, and there is no connection with the other main wire
(2) until the current leaves the lamp. If at any other place a connec-

254 PROJECTION MICROSCOPE [ CH. IX a

tion were made with (2) a short circuit would be formed. The
absolutely necessary parts in the installation of a lantern are : the
main wire conveying the current to the apparatus (i), the main wire
conveying the current from the apparatus (2) ; some resistance or a
rheostat (3) somewhere in the circuit ; and the ordinary lantern or
the projection microscope (5 or 6), (see A of Fig. 204).

A rheostat of some form is absolutely necessary. This part of
the outfit should be adjustable and thus enable one to use a current
ranging from 8 to 20 amperes. If a fixed rheostat is used probably
one giving about 12 amperes would be most generally satisfactory.
It must be remembered that the amount of current at a given moment
depends upon the voltage and also upon the amount of current being
used by others on the line. For really satisfactory projection one
must have an adjustable rheostat. It is also_very desirable to have an
ammeter in the circuit, then the current may be regulated with some
intelligence, and one can determine whether defects in the illumina-
tion are due to insufficient current or to some other cause. If the
amount of current is shown to be sufficient by the ammeter, the
cause of defective projection will be known to be somewhere in the
apparatus and it can be hunted down and corrected. Without an
ammeter one is tempted to increase the current for every defect in the
projection. One must remember also that the current is only one of
the factors in successful projection.

While a rheostat is absolutely necessary one can omit the am-
meter. If it is omitted the main conducting wire (i) would take the
course indicated by the dotted line just at the right of the ammeter
(4). It should be said that the arrangement of the wires and the
rheostat and ammeter in the diagram is a good one, but not the only
arrangement. The real point to remember in installing the apparatus
is that the current in making the circuit from (i) to (2) must pass
through the various pieces of apparatus in turn, and not have it pos-
sible for the current to make any short cuts (short circuits).

§403. Using two Lanterns. — (Fig. 204 A). If two lanterns
are to be used alternately the wiring should be as shown in Fig. 206.
For using two lanterns in this way the most convenient switch is the
double pole double throw form (7). The two main wires (-f-i and
—2) are connected with the binding posts at the base of the switch
blades.

Either lamp may be put in the circuit by turning the lever toward

CH.IXa\ PROJECTION MICROSCOPE 255

that lamp until connection is made. This arrangement enables one
to use either lamp at will.

A fuse block just before the switch is very desirable. The fuses
should carry from 20 to 25 amperes of current.

§404. Arrangement of the Carbons. — Originally arc lamps for
use with the lantern had the carbons both vertical. The "projector"
lamps used at sea, had, however, the carbons inclined at an angle of
30 or 40 degrees from the vertical. Lewis Wright (Optical Projection,
p. 163,) states that at his urgent request a projection arc lamp for
micro-projection was made with inclined carbons. Certain it is that
all projection lamps have now the inclined carbons. The angle of
inclination varies with different makers. The lamps furnished by
Zeiss and Reichert with their apparatus have the carbons at 40 degrees
from the vertical ; Behrens uses and recommends 45 degrees. Barnard
and Carver (J. R. M. S., 1898, p. 170) found by a careful series of
experiments that an angle of about 27 degrees gave the most satis-
factory light.

Within the last few years lamps have been constructed to hold the
carbons at right angles, the horizontal carbon serving for the illumi-
nation. Its brilliant crater faces the lamp condenser, and it is believed
that the illumination with this arrangement is more satisfactory than
with inclined carbons.

However the carbons are arranged in the lamp success is attained
only when the crater at the positive pole faces the condenser and is
on its principal axis, and the negative carbon does not get in the way
and thus form a shadow. The proper appearance is shown in Fig.
205 E of the 90 degree carbons, and in Fig. 207 of the more common
inclined carbons. One can get the right arrangement of the carbons
only by experience. The lamp should be adjustable so that the posi-
tion of the whole lamp may be changed vertically or horizontally to
ensure perfect centering, and then one must be able to change the
relative position of the carbons.

New carbons are very blunt and in many cases need readjusting
after they have become sharpened by wear. Their correct position
and amount of separation for a 12 to 15 ampere current is shown in
Fig. 204.

The best method for determining the character of the crater is to
project the image of the two carbons at the arc on the screen with a
low objective (50 to 75 mm. equiv. focus). One can then mutually

PROJECTION MICROSCOPE

{_CH. IX a

arrange the carbons until the best crater is obtained. (See Figs.
205 K, 207). One must always remember in working with the lan-
tern or projection microscope that the crater should be in the upper
carbon (See Fig. 207 and §406).

§ 405. Kind of Carbons to Employ. — The so-called soft cored
carbons are the most satisfactory. The crater does not shift around
on the end of the carbon so markedly as with hard carbons. The soft

FIG. 207

FIG. 207. Front and side views of the carbons of an arc light with inclined
carbons, -j- and — indicate the positive and negative poles. Compare Figs. 204,
206.

A is a side view showing the carbons in section at an angle of 30 degrees from
the vertical and the negative ( — ) or lower carbon slightly in front of the positive
( _|_) Or upper carbon. The carbons have soft cores.

B is a front view of the carbons as seen projected on the screen with a 42 mm.
objective. It is a projection of the real image of the carbons formed by a special
achromatic condenser next the object. This figure shows that the source of light
is the crater in the positive (-f) or upper carbon; it shows also that the lower
carbon is slightly below the upper carbon as well as slightly in front. This avoids
a shadow from the lower carbon.

In the center of the crater is shown a slight shadow. This is due to the pit
formed in the soft core of the carbon.

CH.IXa] PROJECTION MICKOSCOPE 257

core seems to furnish a kind of guide. In some lamps the two carbons
are of equal size while in others they differ in diameter. In such
cases one must learn from the makers which carbon is to be positive
and which negative. In the lamp shown in Fig. 204, B, C, D, (A. T.
Thompson & Co.'s), the upper or positive carbon is the smaller (i. e.
upper carbon, n mm.; lower carbon 13 mm. in diameter).

§ 406. How to Make the Upper Carbon Positive. — In most
cases one does not know which of the wires in a circuit is positive and
which negative. The simplest way to proceed is to make the connec-
tions as shown in Fig. 206 without regard to which is positive and
which negative. When all is complete turn on the current and allow
it to pass through the lamp for a few moments and then open the
switch. The carbons will glow for some time and the positive one will
be much brighter than the negative one. If the upper carbon is the
bright one then the current passes through the lamp in the right direc-
tion, but if the lower carbon is the bright one then the current passes
through the lamp in the wrong direction and the wires must be
changed. That is, in the diagram, the wire marked ( — 2) is in place
of the one marked (+ i). Very often the simplest way to make the
correction is to change the position of the wires passing from the bind-
ing posts of the switch to the lamp. If one uses a plug to connect
with the main line all that is necessary is to turn it the other side up if
the lower carbon is positive, or if the plug is non-reversible then the
wires must be reversed. It is well to mark the wires in some way after
one determines which is positive and which negative.

§407. Stage of the Projection Microscope. — This should be
large (about 10 x 12 cm.) and independent of the rest of the appa-
ratus. It should be freely movable toward or from the lamp conden-
ser so that objects of various sizes may be completely illuminated (see
Fig. 209). It should also be adjustable vertically so that it may be
centered with the other pieces of apparatus (Fig. 208 C.). There
should be available an attachable mechanical stage with a large range
of movement both vertically and horizontally (Figs, 67 — 69, 204 B.
and D.). A specimen cooler in addition to the large water bath is
also desirable (Fig. 204 B, C, D, Fig. 208 G.).

§ 408. Openings in the Stage. — For the projections desirable
in a modern laboratory of Histology and Embryology it is necessary
to be able to project objects varying greatly in size, i. e. from less
than i millimeter to 50 or more millimeters. In order to accomplish

258

PROJECTION MICROSCOPE

[CH.IXa

CH. IX a] PROJECTION MICROSCOPE 259

FIG. 208 A. — H. Diagrams of the stage and its parts, its grooved block and
the bed-piece with Vs. Half actual size.

A. Large square diaphragm fitting the still larger square diaphragm B. ; C.
Vertical stem fitting into the socket formed in part by the grooved block D. ; E.
Bed-piece with V's which fit the grooves of the blocks carrying the various parts,
i. e. microscope, stage and condenser (compare Fig. 204}. The bed-piece is like a
lathe bed and is of cast iron. The grooves and V's are accurately worked out on a
machine in Sibley College.

F. Diaphragm with circular opening ; G. Specimen cooler in position ; H.
sectional view showing the thickness of the stage and groove, and V, which serve
to hold the diaphragms in position.

this it is necessary to have the stage supplied with changable dia-
phragms to accommodate any specimen which one may desire to pro-
ject. This has been simply done in the apparatus shown in Fig. 204.
The thickness of the stage, the diaphragms and the methods of secur-
ing the diaphragms are indicated in Fig. 208 H. With this apparatus
(Fig. 204 B and Fig. 208) objects as large as lantern slides may be
projected.

§409. Objective Support and Focusing Arrangement.—
This should be independent of the stage and, for all but the lowest
objectives, should have fine and coarse adjustment as in the best
microscopes. As the instrument is horizontal the fine adjustment
should be strong enough to move the apparatus without the aid of
gravity. As with the stage, this part should be freely movable along
the axis of the whole apparatus so that it may be put in the correct
position for high and for low powers and the coarse and fine adjust-
ment used only for the final focusing. It must also be adjustable
vertically so that it may be accurately centered.

A triple or quadruple nose-piece is desirable so that one may turn
quickly from one power to another. Behind the nose-piece should be
a blackened metal screen about 15 cm. in diameter to cut off stray
light. The tube should be large and short so that it may not restrict
the field (2 cm. in diameter and 10 cm. in length).

There should be a draw-tube with proper attaching screw so that
it can be put in place when oculars or other apparatus needing the
draw-tube are to be employed.

§ 410. Support for very Low Power Projection. — For pro-
jecting objects of 40 to 60 millimeters in diameter a photgraphic or
planar objective of about 100 mm. focus answers admirably. This
should be mounted on a separate support and properly centered. Then

260 PROJECTION MICROSCOPE [_CH. IX a

when it is to be used the ordinary objective support and focus ar-
rangements are removed bodily and this apparatus put in place (Fig.
204, B). It is focused by sliding it along the bed-plate.

By having an ordinary lantern slide carrier in position next the
condenser one may use this apparatus also for lantern slide projection
by removing both the microscope and the stage and putting in place a
block carrying the ordinary projection objective. A double lantern is,
however, more satisfactory as no time is lost in making the change of
apparatus, and both lanterns can be in perfect adjustment for the special
purpose in hand.

§411. Water Baths for Removing Excessive Heat. — For
removing the excessive heat of the light beam tanks of water are
placed in its path. In the apparatus shown in Fig. 204 the main tank
is between the condenser lenses (see also Fig. 209 (5) and Fig. 213 A).
In Fig. 205 D the water bath has been removed. In addition to this
large water bath a special one called a specimen cooler is placed imme-
diately under the stage (Fig. 204 C, D, Fig. 208 G). For lower
powers where the specimen is near the condenser and hence in a wide
beam the large water bath usually suffices. (See Fig. 209, 2, 3.) If
the object is in or near the focus (Fig. 209 (i)), the heat is concen-
trated as well as the light and the specimen cooler is necessary to
protect the specimen:

In some projection microscopes the cooling tank is wholly free from
the condenser. That is an advantage for the contact of the tank with
the metal of the condenser serves to conduct much heat to the water
and it becomes hot not from the heat absorbed from the light, but
from the heated metal of the condenser mounting. To overcome this
difficulty one may have two cooling cells and when one is heated a
cold one can be put in its place. Sometimes the water is kept cold by
a constant change of the water. This is liable to give rise to currents
and consequently a shimmering or wavering of the light on the screen.

§412. Liquid for Removing Heat. — In the older works a
solution of alum was always advised as the heat absorber. It has
been found by careful quantitative experiments that distilled water is
quite as efficient. It is much cleaner and more transparent than alum
solutions. See Physical Review, vol. i, p. 14.

§413. Objectives to Use in Micro-Projection. Without
oculars the lowest power ordinarly used with the microscope is about
75 mm. focus. From this lowest point one will find objectives of 64,

CH. IX a} PROJECTION MICROSCOPE 261

50, 42, 35, and 25 mm. focus very excellent. Those found most satis-
factory by the writer are the so called projection objectives to be used
without an ocular. For higher powers than 25 mm. the ordinary
objectives used on the microscope are mostly employed and, when
properly selected, are very satisfactory.

For class demonstration, as stated above, one can show to an
audience of 200 with a screen distance of 5 to 8 meters all the details
which show satisfactorily with a 16 mm. objective and low ocular
under the ordinary microscope. One rarely uses with real satisfaction
an objective higher than 3 or 4 mm. equivalent focus. One of from
6 to 10 mm. focus is mostly used. For fine structural details one
must use an ordinary microscope. (Bee the table, § 425 for size of
field and magnification).

For powers above 3 mm. it is more satisfactory to have a screen
distance of 3 to 5 meters or even less. Such demonstrations are only
for small numbers, and they must be near the screen or use opera
glasses.

§414. The Use of Oculars in Micro-Projection. — The writer
has found projection without oculars more satisfactory then by their
use. The field is larger and higher objectives can be used, but the
oculars have the advantage of giving increased magnification so that
one can get the same amplification with lower objectives. A further
advantage is that the outlines of the field will be sharp and the part of
the specimen shown will be practically all in focus. Besides restrict-
ing the field, however, the oculars reduce the light greatly. Without
the ocular the more brilliant image, and the larger field are very
advantageous. With the larger field the whole of many objects may
be seen and the relations of the special part under examination noted
at a glance. It may be repeated also that the micro-planars and
the objectives of low power made especially for projection and photo-
graphy, are designed for use without an ocular. In using these
objectives their iris diaphragm must be opened as fully as possible.

§ 415. Kind of Oculars. — If an ocular is used it may be either
the projection oculars like those for photography (see§ 372, A, B), in
which case the diaphragm of the ocular must be focused on the screen
to get the best results (§ 372, B), or one may use a compensation or an
ordinary huygenian ocular. From the writer's experience an objective
above 8 mm. focus cannot be used satisfactorily for demonstration with
an ocular. If oculars are used in projection it is better to have a screen

262

PROJECTION MICROSCOPE

[CH.IXa

distance not exceeding 5 meters ; 3 meters would be even more satis-
factory. For size of field and magnification see the table § 425.

§ 416. Arrangement of the Parts of the Projection Micro-
scope.— As stated in section 399, all the parts of the apparatus should
be separately adjustable. This is because the arrangement needs to be
somewhat different for different objectives.

FIG. 209

FIG. 209. To show the elements of the illuminating and condensing apparatus
used in Fig. 204, and the position in the cone of light of objects of various sizes-
This figure shows the necessity of moving the stage to accommodate objects of
various sizes and ensure their complete illumination by the entire cone of light from
the condenser.

i. Focus of the condenser where objects for high powers are placed. 2, j. Posi-
tion of larger objects. 4. Front ten's of the condenser. 5. Laige water bath.
6, 7. Two lenses of the condenser next the radiant. 8. The dotted line over the
meniscus represents the sheet of mica which serves to prevent the too rapid heating
of the lenses. This is very satisfactorily held in position by a cap of sheet iron or
copper. ( It is in position in 204 and 205 D. )

The guiding principle is that the specimen should be lighted by a
converging cone of light, and it should be lighted by the entire cone of
light traversing the lamp condenser. If one uses a white card it is
easy to determine the position and size of the cone of light. If it is
too large for the specimen, either the lamp condenser is too near the
radiant or the specimen is too close to the lamp condenser. (Fig. 210.)

If one uses a substage condenser it and the lamp condenser should
be so placed that the entire cone of light traversing the lamp condenser
can enter the substage condenser. If the cone is too large they are
too close together, or the lamp condenser is too near the radiant. If
the cone is too small then the lamp condenser is too far from the
radiant or the substage condenser, or perhaps both faults are present.
One must remember in all his experiments that a converging cone of

CH. IX a\ PROJECTION MICROSCOPE 263

light should be used and not a diverging one. The specimen must
then not be beyond the focus of the lamp condenser.

§417. Centering and Serial Arrangement. — In installing a
projection apparatus like the one in Fig. 204, one can get the proper
adjustments first by making careful measurements. That is the upper
carbon should be opposite the center of the lamp condenser, and the
objective should also be in line with the center of the lamp condenser.
By moving the condenser close to the lamp and raising the carbon in
the position occupied when in actual use one can get the end opposite
the axis of the condenser by mutually adjusting lamp and condenser.
Then by removing the stage the microscope may be brought close to
the condenser and raised or lowered until the objective is opposite the
center. The stage should then be put in position and adjusted so that
the center shall be opposite the objective.

The purpose of all this is to get all the pieces centered to one axis.
If now the current is turned on the slight adjustments necessary for
the final centering may be made by the centering screws of the lamp
(Fig. 205 D (C D) ).

The relative position of the various pieces in a projection micro-
scope along the axis can be determined only by trial. If one remem-
bers that the object is to be illuminated by a converging cone of light
it will aid in getting the parts in the right position. Firstly the lamp
condenser must be at the right distance from the radiant. Fig. 210
shows the appearances in an ordinary magic lantern with the different
relative positions of radiant and condenser. For the condenser here
used (Fig. 204) the crater in the upper carbon should be about 8 cen-
timeters from the mica sheet over the condenser. One can determine
this only by trial. By changing the relative position of condenser to
radiant and the object to the condenser, and then the objective and
object one can find the positions giving the most satisfactory results.
When these are found it is worth while, as shown in the picture, to
make lines on the fixed part of the apparatus indicating them. A
study of Fig. 209 will aid one in understanding the principles involved
in the best relative positions. The actual cone of light from the con-
denser will be easily seen if some dust, like that from a blackboard
eraser is diffused in the air near the front of the condenser.

If a lamp condenser is properly constructed there is no necessity
of using a special substage condenser if the different parts are freely
movable. If, however, one must put the object in the same position

264

PROJECTION MICROSCOPE

[CH. IX a

regardless of its size then the stage must be nearer the condenser and
a special substage condenser used to bring the cone of light from the
condenser to a focus more quickly where one employs high powers for
the projection.

,L ''' A 3. \^^^

FIG. 210

FIG. 210. Arrangement and Centering of the Radiant (Leiss}.

In (/) The Radiant, i. e., the crater (Fig. 209) is too far to the right ;

(2) The crater is too far to the left ;

(j ) The crater is too high ;

(4} The crater is too low ;

(5) 1 he crater is too far from the lamp condenser ;

(6-7) The crater is too near the condenser.

(8) The crater is in the correct position.

As pointed out in the explanation of Fig. 207, there may be a slight central
shadow with soft cored carbons when the lamp and condenser are in the best relative
position.

If one wishes to make micro-projection a success it will be nec-
essary to give the apparatus the requisite time and thought. Try to
understand the conditions of success and continue experimenting until
you have learned to make it possible for the machine to do its best for
you. The satisfaction of showing a class real things is sufficient re-
ward for all the trouble.

§ 418. Screen and Screen Distance. — For a screen nothing is
vSO good as a dead- white, smooth wall. A lusterless, white cloth
screen answers well also. It is an advantage to have this entirely
opaque, so that none of the light can pass through it. One must
remember that the light passing through the minute lenses of the

CH. IX a] PROJECTION MICROSCOPE 265

objective must be spread out over a relatively great space even with
low powers, and over a much greater with high powers, so that one
cannot afford to have any of the light lost by transmission through the
screen.

The distance of the screen from the microscope depends largely on
the size of one's audience. The writer has found a distance of five to
eight meters (26 feet) good for both low and high power projection.
This distance answers well for a class of 200 persons. If oculars are
used with the objective, a screen distance of 3—5 meters is sufficient.

For the minute details of a projected specimen it is recommended
that the audience use opera glasses. These are also useful for focus-
ing the image on the screen.

§419. Darkening the Room. — It is impossible to succeed in
micro-projection unless the room can be made dark, the darker the
better. It is especially important that the screen should be free from
all light except that projected upon it in forming the image.

§420. Enclosing the Projection Apparatus. — It is desirable
to have the projection apparatus enclosed as completely as possible to
avoid diffusing light through the room and thus vitiating the most
careful darkening of all windows and sky lights. It is also desirable
to shut in the light from the apparatus, as it dazzles the eyes of the
operator and of those near it in the audience so that the image on the
screen cannot be satisfactorily seen. Some forms of apparatus are en-
closed in a metal box, others have a frame over them upon which is
spread black cloth like silesia. If this is made fireproof by soaking it
thoroughly in a solution of alum, borax and sodium tungstate it will
not readily catch fire. The cloth should not be too thick, otherwise it
will retain too much heat around the apparatus.

One should remember the fundamental law of vision, viz, that
other things being equal, the clearest images are obtained when no
light reaches the eye except from the object.

§421. Preparations Suitable for Micro-Projection.— As a
generalization it may be said that any specimen which shows clearly
and sharply under the microscope with a 1 6 mm. or lower objective
will also give an excellent projection image. Details which are not
visible with the 16 mm. objective are rarely well brought out with
sufficient clearness on the screen for one or two hundred people to see.

(A) The stains showing best are those which are very transpar-
ent, or pure differential stains like hematoxylin. Admirable results

266 PROJECTION MICROSCOPE \CH. IX a

have been obtained with hematoxylin and eosin, and the various car-
mines. Every method of staining which gives either sharply differen-
tiated results or transparent colors produces preparations adapted to
projection. A weak, or washed out appearance under the microscope
is sure to be even less satisfactory on the screen.

(B) The thickness of the sections may vary from i/* to 40/4. But
one must remember that thick sections are adapted for low powers
only, while thin sections, if well stained, may be used with both high
and low objectives.

The size of the object which one wishes to project determines the
objectives to be used. By consulting the table one can get a fair idea
of the size of object which each objective will satisfactorily project.
An excellent plan to follow is that for ordinary microscopic study (see
p. 102), that is, use first a low power to show the object as a whole,
then a higher one for details. .

§ 422. Projection of Living Objects. — If living objects are to
be used with the projection microscope it is nececsary to eliminate
more of the heat rays than a single water bath can absorb. The speci-
men cooler will serve for a short demonstration, but if one wishes to
have the specimen under the instrument for 10 minutes or more it will
be necessary to introduce a second large water tank in front of the lamp
condenser and to use the specimen cooler also. For blood circulation
and living phenomena generally the microscope itself is much more
satisfactory than the projection microscope.

§ 423. A practical suggestion is made by L,ewis Wright in his book
on optical projection, and that is to warm the objective before using it
for showing the circulation of the blood or in any case when a moist
object is under it. If the objective is cold the vapor from the object
will be condensed on the objective and make satisfactory projection
impossible.

§ 424. Masks for Projection Preparations. — The light used for
projection is so brilliant that it is practically impossible to arrange the
object under the objective with rapidity and certainty unless there is
some kind of guide. The best one found so far is a mask on the back
of the slide with an opening for the preparation to be shown. This
mask should be made of black paper. One can cut the holes in it with
scissors or with ticket punches. With the specimens properly masked,
and the parts of the apparatus lustreless black, the operator can work
with rapidity and also with comfort. (Fig. 211.)

CH. IXa\

PROJECTION MICROSCOPE

267

FIG. 211

FIG. 211. Slide of several sections with a black mask, perforated over the sec-
tions to be demonstrated with the projection microscope. This mask is put on the
back of the slide and not on the cover-glass.

Unless one has a mask something like this the light is so dazzling that it is
almost impossible to find the proper sections. It is easily removed by placing the
slide on wet blotting paper.

FIG. 212 A. Leitz Large Micro- Projection Apparatus (cuts loaned by Win.
Krafft, N. Y.} In this figure the apparatus is in position for projection with a
projection ocular.

Each part is. independent and capable of special adjustment. (As shown in the
next figure, the microscope may be turned aside, leaving the apparatus for
ordinary lantern slide projection}.

PROJECTION MICROSCOPE

\_CH.IXa

§ 425. Actual Size of Objects for Demonstration. — In the
table below are given the objectives and size of objects which can be
demonstrated with the apparatus shown in Fig. 204. By inspecting
the table one can determine quickly the size of a specimen it is pos-
sible to show as a whole, and also the objective to employ.

The principles involved in the construction of this table are those
given in § 50, § 155. For the low powers a micrometer with coarse
lines is necessary. The diameter of the screen image can be meas-
ured off directly, with a tape measure or a measuring rod. It is of
course the product of the size of the actual field and the magnification.

TABLE SHOWING THE OBJECTIVES, OCULARS, ACTUAL SIZE

OF THE FIELD, MAGNIFICATION AND DIAMETER OF

THE FIELD ON THE SCREEN WITH THE

APPARATUS IN FIG. 204.

Distance of the microscope from the screen, 8 meters (26 -f feet}. Arc lamp
for illumination. Constant electric current of no volts at the dynamo : 12 amperes
going through the lamp.

OBJECTIVES

OCULARS

ACTUAL
DIAMETER OF
FIELD

MAGNIFICA-
TION

DIAMETER OF
THE FIELD ON
THE SCREEN

105 mm.

None

50. mm.

80 diam.

4000 mm.

64 mm.

27.5

116

3190

50

12.85

207.5

2666+

42

23-

155

3565

24

ii. 5

326

3749

16

6.

504

3024

16

Projection X2

1-4

980

1372

16

X2*

3-25

930

3022+

16

X4

1.25

1970

2462+

8

None

2.625

990

2599—

8

Projection X2

.625

2002

1251

8

X2*

1-5

1940

2910

8

X4

.65

4060

2639

12.5

None

4-25

600

2550

6

2.25

1 120

2520

5

1-5

I600

2400

4

1.5

1680

2520

3

•9

2680

2412

2

• 5375

4OOO

2150

CH. IXa\

PROJECTION MICROSCOPE

FIG. 212 B. Leitz large projection apparatus with the microscope turned aside
and the 300 mm. equivalent focus objective in place for projecting a lantern slide.

There are three objectives on a large revolving nose-piece. These serve for
condensers for different powers in micro-projection, and when the microscope is
turned aside, for projecting lantern slides and other large objects.

FIG 2 f3 A. Sectional view of the condenser
system and water bath of the Bausch & Lomb
Optical CoSs Projection apparatus (from the
Journal of Applied Microscopy, VI, /poj).
This condenser system is used also in the ap-
paratus show in Fig. 204.

2670

PROJECTION MICROSCOPE

FIG. 213 B. The Micro- Projection apparatus of the Bausch & Lomb Optical
Co. (From the Jour. Ap. Micr. Vol. VI.}

FIG 213 C. End view of the Bausch & Lomb Projection apparatus showing
the means by which with the same instrument either the microscope or the ordi-
nary projection lens for lantern slides can be brought into position. In this figure
the microscope is in position. (From the Journal of Applied Microscopy,
Vol. VI.)

CH.IXa}

PROJECTION MICROSCOPE

26yd

FIG 213 D. The Bausch & Loinb Optical Co* s projection apparatus for objects
in liquids, etc. As shown the direction of the beam from the lamp is reflected up-
ward by a 45 degree mirror and after traversing the object and the microscope or
ordinary lantern objective, it is again changed go degrees by means of a prism and
passes off horizontally to the screen. From the prefection of the reflecting appliances
very little light is lost. (From the Jour. Ap. Micr. Vol. VI}

§426. How to demonstrate with the Micro-Projection Ap-
paratus.— Microscopical preparations are not so easily used as are lan-
tern slides. The writer has found that the most successful method is
for the teacher himself to stand by the apparatus, insert the specimens,
and find exactly what he wishes his pupils to see. Then to point
them out a bamboo fish pole with sharp end is used. This should be
2-3 meters long and if held out in the diverging cone of light leaving
the microscope, a sharp shadow will be cast upon the image. With
this pointer one can indicate the part to be demonstrated even more
satisfactorily than as if he pointed them out directly on the screen.

§-427. Cleaning the Glass Surfaces of the Micro-projection
Apparatus. — Inasmuch as it is so difficult to make the light sufficienly
brilliant for micro-projection, it is of the greatest importance that all

26ye

PROJECTION MICROSCOPE

\_CH.IXa

glass surfaces be kept as clean as possible. The lenses of the lamp
condenser should be carefully wiped occasionally ; and the water bath
should be opened and the plane glass faces thoroughly cleaned. It
is desirable to soak them in the cleaning mixture for glass. There is
always a certain amount of deposit on the glass even though distilled
water is used. Every grade of opacity renders the image on the screen
less excellent. Cleanliness is one of the most important requirements
for successful micro-projection.

Bach preparation should be wiped off before it is put in position
on the stage. Any particles of dust are painfully evident in the pro-
jected image.

FIG. 214 A

FIG. 214 A. The Combined Projection Lantern, Microscope and Mediascope of
Williams, Brown and Earle (cut loaned by Williams, Brown & Earle}.

As shown in the figure an ordinary lantern slide objective, microscope and a
mediascope are mounted approximately parallel on a single cast iron frame.

A, single arc lamp with a lamp condenser answers for all. It may be moved
opposite the piece of apparatus to be used and thus serve for givivg the proper illum-
ination. It is mechanically centered laterally for each by a click device.

The mediascope, A. is a combination of achromatic lenses of large aperture
capable of projecting objects 30 mm. or more in diameter. The objective is made
adjustable in focus so that the screen may be filled with the image of objects which
vary considerably in size.

CH. IX a}

PROJECTION MICROSCOPE

26yf

FIG. 214

FIG. 214. Zeiss Epidiascope for Opaque Objects, and for Transparent Objects
in a Horizontal Position (Zeiss' Special Catalog}.

As shown in this figure the apparatus is set up for opaque objects. For trans-
parent objects M2 ( mirror 2) is removed when the light striking M^ is reflected to
M* and thence up through the object to M1 and to the screen.

Commencing at the right : R. Parabolic reflector, which projects the light from
the crater through ( W] the water bath to M2 the mirror which is at the proper an-
gle for reflecting the light down upon the opaque object. From the opaque object
the light is irregularly reflected through the objective to Ml. M1 serves to reflect
the rays from the objective to the screen.

V. Ventilator. M* and M* are mirrors for use in reflecting the light through
horizontal transparent objects.

This apparatus is designed to project opaque objects as large as 22 centimeters
in diameter, at a magnification of 5 to 10 with a jo ampere current. For a smaller
object one may magnify as high as 25 diameters. With a 50 ampere current and a
larger reflector the magnification may be from 14 up to j/ diameters.

PROJECTION MICROSCOPE

\CH. IX a

PROJECTION OF OPAQUE OBJECTS

$ 428. Episcope. For the projection of opaque objects like anatomical prep-
arations, figures in books, coins or indeed any opaque object an apparatus on the
principle of the one figured (Fig. 214) is used. That is, a powerful light is thrown
upon the opaque object and the rays reflected from the object are then projected
upon the screen by an objective as for a lantern slide. As the objects are mostly
in a horizontal position the objective points directly upward, and the rays from it
must be made horizontal by means of a 45 degree mirror or prism.

This apparatus is very old. Its first name was "aphengescope" or opaque lan-
tern. Now it is called an episcope, or megascope, and if for both opaque and
transparent objects (Fig. 214) it is designated as an epidiascope.

FIG. 214 B.

FIG. 214 B. Spencer- Winkel Mechanical Stage, New Form. In this new
form of the Spencer- Winkel mechanical stage the milled heads for working the
screws are placed close together so that they can be turned without changing the

CH. IX a} PROJECTION MICROSCOPE

position of the hand. It is a very convenient form of attachable mechanical stage
on account of its wide range of movement and because it receives double slides,
50x76 mm. and those of standard size.

ACKNOWLEDGEMENT

The Author wishes to express his appreciation of help obtained from Sibley
College in designing and constructing the lathe bed and the movable pieces carry-
ing the parts of the projection microscope shown in Fig. 204. Thanks are
especially due to J. L. Morris, Sibley Professor of Practical Mechanics and
Machine Construction for the interest shown and the very practical help in direct-
ing the work. Thanks are also due to Mr. James Wiseman, Foreman in the
Machine Shop for the accuracy and excellence with which the work was carried
to completion.

BLACKENING TABLES

As stated in the preceding pages it is a great advantage to have everything
dead black in connection with the Projection Microscope. It is also desirable to
have photographic tables and laboratory tables black. During the last few years
an excellent method of dying wood with anilin black has been devised. This
black is lustreless, and it is indestructible. It can be removed only by scraping
off the wood to a point deeper than the stain has penetrated.

It must be applied to unwaxed or unvarnished wood. If wax, paint or var-
nish has been used on the tables, that must be first removed by the use of caustic
potash or soda or by scraping or planing. Two solutions are needed :

SOLUTION A

Copper sulphate 125 grams

Potassium chlorate or permanganate 125 grams

Water icoo cc.

Boil these ingredients in an iron kettle until they are dissolved. Apply two
coats of the hot solution. Let the first coat dry before applying the second.

SOLUTION B

Anilin Oil 120 cc.

Hydrochloric Acid 180 cc.

Water icoo cc.

Mix these in a glass vessel putting in the water first. Apply two coats without
heating, but allow the first coat to dry before adding the second.

When the second coat is dry, sand paper the wood and dust off the excess
chemicals. Then wash the wood well with water. When dry sand paper the
surface and then rub thoroughly with a mixture of equal parts turpentine and
linseed oil. The wood may appear a dirty green at first but it will soon become
ebony black. If the excess chemicals are not removed the table will crock. An
occasional rubbing with linseed oil and turpentine or with turpentine alone will
clean the surface. This is sometimes called the Danish method, Denmark black
or finish. See Jour. Ap. Micr., Vol. I, p. 145; Bot. Zeit., Vol. 54, p. 326, Bot.
Gazette, Vol. 24, p. 66, Dr. P. A. Fish, Jour. Ap. Micr., Vol. VI., pp. 211-212.

CHAPTER X

THE ABBE TEST PLATE AND APERTOMETER ; EQUIVA-
LENT FOCUS OF OBJECTIVES AND OCULARS ; DRAW-
INGS FOR PHOTO-ENGRAVING ; WAX MODELS

\ 429. On the Method of Using Abbe's Test-Plate. — This test-plate is in-
tended for the examination of objectives with reference to their corrections for
spherical and chromatic aberration and for estimating the thickness of the cover-
glass for which the spherical aberration is best corrected.

"The test-plate consists of a series of cover-glasses ranging in thickness from
0.09 mm. to 0.24 mm., silvered on the under surface and cemented side by side on
a slide. The thickness of each is written on the silver film. Groups of parallel
lines are cut through the film and these are so coarsely ruled that they are easily
resolved by the lowest powers, yet from the extreme thinness of the silver they
form a very delicate test for objectives of even the highest power and widest
aperture. To examine an objective of large aperture the plates are to be focused
in succession observing each time the quality of the image in the center of the
field and the variation produced by using alternately central and very oblique
illumination. When the objective is perfectly corrected for spherical aberration
for the particular thickness of cover-glass under examination, the contour of the
lines in the center of the field will be perfectly sharp by oblique illumination
without any nebulous doubling or indistinctness of the minute irregularities of
the edges. If after exactly adjusting the objective for oblique light, central
illumination is used no alteration of the adjustment should be necessary to show
the contours with equal sharpness. ' '

"If an objective fulfills these conditions with any one of the plates it is free
from spherical aberration when used with cover-glasses of that thickness : on the
other hand if every plate shows nebulous doubling or an indistinct appearance of
the edges of the silver lines, with oblique illumination, or if the objective requires
a different adjustment to get equal sharpness with central as with oblique light,
then the spherical correction is more or less imperfect."

"Nebulous doubling with oblique illumination indicates overcorrection of the
marginal zone, want of the edges without marked nebulosity indicates under-
correction of this zone ; an alteration of the adjustment for oblique and central
illumination, this is, a difference of plane between the image in the peripheral
and central portions of the objective points to an absence of concurrent action of
the separate zones, which may be due to either an average under or overcorrection
or to irregularity in the convergence of the rays. ' '

"The test of chromatic correction is based on the character of the color bands,
which are visible by oblique illumination. With good correction the edges of the

CH. X}

TEST PLATE AND APERTOMETER

269

silver lines in the center of the field should show but narrow color bands in the
complementary colors of the secondary spectrum, namely, on one side yellow-
green to apple-green on the other violet to rose. The more perfect the correction
of the spherical aberration the clearer this color band appears."

"To obtain obliquity of illumination extending to the marginal zone of the
objective and a rapid interchange from oblique to central light Abbe's illuminat-
ing apparatus is very efficient, as it is only necessary to move the diaphragm in
use nearer to or further from the axis by the rack and pinion provided for the
purpose. For the examination of immersion objectives, whose aperture as a
rule is greater than 180° in air and those homogeneous-immersion objectives,
which considerably exceed this, it will be necessary to bring the under surface of
the Test-plate into contact with the upper lens of the illluminator by means of a
drop of water, glycerin or oil."

"In this case the change from central to oblique light may be easily effected by
the ordinary concave mirror but with immersion lenses of large aperture it is im-
possible to reach the marginal zone by this method, and the best effect has to be
searched for after each alteration of the direction of the mirror."

"For the the examination of objectives of smaller aperture (less than 4o°-5o°)
we may obtain all the necessary data for the the estimation of the spherical and
chromatic corrections by placing the concave mirror so far laterally, that its edge
is nearly in the line of the optic axis the incident cone of rays then only filling
one-half of the aperture of the objective. The sharpness of the contours and the
character of the color bands can be easily estimated. Differences in the thickness
of the cover-glass within the ordinary limits are scarcely noticeable with such
objectives."

"It is of fundamental importance in employing the test as above described to
have brilliant illumination and to use an eye-piece of high power."

"When from practice the eye has learnt to recognize the finer differences in
the quality of the contour images this method of investigation gives very trust-
worthy results. Differences in the thickness of cover glasses of o.oi or 0.02 mm.
can be recognized with objectives of 2 or 3 mm. focus."

"With oblique illumination the light must always be thrown perpendicularly
to the direction of the lines.

FIG. 215. The Abbe Test Plate.

"The quality of the image outside the axis is not dependent on spherical and
chromatic correction in the strict sense of the term. Indistinctness of the con-
tours towards the borders of the field of view arises as a rule, from unequal mag-
nification of the different zones, of the objective ; color bands in the peripheral

270

TEST PLATE AND APERTOMETER

\_CH.X

portion (with good color correction in the middle) are caused by unequal magnifi-
cation of the different colored images."

"Imperfections of this kind, improperly called "curvature of the field," are
shown to a greater or less extent in the best objectives, where the aperture is con-
siderable."

FIG. 216. Abbe Apertometer.

\ 430. Determination of the Aperture of Objectives with an Apertometer. —
Excellent directions for using the Abbe apertometer may be found in the Jour.
Roy. Micr. Soc., 1878, p. 19, and 1880, p. 20 ; in Dippel, Zimmermann and Czapski.
The following directions are but slightly modified from Carpenter-Dallinger, pp.
394-396. The Abbe apertometer involves the same principle as that of Tolles, but
it is carried out in a simpler manner ; it is shown in Fig. 216. As seen by this figure
it consists of a semi-circular plate of glass. Along the straight edge or chord the
glass is beveled at 45°, and near this straight edge is a small, perforated circle, the
perforation being in the center of the circle. To use the apertometer the micro-
scope is placed in a vertical position, and the perforated circle is put under the mi-
croscope and accurately focused. The circular edge of the apertometer is turned
toward a window or plenty of artificial light so that the whole edge is lighted.
When the objective is carefully focused on the perforated circle the draw-tube is
removed and in its lower end is inserted the special objective which accompanies
the apertometer. This objective and the ocular form a low power compound mi-
croscope, and with it the back lens of the objective, whose aperature is to be meas-
ured, is observed. The draw-tube is inserted and lowered until the back lens of
the objective is in focus. "In the image of the back lens will be seen stretched
across, as it were, the image of the circular part of the apertometer. It will ap-
pear as a bright band, because the light which enters normally at the surf ace is re-
flected by the beveled part of the chord in a vertical direction so that in reality a
fan of 1 80° in air is formed. There are two sliding screens seen on either side of
the apertometer ; they slide on the vertical circular portion of the instrument.
The images of these screens can be seen in the image of the bright band. These
screens should now be moved so that their edges just touch the periphery of the back
lens. They act, as it were, as a diaphragm to cut the fan and reduce it, so that its
angle just equals the aperature'of the objective and no more." "This angle is
now determined by the arc of glass between the screens ; thus we get an angle in
glass the exact equivalent of the aperature of the objective. ' As the numerical ap-
ertures of these arcs are engraved on the apertometer they can be read off by inspec-
tion. Nevertheless a difficulty is experienced, from the fact that it is not easy to

CH. X} TEST PLATE AND APERTOMETER 271

determine the exact point at which the edge of the screen touches the periphery
of the back lens, or as we prefer to designate it, the limit of aperture, for curious
as the expression may appear we have found at times that the back lens of an ob-
jective is larger than the aperture of the objective requires. In that case the
edges of the screen refuse to touch the periphery. ' '

In determining the aperture of homogeneous immersion objectives the proper
immersion fluid should be used as in ordinary observation. So, also, with glycerin
or water immersion objectives.

\ 431. Testing Homogeneous Immersion Liquid. — In order that one shall
realize the full benefit of the homogeneous immersion principle it is necessary
that the homogeneous immersion liquid shall be truly homogeneous. In order
that the ordinary worker may be able to test the liquid used by him, Professor
Hamilton L. Smith devised a tester composed of a slip of glass in which was
ground accurately a small concavity and another perfectly plain slip to act as
cover. (See Proc. Amer. Micr. Soc.; 1885, p. 83). It will be readily seen that
this concavity, if filled with, air or any liquid of less refractive index than glass,
will act as a concave or dispersing lens. If filled with a liquid of greater refractive
index than glass, the concavity would act like a convex lens, but if filled with a
liquid of the same refractive index as glass, that is, liquid optically homogeneous
with glass, then there would be no effect whatever.

In using this tester the liquid is placed in the concavity and the cover put on.
This is best applied by sliding it over the glass with the concavity. A small
amount of the liquid will run between the two slips, making optical contact on
both surfaces. One should be careful not to include air bubbles in the concavity.
The surfaces of the glass are carefully wiped so that the image will not be ob-
scured. An adapter with society screw is put on the microscope and the objective
is attached to its lower end. In this adapater a slot is cut out of the right width
and depth to receive the tester which is just above the objective. As object it
is well to employ a stage micrometer and to measure carefully the diameter of the
field without the tester, then with the tester far enough inserted to permit of the
passage of rays through the glass but not through the concavity, and finally the
concavity is brought directly over the back lens of the objective. This can be
easily determined by removing the ocular and looking down the tube.

Following Professor Smith's directions it is a good plan to mark in some way
the exact position of the tube of the microscope when the micrometer is in focus
without the tester, then with the tester pushed in just far enough to allow the light
to pass through the plane glass and finally when the light traverses the concavity.
The size of the field should be noted also in the three conditions (\ 50-52. )

It will be seen by glancing at the following table that whenever the liquid in the
tester is of lower index than glass, that the concavity with the liquid acts as a
concave lens, or in other words like an amplifier (p. 109), and the field is smaller
than when no tester is used. It will also be seen that as the liquid in the concav-
ity approaches the glass in refractive index that the field approaches the size
when no tester is present. It is also plainly shown by the table that the greater
the difference in refractive index of the substance in the concavity and the glass,
the more must the tube of the microscope be raised to restore the focus.

If a substance of greater refraction than glass is used in the tester the field
would be larger, i. e., the magnification less, and one would have to turn the tube
down instead of up to restore the focus.

272

TEST PLATE AND APERTOMETER

CH. X-]

The table given below indicates the points with a tester prepared by the Gund-
lach Optical Co., and used with a 16 mm. apochromatic objective of Zeiss, X4
compensation ocular, achromatic condenser, i.oo N. A. (Fig. 41) :

Tester and L,iquid in the Concavity

Size of the
Field

Klevation of the Tube
necessary to
Restore the Focus

No tester used

1.825 mm.

Standard position.

Whole thickness of the tester at one end,
not over the cavity

.85 mm. _

No change of focus.

Tester with water . _____ . _ _ .

.075 mm.

Tube raised 3^ mm.

Tester with 95 % alcohol

15 mm.

3 mm.

Tester with kerosene

.4 mm.

2 mm.

Tester with Gundlach Opt. Go's horn, liquid

.825 mm.

TTfTT mm.

Bausch & L/omb Opt. Co.'s horn, liquid
L/eitz' horn, liquid

.825 mm.
.825 mm.

.... T2o°omm.
T27T°7T mm.

Zeiss' horn liquid

.825 mm.

i2rnr mm.

\ 432. Equivalent Focus of Objectives and Oculars. — To work out in proper
mathematical form or to ascertain experimentally the equivalent foci of these
complex parts with real accuracy would require an amount of knowledge and of
apparatus possessed only by an optician or a physicist. The work may be done,
however, with sufficient accuracy to supply most of the needs of the working
microscopist. The optical law on which the following is based is : — "The size of
object and image varies directly as their distance from the center of the lens. ' '

By referring to Figs. 14, 16, 21, it will be seen that this law holds good.
When one considers compound lens-systems the problem becomes involved, as the
centre of the lens systems is not easily ascertainable hence it is not attempted,
and only an approximately accurate result is sought.

| 433. Determination of Equivalent Focus of Objectives. — Look into the
upper end of the objective and locate the position of the back lens. Indicate the
level in some way outside of the objective. This is not the center of the object-
ive but serves as an arbitrary approximation. Screw the objective into the tube
of the microscope. If a Huygenian ocular is used with the ocular micrometer,
screw off the field lens and use the eye-lens only. If a positive ocular is used
no change need be made. Pull out the draw-tube until the distance between the
ocular micrometer and the back lens is 250 millimeters. Use a stage micrometer as
object and focus carefully. Make the lines of the two micrometers parallel
(Fig. 108). Note the number of spaces on the ocular micrometer required to
measure one or more spaces on the stage micrometer. Suppose the two microm-
eters are ruled in TV mm. and that it required 10 spaces on the ocular micrometer
to enclose 2 spaces on the stage micrometer, evidently then 5 spaces would cover
one. The image, A^1 Fig 21 in this case is five times as long as the object, A,B. —
Now if the size of object and image are directly as their distance from the lens it
follows that as the size of object is known (-£§ mm.), that of the image directly
measured (£$ mm.), the distance from the lens to the image also determined in
the beginning, there remains to be found the distance between the objective and
the object, which will represent approximately the equivalent focus. The general
formula is, Object, O: Image, I: [equivalent focus, F 1250. Supplying the known

CH.X] TEST PLATE AND APERTOMETER 273

values, O=T2ff, !=}§ ^en T2o m- : l mm- : : F : 25° whence F=5o mm. That is, the
equivalent focus is approximately 50 millimeters.

\ 434. Determination of Initial or Independent Magnification of the Objec-
tive. — The initial magnification means simply the magnification of the real image
(A'B1, Fig. 21) unaffected by the ocular. It may be determined experimentally
exactly as described in \ 433. For example, the image of the object (T2ff mm.)
measured by the ocular micrometer, at a distance of 250 mm. is yg mm., i. <?., it is
five times magnified, hence the initial magnification of the 50 mm. objective is
approximately five.

Knowing the equivalent focus of ah objective, one can determine its initial
magnification by dividing 250 mm. by the equivalent focus in millimeters. Thus
the initial magnification of a 5 mm. objective is -f- — 50 ; of a 3 mm., -2-jp =83.3 ;
of a 2 mm., *jp= 125, etc.

\ 435. Determining the Equivalent Focus of an Ocular. — If one knows the
initial magnification of the objective (\ 434) the approximate equivalent focus of
the ocular can be determined as follows :

The field lens must not be removed in this case. The distance between the
position of the real image, a position indicated in the ocular by a diaphragm, and
the back lens of the objective should be made 250 mm., as described in \ 433, 434,
then by the aid of Wollaston's camera lucida the magnification of the whole mi-
croscope is obtained, as described in \ 1 60. As the initial power of the objective
is known, the power of the whole microscope must be due to that initial power
multiplied by the power of the ocular, the ocular acting like a simple microscope
to magnify the real image (Fig. 21 ).

Suppose one has a 50 mm. objective, its initial power will be approximately 5.
If with this objective and an ocular of unknown equivalent focus the magnification
of the whole microscope is 50, then the real image or initial power of the objective
must have been multiplied 10 fold. Now if the ocular multiplies the real image
10 fold it has the same multiplying power as a simple lens of 25 mm. focus, for,
using the same formula as before : 0 = 5:1 = 50 :: F: 250 whence F = 25. The
matter as stated above is really very much more complex than this, but this gives
an approximation.

For a discussion of the equivalent focus of compound lens-systems, see
modern works on physics ; see also C. R. Cross, on the Focal Length of Micro-
scopic Objectives, Franklin Institute Jour., 1870, pp. 401-402; Monthly Micr.
Jour., 1870, pp. 149-159 J. J. Woodward on the Nomenclature of Achromatic
Objectives, Amer. Jour. Science, 1872, pp. 406-414 ; Monthly Micr. Jour., 1872, pp.
66-74. W. S. Franklin, method for determining focal lengths of microscope
lenses. Physical Review, Vol. f, 1893, p. 142. See pp. 1119-1131 of Carpenter-
Dallinger for mathematical formulae ; also Daniell, Physics for medical students ;
Czapski, Theorie der optischen Instrumente ; Dippell, Nageli und Schwendener,
Zimmermann. E. M. Nelson, J. R. M. S. 1898, p. 362, 1900, pp. 162-169. Jour.
Quekett Micr. Club, vol. V. pp. 456, 462.

| 436. Drawings for Photo- Engraving. — The inexpensive processes of repro-
ducing drawings bring within the reach of every writer upon scientific subjects
the possibility of presenting to the eye by diagrams and drawings the facts dis-
cussed in the text. Though artistic ability is necessary for perfect representation
of an object, neatness and care will enable anyone to make a simple illustrative draw-
ing, from which an exact copy can be obtained and a plate prepared for printing.

274 APPARA TUS FOR SECTIONING \_CH. X

A careful study of the cuts or plates used to illustrate the same class of facts
as one wishes to show will enable one to produce similar effects. Out-
lines which are transferred to the drawing paper may be obtained by the camera
lucida or from a photograph. The drawing should be made so that it can be
reduced anywhere from one-eighth to one-half. For ordinary photo-engraving
for such line drawings as are used to illustrate this book, use perfectly black
carbon ink. A shaded or wash drawing can be reproduced by the half-tone
process, also photographs as is illustrated by figures 190-191. A crayon drawing
on stipple paper with shadows re-enforced by ink lines and high lights scratched out
with a sharp knife give admirable results for anatomical figures by the half-tone
process. (See for example the work of Max Broedel in Contributions to the Science
of Medicine, (Welch Book) Baltimore, 1900).

For photo-engravings of line work the letters, figures or words used to desig-
nate the different parts can be put on the drawing by pasting letters, etc., of the
proper size in the right position. In preparing the block the photo-engraver
eliminates all shadows and the letters look as if printed on the drawings.

$ 437. Wax Models. — Large wax models of the objects which one studies
tinder the microscope are helpful both to the teacher and to the investigator.
These models are becoming more and more appreciated for embryologic and
morphologic investigations, for, as one can readily appreciate, the effort to produce
a representation of the embryo or organ in three dimensions helps to overcome
difficulties which are almost insurmountable if studied in the sections alone.

They are made from wax plates, the principle involved being that the diame-
ter of the drawing on the wax plate is as much greater than the object as the wax
plate is thicker than the section.

The wax plate is cut with a sharp instrument, following the outlines of the
object which has been traced upon it by the aid of a camera lucida or the projec-
tion microscope. The sections are piled together, some line or lines obtained
from a drawing or photograph of the specimen before it was imbedded and sec-
tioned being used as a guide by which the correct form of the pile of sections can
be tested. Finally the whole is welded into one by the use of hot wax or a hot
instrument. Models which illustrate complex internal structures are difficult to
prepare, but numerous devices will occur to the worker as the representation of
blood vessels and nerves by strings or wires. A large model will need much sup-
port which can be given by wire gauze, wires, pins or paper according to the
special needs.

A practical method for wax modeling was first published by G. Born, Arch. f.
Mikr. Anat., Bd. xxii, 1883, p. 584. The most, detailed statements of improve-
ments of the method have been published by Born (Bohm u. Oppel) 1900, and by
Dr. F. P. Mall and his assistants. See contributions to the Science of Medicine,
pp. 926-1045. Proceedings of the Amer. Assoc. Anatomists, 1901, I4th session
(1900) p. 193.

$ 438. Some Apparatus for Imbedding and Sectioning. — As a supplement to
Chapter VIII, the following figures of imbedding and sectioning apparatus are
appended. It will be noticed that the microtomes are complex and consequently
expensive. One is figured in which the knife is moved by the hands of the oper-
ator (Fig. 217). This form of instrument is excellent, and with it one can do
all kinds of work, both with collodion and paraffin. One cannot work so rapidly

CH.X-]

APPARATUS FOR SECTIONING

275

nor with the same precision. For much of the work one may section free-hand,
without a microtome. Indeed the great basis of histological and embryological
knowledge was gained by studying free-hand sections and dissections. At the
present time there is a strong reaction against the exclusive study of sections,
and a tendency to combine with the serial sections dissections such as the older
anatomists and embryologists made and gained so much from.

FIG. 217. A Microtome for
all kinds of sectioning ; the
knife is guided by the top of the
microtome, but moved by the
hands of the operator (Bausch &
Lomb Optical Co. )

FIG. 218. The Minot microtome for ribbon sections as made by Bausch and
Lomb Optical Co. It is arranged for sections from /// to 25^1 and any intermedi-
ate thickness.

276

APPARATUS FOR SECTIONING

[ CH. X

FIG. 219. The Minot microtome for ribbon sections as made by the Franklin
Laboratory Supply Co., Boston. This is to be made for 2ju, 6ju, ion, 141*, 20^1, and,
30)JL. sections.

FIG. 220.

FIG. 221. A.

CH.X}

APPARATUS FOR SECTIONING

277

FIG. 221. B.

FIG. 221. C

A B

FIG. 222.

FIG. 220-222. A paraffin holder clamp and a razor support for the Minot Mi-
crotome. ( Trans. Amer. Micr. Soc., igoi}.

FIG. 220. Clamp for the paraffin block holder. In A it is shown in section, in
a side view. With this clamp one can use stove bolts as well as the expensive par-
affin holders furnished with the instrument. A laboratory can have as many par-
affin block holders as necessary without undue expense. '

FIG. 221. Razor Support and Razor.

(A] Support with heavy base and vertical piece. The base should be capable
of moving endwise one or two centimeters to bring the opening in the vertical part
opposite the paraffin block.

(B} Front piece to the razor (see Fig. 222 A ).

( O Razor with straight back and edge. By moving this back and forth on
the support nearly the entire cutting edge can be utilized.

FIG. 222. The knife support of the microtome with the razor support and
razor in position.

(A] Front view ; (B) Back view. In the inclination of the knife toward the
paraffin block is shown.

278

APPARA TVS FOR SECTIONING

\_CH.X

FIG. 223. Sliding microtome -adapted especially for collodion sectioning. ( The
Bausch & Lomb Optical Co.).

CH. X]

APPARA TVS FOR SECTIONING

279

FIG. 224 Paraffin dish for infiltrating in the Lillie oven.
per and as shown has a handle for ease in transference.
dish in section. (Jour. Appl. Micr. 1809, p. 266).

It is made of cop-
A the whole dish, B the

FIG. 225. The Lillie compartment, paraffin oven for infiltrating tissues with
paraffin. Various sizes of this are made ( 8, 16 and 24 compartments). Except for
the largest laporatories the one with 16 compartments and trays will be found of suf-
ficient capacity. ( Bausch & Lomb Optical Co. ).

280

APPARATUS FOR SECTIONING

\CH:X

FIG. 226. Circulation board, especially for Necturus. This is prepared from
a board about 8 x 20 centimeters. Near one edge it has a hole for a perforated cork.
On the top of the cork is cemented a thick cover-glass with shellac or rubber cement.
The cork can be raised or lowered in the board. The gills of Necturus or the
web of a frog"1 s foot can be spread out on glass over the cork. (four. Appl. Micr. ,
1898, p. 131.

FIG. 227. Copper can with screw top for collecting
embryologic material and small aquatic animals. It was
especially designed for collecting with a bicycle (Jour.
Appl. Micr., 1898, p. 131).

FIG. 228. Egg pipette. This is made by putting a
short piece of soft rubber tubing over the end of a glass
pipette with rubber bulb. With this one can handle the
eggs both fresh and hardened without any degree of in-
jury. (Jour. Appl. Micr. 1898, p.

FIG. 227.

FIG. 229. Washing apparatus for tissues fixed in osmic and chromium mix-
tures. As shown in the figure the apparatus is connected with the water pipe by a
small side cock. It is composed of a double vessel, the inner one being made of per-
forated brass. There are special perforated -dishes to insert in the little compart-
ments. This apparatus is convenient for washing cover-glasses, for the washing
out for iron hematoxylin, etc. The deeper box at the right answers for the slide
baskets or holders (Fig.

Fig. 223a. The Spencer Lens Company's New Table Micro-
tome for hand sectioning. The vertically moveable socket which
holds the object clamp is held by hardened steel pivot screws in
two vertically swinging arms similarly attached to the main frame,
providing a movement upon the parallelogram principle, regulated
by micrometer screw with graduated disc and index plate. Glass
surface plates for travelling ways for the knife.

Ji

Fig. 223b. The Spencer Lens Company's New Automatic
Laboratory Microtome, with parallelogram principle, giving a
curvilinear knife movement similar to that of free hand section-
ing; with automatic feed and with independent crank for quickly
raising or lowering the object. The convenient drip pan may be
quickly unscrewed. Furnished when desired with Gaylord's
freezing attachment for CO2 .

The joints are provided with hardened steel pivot screws and
with check nuts. Lubrication not required.

CH.X]

APPARATUS FOR SECTIONING

281

FIG. 229 A. — Same as the preceding with the inner, perforated box on edge.

Name of Article

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From

Cost,

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toned }

Address • m

Order No. I -I 3 ~ Date JLlA.<\AJU [tf^ \

Date of receipt of Articles Hx\A_^/%A^ *£..|...|..£.<t.X.

Remarks

S

NEW YORK STATE

DEP'T. ... UtLt5,.tr.Q~^.OC£./rf VETERINARY COLLEGE,

CORNELL UNIVERSITY.

FIG. 230. An inventory card for the property in a department. The cards are
the standard sizefpr libraries. (Jour. Appl. Micr. , 1898, p. 124}.

BIBLIOGRAPHY

The books and periodicals named below in alphabetical order pertain wholly or in part to the
microscope or microscopical methods. They are referred to in the text by recognizable abbrevi-
ations.

For current microscopical and histological literature, the Journal of the Royal Microscopical
Society, the Zoologischer Anzeiger, and the Zeitschrift ftir wissenschaftliche Mikroskopie, Ana-
tomischer Anzeiger, Biologisches Centralblatt and Physiologisches Centralblatt, the Journal of
Applied Microscopy and laboratory methods and the smaller microscopical journals taken to-
gether furnish nearly a complete record.

References to books and papers published in the past may be found in the periodicals just
named, in the Index Catalog of the Surgeon General's library ; in the Royal Society's Catalog of
Scientific Papers, and in the bibliographical references given in special papers. A full list of peri-
odicals may also be found in Vol. XVI of the Index Catalog.

BOOKS.

Adams, G. — Micrographia illustrata, or the microscope explained, etc. Illustrated. 4th edi-
tion, I,ondon, 1771. Also Essays, 1787.

AngstrOm. — Recherches sur le spectre solaire, spectre normal du soleil. Upsala, 1868.

Anthony, Wm. A., and Brackett, C. F.— Elementary text- book of physics. 7th ed. Pp. 527, 165
Fig. New York, 1891.

Barker' — Physics. Advanced course. Pp. 902, 380 Fig. New York, 1892.

Bausch, E. — Manipulation of the Microscope. A manual for the work table and a text-book
for the beginners in the use of the microscope, illustrated. New Edition, 1901. Rochester, N. Y.

Beale, I,. S.— How to work with the microscope. 5th ed. Pp. 518, illustrated. lyondon, 1880.
Structure and methods.

Beauregard H., et Galippe, V. — Guide de 1'eleve et du praticien pour les travaux pratiques de
micographie, comprenant la technique et les applications du microscope & 1'histologie vegetale,
h, la physiologic, a. la clinique, & la hygiene et & la medicine legale. Pp. 904, 570 Fig. Paris, 1880.

Behrens, H — Mikrochemische Technik, 2d ed. Pp. 68. Hamburg, 1900.

Behrens, T. H. — Anleitung zur microchemischen Analyse der wichtigsten organischen Ver-
bindungen. Hamburg, 1895-1897.

Behrens, T. H. — Transl, by J. W. Judd. A manual of microchemical analysis with an intro-
ductory chapter by J. W. Judd. I,ondon, 1894.

Behrens, W.— The microscope in botany. A guide for the microscopical investigation of
vegetable substances. Translated and edited by Hervey and Ward. Pp. 466, illustrated. Boston,
1885.

Behrens, W.— Tabellen zum Gebrauch, bei mikroskopischen Arbeiten.' 3d edition. Pp. 237.
Braunschweig, 1898.

Behrens, W., Kossel, A., und Schiefferdecker, P.— Das Mikroscop und die Methoden der
mikroskopischen Uuntersuchung. Pp. 315, 193 Fig. Bravtnschweig, 1889 +.

Boehm, A. A. und von Davidoff, M. — A text-book of Histology, including microscopic tech-
nique, from the 2d German edition. Translated by H. H. Gushing and edited by G. C. Huber.
Pp. 501, illustrated. Philadelphia and I,ondon, 1900.

- Boehm, A. und Oppel, A. — Taschenbuch der mikroskopischen Technik, 4th ed. with direc-
tions by Born for making wax models. Pp. 240. Mtinchen, 1900.

Brewster, Sir David. — A treatise on the mikroscope. From the 7th ed. of the Encyc. Brit.,
with additions. Illustrated, 1837.

Brewster, .Sir David. — A treatise on optics. Illustrated. New edition. I,ondon, 1853.

Browning, J. — How to work with the micro-spectroscope. L,ondon, 1894.

BIBLIOGRAPHY 283

Bousfield, E. C. — Guide to photo-micrography. 2d ed. Illustrated. London, 1892.

Carnoy, J. B. L,e Chanoine. — La Biologic Cellulaire ; Etude comparee de la cellule dans les
deux regnes. Illustrated (incomplete). Paris, 1884. Structure and methods.

Carpenter, W. B. — The microscope and its revelations. 6th ed. Pp. 882, illustrated. London,
and Philadelphia, 1881. Methods and structure.

Carpenter- Dallinger. — The microscope and its revelations, by the the late William B. Carpen-
ter. 8th edition, in which the ist seven and the 23d chapters have been entirely re-written, and
the text throughout reconstructed, enlaiged and revised by the Rev. W. H. Dallinger. 22 plates
and nearly 900 wood engravings. Pp. 1181. London and Philadelphia, 1901.

Chamberlain, C. J. — Methods in plant histology, Illustrated. Chicago, 1901.

Clark, C. H. — Practical methods in microscopy. Illustrated. Boston, 1894.

Cooke, M. C. — One thousand objects for the microscope. Pp. 123. London, no date. 500 fig-
ures and brief descriptions of pretty objects for the microscope.

Crookshank, E. M. — Photography of bacteria. London and New York, 1887.

Cross and Cole.— Modern microscopy for beginners. Part I. The microscope and instruc-
tions for its use. Part II. Microscopic objects, how prepared and mounted. Illustrated. 2d
edition, London, 1895.

Czapski, Dr. Siegfried. — Theorie der optischen Instrumente nach Abbe. Illustrated. Bres-
lau, 1893.

Dana, J. D. — A system of mineralogy. Illustrated. 6th ed. New York, 1892.

Daniell, A. — A text-book of the principles of physics. Illustrated. 3d ed. London, 1895.

Daniell, A. — Physics for students of medicine. Illustrated. London and New York, 1896.

Davis, G. — Practical microscopy. 3d ed. Illustrated. London, 1895.

Dippel, L. — Das Mikroskop und seine Anwendung. Illustrated. Braunschweig, 1898.

Dodge, Charles Wright. — Introduction to elementary practical biology ; a laboratory guide for
high school and college students. New York, 1894.

Eberth, C. J. — Friedlander's mikroskopische Techtiik zum gebiauche bei mediciiiischen und
pathologische-anatomischen Untersuchungen. Pp. 359, illustrated. Berlin, 1900.

Ebner, V. V. — Untersuchungen tiber die Ursachen der Anisotropie organischer Substanzen.
Leipzig, 1882. Large number of references.

Ellenberger, W. — Handbuch der vergleichenden Histologie und Physiologic der Haussauge-
thiere. 2d edition. Berlin, 1901 -r.

Fol, H. — Lehrbuch der vergleichenden mikroskopischen Anatomic, mit Einschluss der ver-
gleichenden Histologie und Histogenie. Illustrated (incomplete). Leipzig, 1884. Methods and
structure.

Foster, Frank P. — An illustrated encyclopaedic medical dictionary, being a dictionary of the
technical terms used by writers on medicine and the collateral sciences in the Latin, English,
French and German languages. Illustrated, four quarto volumes. 1888-1893.

Fraenkel und Pfeiffer.— Atlas der Bacterien-Kunde. Berlin, 1889+.

Francotte, P. — Manuel de technique microscopique. Pp. 433, no Fig. Brussels, 1886.

Francotte, P.— Microphotographie appliquee a Thistologie, 1'anatomie comparee et 1'embryolo-
gie. Brussels, 1886.

Frey, H.— The microscope and microscopical technology. Translated and edited by G. R.
Cutter. Pp. 624, illustrated. New York, 1880. Methods and structure.

Gait, H. — The microscopy of the starches illustrated by photomicrographs. London, 1900.

Gamgee, A. — A text-book of the physiological chemistry o.f the animal body. Part I, pp. 487,
63 Fig. London and New York, 1880. Part, II, 1893.

Gebhardt, W. — Die mikrophotographische Aufnahme gefarbter Praparate. Illustrated.
Miinchen, 1899.

Giltay, Dr. E.— Sieben Objecte unter dem Mikroskop. Einf uhrung in die Grundlehren der
Mikroskopie. Leiden, 1893. This is also published in the Holland (Dutch) and French language.

Goodale, G. L- Physiological botany. Pp. 499 + 36, illustrated. New York, 1885. Structure
and methods.

284 BIBLIOGRAPHY

Griffith and Henfrey. — The Micrographic Dictionary ; a guide to the examination and investi-
gation of the structure and nature of microscopic objects. Fourth edition, by Griffith, assisted by
Berkeley and Jones. L,ondon, 1883.

Gould, G. M. — The illustrated dictionary of medicine, biology and allied sciences. Illustrated,
5th ed., Philadelphia, 1900. This is recognized as the best single volume medical dictionary. It is
especially satisfactory for the worker with the microscope.

Hager, H. und Mez. C. — Das mikroskop und Seine Anwendung. 8th ed., revised and en-
larged. Pp. 335. Illustrated. Berlin, 1899.

Halliburton, W. D. — A text-book of chemical physiology and pathology. Pp. 874, 104 illus-
trated. Condon and New York, 1891.

Hanausek, T. F. — L,ehrbuch der mikroskopischen Technik. Illustrated. Stuttgart, 1900.

Harker, H. — Petrology for students ; an introduction to the study of rocks under the micro-
scope. Cambridge, 1895.

Harting, P. — Theorie and algemeine Beschreibung des Mikroscopes. 2d ed. 3 vols. Braun-
schweig, 1866.

Herzfeld, T, J. — The technical testing of yarns and textile fabrics with reference to official
specifications. Translated by C. Salter. Illustrated. L,ondon, 1898.

Hogg, J.— The microscope, its histoiy, construction and application, isth edition, illustrated.
Pp. 704. L,ondon and New York, 1898. Much attention paid to to the polariscope.

Huber, G. C. — laboratory work in histology, 3d revised and enlarged edition. Pp. 204, illus-
trated. Ann Arbor, 1900.

James, F. I,., — Elementary microscopical technology. Part I, the technical history of a slide
from the crude material to the finished mount. Pp. 107, illustrated. St. L,ouis, 1887.

Jeserich, P. — Die mikrophotographie auf Bromsilbergelatine bei nattlrlichem und Ktinstli-
chem I^ichte. Figs, and Plates. Pp. 245. Berlin, 1888.

Jtlptner, Hanns Freiherr von. — Grtindzuge der Siderologie, Erster Teil, Leipzig, 1900. Full
discussion of theory of solution, chemidal constitution, micrography, etc., of iron alloys and
slags. Illustrated. Bibliography very complete. Part II not yet issued. "Will deal with relation
between chemical constitution, mechanical treatment, microscopic structure," etc.

Kahlden, C. von. — Technik der histologischen Unterschung pathologisch — anatomischer
Praparate. 6th revised and enlarged edition. Pp. 174. Jena, 1900.

Kaiser, W. — Die Technik des modernen Mikroskopes. 2d edition. Illustrated- Wien, 1901.

King, J. — The microscopist's companion. A popular manual of practical microscopy. Illus-
trated. Cincinnati, 1859.

Klement et Renard. — Reactions microchemiques a cristaux et leur application en analyse
qualitative. Pp. 126, 8 plates. Bruxelles, 1886.

Kraus, G. — Zur Kentniss der Chlorophyllfarbstoffe. Stuttgart, 1872.

L,ankaster, E. — Half-hours with the microscope ; a popular guide to the use of the microscope
as a means of amusement and instruction. . 2oth ed. Pp. 150. I,oiidon, 1898.

L,atteux, P. — Manuel de technique microscopique. 3d ed. Paris, 1887.

I,e Conte, Joseph. — Sight — an exposition of the principles of monocular and binocular vision.
Pp. 275, illustrated. New York, 1881.

Ivee, A. B. — The microtomist's vade-mecum. A hand-book of the methods of microscopic
anatomy. 5th ed. Pp. 532. Philadelphia, 1900.

L,ehmann, C. G. — Physiological chemistry. 2 vols. Pp. 648+547, illustrated. Philadelphia,
1855-

L,ehmann, O. — Molekularphysik mit besonderer Berilcksichtigung mikroskopischer Unter-
suchungen und Anleitung zu solchen, sowie einem Anhang liber mikroskopische Analyse. 2 vols.
Illustrated. Leipzig, 1888-1889.

lyeiss, C. — Die optischen Instruments der Firma R. Fuess, deren Beschreibung. Justierung
und Anwendung. Pp. 397, illustrated. Iyeipzig, 1899.

L,ockyer, J. N. — The spectroscope and its application. Pp. 117, illustrated. l,oiidon and New
York, 1873

BIBLIOGRAPHY 285

lAiquer, L,. Me. I. — Minerals in Rock Sections. Practical methods of identifying minerals in
rock sections with the microscooe.

M'Kendrick, J. G. — A text-book of physiology. Vol. I, general physiology. Pp. 516, illus-
trated. New York, 1888.

Mace, E. — I,es substances alimentaire etudies au microscope surtout au point de vue de leurs
alterations et de leur falsifications. Illustrated. Paris, 1891.

Macdonald, J. D. — A guide to the microscopical examination of drinking water. Illustrated.
London, 1875. Methods and descriptions.

MacMunn, C. A.— The spectroscope in medicine. Pp. 325, illustrated. Condon, 1885.

Marktanner-Turneretscher, G. — Die Mikro-Photographie als Hilfsmittel naturwissenchaft-
licher Forschung, Pp. 344, illustrated. Halle, a, S., 1890.

Martin. John H. — A manual of microscopic mounting with notes on the collection and exam-
ination of objects. 2d ed. Illustrated. Condon, 1878.

Mason, John J. — Minute structure of the central nervous system of certain reptiles and
batrachians of America. Illustrated by permanent photo-micrographs. Newport, 1879-82.

Matthews, C. G.. and I,ott, F. E. — The microscope in the brewery and the malt house.
Illustrated. London, 1889.

Mayall, John, Jr. — Cantor lectures on the microscope, delivered before the society for the
encouragement of arts, manufacturers and commerce. Nov.-Dec., 1885. History of the micro-
scope, and figures of many of the forms used at various times.

Meyer, A. Die Grundlagen und die Methoden fttr die mikroskopische Untersuchung von
Pflanzenpulvern. Pp. 258, illustrated. Jena, 1901.

Moeller, J. — Leitfaden zu mikroskopisch-pharmakgnostischen Ubungen. Pp. 336, illus-
trated. Wien, 1901.

Moitessier, A. — L,a photographic appliquee aux recherches micrographiques. Paris, 1866.

Morel et Soulie. — Manuel de technique microscopique. Paris. 1899.

Nageli und Schwendener.— Das Mikroskop, Theorie und Anwendung desselben. 2d ed. Pp.
647, illustrated. Leipzig, 1877.

Neuhauss, R.— Lehrbuch der Mikro-photographie. Pp. 266. Illustrated. 2d ed. revised.
Braunschweig, 1898.

Nichols, E. I,.— The outlines of physics. Illustrated, N. Y., 1897.

Nichols, E. L. and Franklin W. S.— The Elements of Physics ; Light and Sound. Pp. 201,
illustrated. New York and London, 1897.

Pappenheim, A. — Grundriss der Farbenchemie zum Gebrauch bei mikroscopischen Arbei-
ten. Pp.476. Berlin, 1901.

Petri, R. J. — Das Mikroskop von seinen Anfangen bis zur jetzigen Vervollkommung filr alle
Freunde dieses In stiuments. Pp.248. Illustrated. Berlin, 1896.

Phin, J. — Practical hints on the selection and use of the microscope for beginners. 6th ed.
Illustrated. New York, 1891.

Posselt, E. A. — The structure of fibres, yarns and fabrics. Illustrated. Philadelphia and
London, 1891.

Preyer, W. — Die Blutkrystalle. Jena, 1871. Full bibliography to that date.

Pringle, A.— Practical photo-micrography. Pp. 193, illustrated. New York, 1890.

Procter, R. A. — The spectroscope and its work. London, 1882.

Quekett, J. — A practical treatise on the use of the microscope, including the different methods
of preparing and examining animal, vegetable and mineral structures. Pp. 515, 12 plates. 2d ed.
London, 1852.

Rafter, G. W— The Microscopical Examination of Potable Water. Pp. 160, New York, 1892.

Ranvier, I,. — Traite technique d'histologie. Pp. 1109, illustrated. Paris, 1875-1888. Structure
and methods. Also German translation, 1888.

Reeves, J. E. — A hand-book of medical microscopy, including chapters on bacteriology, neo-
plasms and urinary examination. Illustrated. Philadelphia, 1894.

286 BIBLIOGRAPHY

Reference Hand-Book of the medical sciences. Albert H. Buck, editor. 8 quarto vols. Illus-
trated with many plates, and figures in the text. New York, 1885-1889. Supplement, 1893. New
edition, 1901 + .

Richardson, J. G. — A hand-book of medical microscopy. Pp. 333, illustrated. Philadelphia,
1871. Methods and descriptions.

Robin, ^Ch. — Traite du microscope et des injections. 2d ed. Pp. noi, illustrated. Paris, 1877.
Methods and structure.

Roscoe, Sir Henry. — lectures 011 spectrum analysis. 4th ed. I,ondon, 1885.

Rosenbusch, K. H. F. translated by Iddings, P. — Microscopical physiography of the rock
making minerals. Illustrated. New York, 1889.

Ross, Andrew.— The microscope. Being the article contributed to the "Penny Cyclopaedia."
Republished in New York in 1877. Illustrated.

Rusby, H. H. and Jeliffe, S. E. — Morphology and Histology of Plants designed especially as a
guide to plant analysis and classification, and an introduction to pharmacognosy and vegetable
physiol. Pp. 378, illustrated. New York, 1899.

Rutherford, W.— Outlines of practical histology. 2d. ed. Illustrated. Pp. 194. Condon and
Philadelphia, 1876. Methods and structure.

Sayre, I,. E. — Organic Materia Medica and Pharmacognosy. An introduction to the vegetable
kingdom and the vegetable and animal drugs. Chapters on insects injurious to drugs and on
pharmacal botany. Pp. 220, Detroit, 1880.

Schafer, E. A. — A course of practical histology, being an introduction to the use of the micro-
scope. Pp. 304, 40 Fig. Philadelphia, 1877. Methods.

Schellen, H. — Spectrum analysis, translated by Jane and Caroline L,assell. Edited with notes
by W. Huggins. 13 plates, including Angstrom's and Kirchhoff 's maps. L,ondon 1885.

Schimper, A. F. W. — Anleitung zur mikroskopischen Untersuchurigen der vegetabilischen
Genussmittel. Pp. 158, illustrated. Jena, 1900.

Schneider, A. — Microscopy and micro-technique. Pp. 189, illustrated. Chicago, 1899.

Sedgwick and Wilson. — General biology. 2d ed. Illustrated. New York, 1895.

Seiler, C. — Compendium of microscopical technology. A guide to physicians and students in
the preparation of histological and pathological specimens. Pp. 130, illustrated. New York, 1881.

Silliman, Benj., Jr, — Principles of physics, or natural philosophy. 2d ed., rewritten. Pp. 710,
722 illustrations. New York and Chicago, 1860.

Spitta, E. J. — Photomicrography. 4°. Pp. 163. Illustrated by half-tone reproductions from
original negatives. Text illustrations. L,ondon, 1899.

Starr, Allen M., with the cooperation of Oliver S. Strong and Edward Learning. — An atlas of
nerve-cells. Columbia University Press, New York, 1896. The atlas consists of text, diagrams,
and some of the best photo-micrographs that have ever been published.

vSternberg, G. M. — Photo-micrographs, and how to make them. Pp. 204, 20 plates. Boston,
1883.

Stoehr, S. P. — Text-book of histology including the microscopic technique, 3d American from
8th German edition. Translated by Dr. Emma Bilstein and edited by Dr. A. Schaper. Pp. 432,
illustrated. Philadelphia, 1901.

Stokes, A. — Aquatic microscopy for beginners, or common objects from the ponds and ditches.
Illustated. Portland, Conn., 1896.

Suffolk, W. T. — On microscopical manipulation. 2d ed. Pp. 227, Illustrated. L,ondon, 1870.

Szymonowicz, I,. — I^ehrbuch der Histologie-einschliesslich der mikroskopischen Technik.
Illustrated. Wtirzburg, 1900.

Thomas, Mason B., and Wm. R. Dudley. — A laboratory manual of plant histology. Illus-
trated. Crawfordsville, Ind., 1894.

Trelease, Wm. — Poulsen's botanical micro-chemistry ; an introduction to the study of veget-
able histology. Pp. 118. Boston, 1884. Mehods.

Trutat, M. — L,a photographic appliquee & histoire naturelle. Pp. 225, illustrated. Paris,
1884.

BIBLIOGRAPHY 287

Valentin, G. — Die Untersuchung der Pflanzen und der Thiergewebe in polarisirtem Licht.
Leipzig, 1861.

Van Heurck, H — The microscope. Illustrated. London and New York, 1898.

Vierordt. Die quantitative Spectralanalyse in ihrer Anwendung auf Physiologic, 1876.

Vogl, A. E. — Die wichtigsten vegetablischen Nahrungs und Genussmittel. Pp. 575., illus-
trated. Wien, 1899.

Vogel, Conrad.— Practical pocket-book of photography. Pp. 202, Figs. London, 1893.

Vogel. H. W.— Practische Spectralanalyse irdischer stoffe ; Anleitung zur Benutzung der
Spectralapparate in der qualitativen und quantitativen chemische Analyse organischer und un-
organscher Korper. 2d ed. Figs. Berlin 1889.

Wall, O. A.— Notes on Pharmacognosy.— Pp. 180, illustrated. St Louis, 1897. m

Weinschenk, E. Anleitung zum Gebrauch des Polarisationsmikroskopes. Pp. 123, illus-
trated. Freiburg. 1901.

Wethered, M. — Medical microscopy. Pp. 406, Figs. London and Philadelphia, 1892.

Whipple, G. C. — The Microscopy of Drinking Water. Pp. xii -f- 300. Illustrated. New York,
1899.

Whitman, C. O. — Methods of research in microscopical anatomy and embryology. Pp. 255,
illustrated. Boston, 1885.

Wilder and Gage. — Anatomical technology as applied to the domestic cat. An introduction to
human, veterinary and comparative anatomy. Pp. 575, 130 Fig. 2d ed. New York and Chicago,
1886.

Wilson, Edmund, B., with the cooperation of Edward Learning. — An atlas of fertilization
and karyokinesis. Columbia University Press, New York, 1895. This atlas marks an era in
embryological study. It has admirable texts and diagrams, but the distinguishing feature is the
large number of almost perfect photo-micrographs.

Wood, J. G. — Common objects for the microscope. Pp. 132. London, no date. Upwards of
400 figures of pretty objects for the microscope, also brief descriptions and direction for prepa-
rtions.

Wormly, T. G. — The micro-chemistry of poisons. 2d ed. Pp. 742, illus. Philadelphia, 1885.

Wright, Lewis. — Optical Projection, a treatise on the use of the lantern in exhibition and
scientific demonstration. Pp. 425, illustrated. London, 1891. (Beginners will find this book
very helpful).

Wythe, J. H. — The microscopist ; a manual of microscopy and a compendium of microscopi-
cal science. 4th ed. Pp. 434, 252 Fig. Philadelphia, 1880

Zimmermann, Dr. A. — Das Mikroskop, ein Leitfaden der wissenschaftlichen Mikroskopie.
Illustrated. Leipzig und Wein, 1895.

See also Watt's chemical dictionary, and the various general and technical encyclopedias.

PERIODICALS*

The American journal of anatomy (including histology, embryology and cytology). Balti-
more, 1901 + .

The American journal of medical research, Boston, 1901 + .

The American Journal of physiology. Boston, 1896+.

The American journal of microscopy and popular science. Illustrated. New York, 1876-1881.

The American monthly microscopical journal. Illustrated. Washington, D. C., 1880 + .

American naturalist. Illustrated. Salem and Philadelphia, 1867-!-.

American quarterly microscopical journal, containing the transactions of the New York
microscopical society. Illustrated. New York, 1878-4-.

American microscopical society, Proceedings. 1878-1894 ; Transactions, 1895+.

*NOTE— When a periodical is no longer published, the dates of the first and last volumes are
given ; but if still being published, the date of the first volume is followed by a plus sign.

See Vol. XVI of the index Catalog of the Library of the Surgeon General's office for a full
list of periodicals.

288 BIBLIOGRAPHY

Anatomischer Anzeiger. Centrablatt ftlr die gesammte wissenchaftliche Anatomic. Amt-
liches Organ der anatomischen Gesellschaft. Herausgegeben von Dr. Karl Bardeleben. Jena,
1886+. Besides articles relating to the microscope or histology, a full record of current anatomi-
cal literature is given.

Annales de la societe beige de microscopic. Bruxelles, 1874 f .

Archives d'Anatomie microscopique. Illustrated. Paris, 1897. (Balbiani et Ranvier).

Archiv ftir miroscopische Anatomic. Illustrated. Bonn, 1865+.

Centrablatt ftir Physiologic. Unter Mitwirkung der physiologischen Gesellschaft zu Berlin
Herausgegeben von S. Exner und J. Gad. Leipzig and Wien. 1887 + . Brief extracts of papers
having a physiological bearing. Full bibliography of current literature.

English mechanic. L,ondon, 1866+ . Contains many of the papers of Mr. Nelson on light-
ing, photo-micrography, etc.

Index medicus. New York, 1879+. Bibliography, including histology and microscopy.

International journal of micioscopy and popular science. L,otidon, 1890+.

Journal of anatomy and physiology. Illustrated. L,ondon and Cambridge, 1867+.

Journal of Applied Microscopy and Laboratory methods. Illustrated. Rochester, N. Y.,
1898+ .

Journal de micrographie. Illustrated. Paris, 1877-1892.

Journal of microscopy and natural science. L,ondon, 1885 + .

Journal of the New York microscopical society. Illustrated. New York, 1885+.

Journal of physiology. Illustrated. L,ondon and Cambridge, 1878+.

Journal of the American chemical society. New York, 1879+.

Journal of the chemical society. L/ondon, 1848 + .

Journal of the royal microscopical society. Illustrated. L,ondon, 1878+. Bibliography of
works and papers relating to the microscope, microscopical methods and histology. It also in-
cludes a summary of many of the papers.

Journal of the Quekett microscopical club. L/ondon, 1868 + .

The L,ens, a quarterly journal of microscopy, and the allied natural sciences, with the tran-
sactions of the state microscopical society of Illinois. Chicago, 1872-1873.

The Metallographist, a quarterly publication devoted to the study of metals with special
reference to their physics, microstructure, their industrial treatment and application. Illustrated
especially by photo-micrographs of metals and alloys. Boston, 1898+.

The Microscope. Illustrated. Washington, D. C., 1881-1897.

Microscopical bulletin and science news. Illustrated. Philadelphia, 1883 + . The editor, Ed-
ward Pennock introduced the term "par-focal " for oculars (see vol. iii, p 31).

Monthly microscopical journal. Illustrated. L,ondon, 1869-1877.

Nature. Illustrated. London, 1869+.

The Observer. Portland, Conn., 1890-1897.

Philosophical Transactions of the Royal Society of L,ondon. Illustrated. L,ondon, 1665+.

Proceedings of the American microscopical society, 1878+.

Proceedings of the Royal Society. London, 1854+.

Quarterly journal of microscopical science. Illustrated. L,ondon, 1853 + .

Science Record. Boston, 1883-4.

Zeitschrift f. Angewandte. Mikroskopie. 1898+.

Zeitschrift f tir Instrumentenkunde. Berlin, 1881+.

Zeitschrift ftlr physiologische Chemie. Strassburg, 1877+.

Zeitschrift ftir wissenschaftliche Mikroskpie und ftlr mikroskopische Technik. Illustrated.
Braunschweig. 1884 + . Methods, bibliography and original papers.

Besides the above-named periodicals, articles on the microscope or the application of the
microscope appear occasionally in nearly all of the scientific journals. One is likely to get
references to these articles through the Jour. Roy. Micr. Soc. or the Zeit. wiss. Mikroskopie.
Excellent articles on Photo-micrography occur in the special Journals and Annuals of Photog-
raphy.

BIBLIOGRAPHY 288 a

ADDITIONAL BIBLIOGRAPHY

Bagshaw, W. — Elementary Photomicrography. Illustrated. Pp. 70. I^ondon, 1902.

Bausch, Edward. — Use and care of the Microscope (Extracts from Manipulation of the Micro-
scope). Rochester, 1902. This booklet should be in the hands of every beginner in microscopy.
It is furnished free to laboratory teachers.

Beck and Andrews. — Photographic lenses. I^ondon, 1903. Many illustrations showing vari-
ous forms of photographic objectives and the work for which they are adapted.

Cabot, R. C. — A guide to the clinical examination of the blood for diagnostic purposes. 3d
edition, Illustrated. New York, 1903.

Encyclopadie der Mikroskopischen Technik. Herausgegeben von : Ehrlich, Krause, Mosse,
Rosin und Weigert. Pp. 1400, Berlin and Vienna, 1903.

Ewing, James.— Clinical Pathology of the Blood. 2d Edition. Illustrated. Philadelphia, 1903;

Gay lord and Aschoff.— The Principles of Pathological Histology, Philadelphia, 1901. The
illustrations are mostly photo-micrographs. Part I gives a resume of microscopical technique.
Part III is on the principles of optics and photomicrography.

Greenish, H G. — Microscopical examination of food and drugs. Illustrated. Pp. 24 and 321.
I,ondon and Philadelphia, 1903.

Hardesty, Irving. — Neurological Technique. Pp. 183. Chicago and I^ondon, 1902. labora-
tory directions for the dissection of the central nervous system and Bna neurological nomen-
clature.

Havestadt, Dr. H.— Translated by J. D. and Alice Everett. Jena Glass. Illustrated. Pp. xiv
and 419. L,ondon and New York, 1903.

Heisler, J. C. — A text-book of Embryology, for students of medicine. 2d revised edition.
Pp. 405. Illustrated. Philadelphia, 1902.

Hiorns. Arthur H.— Metallography, an introduction to the study of the structure of metals,
chiefly by the aid of the microscope. Pp. 158. Illustrated. I,ondon, 1902.

von HOhnel F. R.— Die Mikroskopie der technisch-verwandten Fasserstoffe. Vienna, 1887.

Kaiserling, C. — I<ehrbuch der Mikrophotographie nebst Bemerkungen tlber VergrOsserungen
und Projection. Illustrated. Pp. 179. Berlin, 1903.

Koristka, Ditta F. — Di Microscopi ed accessor!. Milano, 1902.

L,angley, J. N.— Practical Histology. Illustrated. Pp. 340. lyOndon and New York, 1901.

L,econte, H. — I^es textiles vegetaux, leur examen microchimique. Paris.

L,indner, P. — Atlas der mikroskopischen Grundlagen der Gahrungskunde. Illustrated.
Berlin, 1903.

Mallory, F. B., and Wright, J. H.— Pathological Technique. A practical manual for workers
in pathological histology and bacteriology including directions for the performance of autopsies
and for clinical diagnosis by laboratory methods. 2d revised edition. Pp. 432. Illustrated.
Philadelphia, 1901.

Mann, Gustav. — Physiological Histology. Methods and Theory. Pp. 488. Oxford, 1902.

McMurrich, J. P.— The development of the Human Body. A manual of human embryology.
Illustrated. Pp. 527. Philadelphia, 1902.

Mell, P. H.— Biological laboratory Methods. Illustrated. Pp. 321. New York and Condon,
1902.

Minot, Charles S.— A laboratory text-book of Embryology. Illustrated. Pp. 380. Phila-
delphia, 1903. Full and satisfactory methods for Embryological work.

Molisch, H. — Grundriss einer Histochemie der pflanzlichen Genussmittel. Jena 1891.

Moore, V. A. — laboratory Directions for beginners in Bacteriology. Illustrated. 2d edition.
Boston, 1900.

Nelson, Edward M. — A bibliography of works (dated not later than 1700) dealing with the
microscope and other optical subjects. Journal of the Royal Microscopical Society for the Year
1902, pp. 20-23.

288 b BIBLIOGRAPHY

Oertel. T. E.— Medical Microscopy. Illustrated. Philadelphia, 1902,

Williams, H. U.— Bacteriology. 4th edition. Illustrated. Philadelphia, 1903.

Piersol, G. A.— Text-book of Normal Histology including an account of the development of
the tissues and of the organs. Also technique. Illustrated. Pp. 439. Philadelphia, 1893-1899.

Pozzi-Escot. — Analyse microchimique. Paris.

Reference Handbook of the Medical Sciences. 8. vols. 4°. New edition completely revised
and rewritten. New York, 1900-1905.

Schaefer, E. A.— The essentials of Histology, descriptive and practical, for the use of students.
6th edition, revised and enlarged. Illustrated. Pp. 416. Methods are given also.

Szymonowicz, I,, translated by MacCallum, J. B.— A text-book of Histology and Microscopic
Anatomy of the human body, including microscopic technique. Illustrated. Pp. 435. Phila-
delphia and New York, 1902.

Walmsley, W. H.— The A, B, C of Photo-Micrography. A practical handbook for beginners.
Plates and text figures. New York, 1902.

PERIODICALS

The American Journal of Anatomy, Baltimore, 1901 +. The American Journal of Anatomy
including Histology, Embryology and Cytology was established by seven universities,— Harvard,
Johns Hopkins, Columbia, Pennsylvania, Michigan, Cornell and Chicago. It has an editorial
board of 10, viz.

I^ewellys F. Barker, Chicago University ; Thomas Dwight, Harvard University ; Simon H.
Gage, Cornell University ; G. Carl Huber, Michigan University ; George S. Huntington, Columbia
University ; J. Playfair McMurrich, Univ. Michigan ; Franklin P. Mall, Johns Hopkins Univer-
sity ; Charles S. Minot, Harvard University ; George A. Piersol, Univ. Pennsylvania ; Henry
McE. Knower, Secretary, Johns Hopkins University. There are also over 60 collaborators from
different institutions.

The Journal of Comparative Neurology and Psychology, Granville, O. 1900+. This was
originally the Journal of Comparative Neurology. It has been reorganized and now has an
editor, Dr. C. I,. Herrick. a managing editor, C. Judson H errick, of Denison University, and two
associate editors. O. S. Strong of Columbia University, and Robert M. Yerkesof Harvard Univer-
sity. Its collaborators represent 20 other institutions.

The Journal of Experimental Zoology. Baltimore, 1904+. This new journal has u editors
representing 6 Universities : Johns Hopkins, 2, — W. K. Brooks and R. G. Harrison ; Harvard
University, 2, — G. H. Parker and W. E. Castle ; University of Pennsylvania, 2, — E. G. Conklin
and H. S. Jennings ; Columbia University, i, — E. B. Wilson ; Chicago University, 2, C. O. Whit-
man and C. B. Davenport ; Bryn Mawr College, i, T. H. Morgan. Besides the editors there
are many collaborators from different universities.

INDEX

Abbe apertometer, 270-272.

Abbe camera lucida, 123-132.

Abbe condenser or illuminator, 45-50.

Abbe's test-plate, method of using, 268-
270.

Aberration, chromatic, 4, 5 ; by corer-
glass, 55 ; spherical, 4, 5, 268.

Absorption spectra, 136-139, 145-150.

Acetelyne light, 37, 51, 229.

Achromatic condenser, 42-43, 256 ; objec-
tives, u, 64 ; oculars, 22.

Achromatism, 12.

Actinic focus, 226 ; image, 221.

Adjustable objectives, n, 12, 54-57; ex-
periments. 54 ; and micrometry, 118 ;
and photo-micrography, 234.

Adjusting collar, graduation of, 55.

Adjustment, of analyzer, 151 ; .coarse or
rapid, and fine, 63, Frontispiece ; of
objective, n, 12, 54; of objective for
cover-glass, specific directions, 55 ;
testing, 63.

Aerial image, 30, 31, 230.

Air bubbles, 94, 95.

Albumen fixative, Mayer's, 198.

Alcohol, absolute, 198 ; ethylic, 198 ;
methyl, 198, 203.

Alcoholic dye, staining sections with,
189.

Alum solution, 198.

Amici prism, 134, 140.

Amplifier, 109.

Amplification of microscope, 103.

Analyzer, 141, 151.

Anastigmatic objective, 208, 212.

Angle, of aperture, 15, 16 ; of carbons for
arc lamps, 251-252 ; critical, 54.

Angstrom and Stokes' law of absorption
spectra, 138.

Angular aperture, 15, 16.

Anisotropic, 152.

Apertometer, Abbe's, 270.

Aperture of objective, 15, 21, 270; angu-
lar, 15, 16 ; formula for, 16-18 ; of
illuminating cone, 45 ; numerical, 17,
19, 270 ; numerical of condenser, 44 ;
significance of, 20.

Aphengescope, 267.

Aplanatic cone, 46 ; objectives, n ; ocu-
lar, 22 ; triplet, 7.

Apochromatic condenser, 42 ; objectives,
12, 64, 214.

Apparatus and material, i, 34, 90, 103,
122, 134, 205, 243 ; for micro-projec-
tion, 256, 257 ; for photography, 233 ;
for photo-micrography, 205.

Appearances, interpretation, 90-102.

Arc lamp, 37, 229, 250-255 ; continuous
current, 249.

Arrangement of condenser, 46 ; of lamp,
bull's eye and microscope, 51 ; mi-
nute objects, 204; serial sections,
192 ; tissue for sections, 191.

Artifacts, 91.

Artificial illumination, 37, 48, 50 ; for
photo-micrography, 229.

Avoidance of diffusion currents, 180 ; of
distortion, 124.

Axial light, 36 ; experiments, 40 ; with
Abbe illuminator, 48.

Axial, point, 15 ; ray, 36.

Axis, optic, 2, 3, 10 ; of illuminator, 49 ;
secondary, 3, 6, 49.

! Back-ground for photographing, 209.
Back combination or system of objective,

9-12.

I Bacterial cultures, photographing, 241-
242.

Balsam, 199 ; bottle, 183 ; mounting in,
183, 190 ; preparation of, 199 ; re-
moval from lenses, 61 ; nat.ural, 190,
199 ; neutral, 199 ; removal from
slides, 162 ; xylene, 199.

Bands, absorption, 137.

Base of microscope, Frontispiece.

Bath, water, 255.

Bed, camera, 207.

Benzin, removing from sections, 189.

Biaxial crystals, 154.

Bibliography, 26, 33, 99, 101, 121, 133,
150. I55> 157. 158,159, 196, 204, 220,
240, 242, 251, 258, 267, 273, 274, 282.

Blocks for collodion, 178 ; for shell vials,
174.

Blood, absorption spectrum of, 146 ; cir-
culation of with micro-projection,
257, 264.

Board, circulation, 280.

Body of microscope, Frontispiece.

290

INDEX

Bottle for balsam, glycerin or shellac,

172 ; reagent, 179.
Bowl, waste, 181.
Box, glass, 181 ; slide, 198.
Brownian movement, 99.
Bubble, air, 94, 95.
Bull's-eye, 51, 217, 227 ; engraving glass

for, 228.

Burning point, 7, 30.
Buxton's photo-micrographic outfit, 237-

238.

Cabinet for microscopical preparations,
196,197.

Calipers, micrometer, 164 ; pocket, 164.

Camera, bed, 207 ; for embryos, 211 ; for
large, transparent sections, 215 ;
photo-micrographic, 222, 225, 235 ;
testing, 223 ; vertical, 205-210.

Camera lucida, Abbe, 122-132 ; Wollas-
ton's, 107, 125.

Canada balsam, 199 ; mounting in, 183,
190 ; preparation of, 199 ; removal
from lenses, 6r ; removal from slides,
162.

Carbol-xylene, 200.

Carbon-monoxide hemaglobin, spectrum
of, 147.

Carbons, for projection apparatus, 250-
255 ; adjusting, 251 ; angle, 251 ;
kinds, 253 ; positive and negative,
252 ; size, 254 ; wear, 253.

Card, catalog, 196 ; centering, 169 ; inven-
tory, 281.

Care of, eyes, 61 ; micro-projection appa-
ratus, 265 ; microscope, mechanical
parts, 59 ; optical parts, 60 ; nega-
tives, 211 ; water immersion objec-
tives, 58.

Carmine, to show currents and pedesis,
99 ; spectrum of, 148.

Carrier, objective, 259.

Castor-xylene clarifier, 200.

Cataloging, formula, 195 ; preparations,
194-196.

Cedar- wood oil, bottle for, 199 ; for clear-
ing, 184-185, 200 ; for oil immersion
objectives, 200.

Cells, deep, thin, 168 ; isolated, prepara-
tion of, 175 ; mounting, 168 ; stain-
ing, 174.

Cement, shellac, 203.

Cementing collodion, 201.

Center, optical, 2, 3.

Centering, and arrangement of illumina-
tor, 43, 47 ; card, 169 ; image of source
of illumination, 44 ; the radiant, 255.

Centimeter rule, 104, 133.

Central .light, 36, 95.
Chamber, moist, 171.

Cheese-cloth for cleaning slides, 161.

Chemical focus, 12 ; rays, 12.

Chemistry, Micro- 155.

Chromatic, aberration, 4 ; correction, n,
12 ; correction, test for, 270.

Circulation^ of blood with micro-projec-
tion, 257, 264 ; board, 226.

Clarifier, 200 ; castor-xylene, 200.

Class demonstrations in histology and
embryology, 243.

Cleaning back lens of objective, 61 ; ho-
mogeneous objectives, 59 ; micro-pro-
, jection apparatus, 265 ; mixtures for
glass, 165 ; sildes and cover-glasses,
161-163 ; water immersion objectives,
58.

Clearer, 173, 183, 190, 200.

Clearing mixture, preparation of, 200 ;
tissues with cedar- wood oil, 184.

Clinical microscope, 243-245.

Cloudiness of objective and ocular, how
to determine, 92 ; removal 60.

Coarse adjustment of microscope, Front-
ispiece ; testing, 63.

Cob- web micrometer, 117.

Collecting can, 280

Collodion, 200 ; for coating glass rod, 97 ;
cementing, 201 ; clarifying, 178 ; cot-
ton, 200 ; for fastening sections to
slide, 1 88 ; hardening, 178 ; method,
176 ; thick, thin, 177, 201.

Color, correct photography, 217 ; correc-
tion, 12 ; images, 54, 58 ; law of, 138 ;
production of, 154 ; screens, 218, 220.

Colored minerals, absorption spectra of,
149 ; specimens, photography of, 218.

Comparison prism, 141, 142 ; spectrum,
142.

Compensating ocular, 24, 25.

Complementary spectra, 139.

Compound microscope, see under micro-
scope.

Concave lenses, 3 ; mirror, use of, 37.

Condenser, 41-50; Abbe, 46, 48-49 ; ach-
romatic; 42, 43, 227, 256 ; apochro-
matic, 42, 227 ; bull's-eye, 51, 227,
228 ; centering, 43, 47 ; illuminating
cone with, 45 ; lamp, for projection,
250 ; mirror for, 48 ; non-achromatic,
46 ; numerical aperture of, 44-46 ;
optic axis of, 43, 47 ; for photo-micro-
graphy, 42, 227, 233 ; standard size
for, 26, 47 ; substage, 42; See also
illuminator.

Condensing lens, 36.

Cone, aplanatic, 46 ; illuminating, 45.

Conjugate foci, 4.

Construction of images, geometrical, 6.

INDEX

291

Continuous, current arc lamp, 249 ; spec-
trum, 137.

Contoured, doubly, 97.

Converging lens, 3-5 ; lens system, 9.

Convex lenses, 3-5.

Cooler, specimen, 257.

Correction, chromatic, or color, 5, 12, 268-
270 ; cover-glass, 55-56 ; cover, tube-
length for, 56-57 ; over and under, 12.

Cotton, collodion, gun, or soluble, 200.

Counterstaining, 189-190.

Cover- glass, or covering glass, 162-163 \
aberration by, 54 ; adjustment, spe-
cific directions, 55 ; adjustment for,
in photo-micrography, 234 ; adjust-
ment and tube-length, 12, 13, 55 ;
anchoring, 170 ; cleaning, 162-163 ;
correction, 55, 56 ; effect on rays from
object, 17, 55 ; gauges, 164-165 ; meas-
urer, 164-165; measuring thickness of,
163 ; non-adjustable objectives, table
of thickness, 14; No. i, variation of
thickness, 164; putting on, 94, 167;
sealing, 169, 170; size of, 164, 162;
thickness of, 13, 14, 163-165, 193 ;
tube-length with, 13, 56, 57 ; wiping,
163 ; with, serial sections, 193.

Crater of carbons, 252.

Critical angle, 54.

Crystals, biaxial, depolarizing, 154; from
frog for pedesis, 100.

Crystallization under microscope, 50, 157.

Crystallography, 155 ; list of substances
for, 156-157.

Currents, diffusion, avoidance of, 180 ;
in liquids, 98.

Cutting sections, 178, 186, 191-192.

Dark-ground illumination, 37, 49-50 ;
with Abbe illuminator, 50 ; with
mirror, 50.

Daylight, lighting with, 35.

Deck-plugs for collodion blocks, 178.

Dehydration, 177, 190.

Demonstration , microscope, 243-245 ;
with micro-projection apparatus, 265.

Depolarizing crystals, 154.

Designation, of oculars, 25 ; of wave
length; 143.

Determination, of, field of microscope,
28 ; equivalent focus, 272-273 ; mag-
nification, 103-109, 273 ; of working
distance, 39.

Diamond, writing, 196.

Diaphragms and their employment, 36-50.

Diffraction, grating, 137 ; illusory ap-
pearances due to, 101.

Diffusion currents, avoidance of, 180.

Direct, light, 35 ; vision spectroscope,
134-

Dispersing prism, 137.

Dissecting microscope, 8, 33, 175, 228.

Dissociator, formaldehyde, 201 ; nitric
acid, 203.

Distance, principal focal, 3. 30; standard
at which the virtual image is meas-
ured, 109 ; working d. of simple mi-
croscope or objective, 39 ; working
d. of compound microscope, n, 34,
39-40.

Distinctness of outline, 96.

Distortion in drawing, avoidance of, 124.

Diverging lens, 3.

Double spectrum, 142 ; vision, 103, 105.

Doubly contoured, 97 ; refracting, 152.

Draw-tube, Frontispiece ; pushing in, 38.

Drawing, with Abbe camera lucida, 129-
.131 ; board for Abbe camera lucida,
129, 130; distortion, avoidance of,
124 ; embryograph for, 132 ; with
microscope, 122; photographic cam-
era for, 132 ; for photo-engraving,
273 ; scale and enlargement of, 131 ;
with simple microscope, 132.

Dry objectives, 10, 16-18 ; light utilized,
17 ; dry mounting, 167 ; numerical
aperture, 16 ; dry plates, discovery by
Maddox, 221.

Dust, of living rooms, examination of,
101 ; on objectives and oculars, how
to determine, 92 ; removal, 60.

Dye, general, staining with, 189 ; aque-
ous, 180, 189 ; alcoholic, 180, 189.

Eccentric diaphragm, 44, 49-50.

Egg pippett, 280.

Electric light, 37, 229, 235, 250.

Embryograph, 132

Embryos, .camera for, 211 photograph-
ing, 211-214; records of, 213.

Engraving glass for bull's-eye condenser,
228

Enlargements, 241.

Eosin, 201.

Epidiascope, 266-267.

Episcope, 267.

Equivalent focal length or focus of objec-
tives and oculars, 10, 25, 272.

Erect image i.

Ether, alcohol, 201 ; sulphuric, 201.

Ethylic alcohol, 198.

Examination of dust of the living rooms,
bread crumbs, corn starch, fibres of
cotton, linen, silk, human and ani-
mal hairs, potatoes, rice, scales of
butterflies and moths, wheat, 101.

INDEX

Experiments, Abbe condenser, 48 ; with
adjustable and immersion objectives,
54 ; compound microscope, 26 ; ho-
mogeneous immersion objective, 58 ;
lighting and focusing, 37 ; in micro-
chemistry, 157 ; with micro spectro-
scope, 145 ; with micro-polariscope,
152 ; in mounting, 166 ; photo-micro-
graphy, 229 ; simple microscope, 6.

Exposure, of photographic plates, 232,
235, 240; with color-screen 220.

Extraordinary ray of polarized light, 150.

Eye and microscope, 6, 9, 32.

Eyes, care of, 6r ; muscae volitantes of,

100.

Eye-lens of the ocular, 22.
Eye-piece, 22 ; micrometer, 1 14.
Eye-point, 7, 22, 123 ; of ocular, demon-
stration, 32.
Eye-shade, adjusting, 62 ; double, 62.

Farrants' solution in mounting objects,
171 ; preparation of, 201.

Fibers, examination of, 101 ; textile, 158.

Field, 28 ; with camera lucida, 107 ; illu-
mination of, 45, 51 ; with orthoscopic
ocular, 23 ; with periscopic ocular,
24 ; of view with microscope, 28, 29,
105, 123-125; size of, with different
objectives and oculars, 28, 29.

Field-lens, of ocular, 22 ; action of, 32 ;
dust on, 92.

Filar, micrometer ocular, 23, 26 ; ocular
micrometer, 117, 118.

Filter, hot, 185.

Filtering balsam, etc., 185, 199.

Fine adjustment, Frontispiece ; testing,

63-

Fir, balsam of, 199.

Fixative, albumen, Mayer's, 198.

Fixing, reagents for, 198 ; tissue, 176.

Focal distance, or point, principal, 30 ;
length equivalent, 10.

Focus, 6; actinic, 226; chemical, 12 ;
conjugate, 4 ; equivalent, of object-
ives and oculars, 10, 25, 272-273 ; op-
tical, 12 ; principal^, 5 ; virtual, 3 ;
visual, 226.

Focusing, 6, 34 ; adjustments, testing, 63;
with compound microscope, 34 ; em-
bryos, 213 ; experiments, 38 ; glass,
209 ; with high objectives, 38 ; with
low objectives, 38 ; objective for mi-
cro-spectroscope, 144 ; for photo-mi-
crography, 206, 213, 230; scale for,
206 ; screen for photo-micrography,
209 ; with simple microscope, 6, 34 ;
slit of micro-spectroscope, 145.

Food, detection of adulteration in, 158.

Form of objects, determination of, 93.

Formal, 201.

Formaldehyde, dissociator, 201 ; for isola-
tion, 173.

Formula, for aperture, 16, 17 ; for catalog-
ing) *95 i f°r refraction, 52-54.

Fraunhofer lines, 137.

Free, hand sections, 274 ; working dist-
ance, 40.

Front combination or lens of objective,
9-11.

Frontal sections, 192.

Function of objective, 29-31 ; of ocular,

Gauge, cover-glass, 164-165.

Gelatin, liquid, preparation of, 203.

Geometrical construction of images, 6.

Glass, cleaning mixture for, 165 ; focus-
ing, 209 ; ground, 29, 209 ; rod appear-
ance under microscope, 96, 97 ; slides
or slips, 161, 162.

Glasses, opera, 262.

Glue, liquid, preparation of, 203.

Glycerin, bottle for, 183 ; mounting ob-
jects in, order of procedure, 170, 201 ;
removal, 61.

Glycerin jelly, mounting objects in, or-
der of procedure, 170-171 ; prepara-
tion of, 201.

Grating, diffraction, 137.

Ground glass, focusing screen, 209 ; pre-
paration of, 29.

Gun cotton, 200.

H

Half-tones from photo-micrographs, 232,

274.
Hardening collodion, 178 ; tissue, 176,

183,

Hematein, 203.
Heinatoxylin, 202 ; stained preparations,

photographing, 218.
Hemoglobin spectrum, 147.
High school microscope, 64, 71-89.
Histology, physiological, 196.
History of photo- micrography, 220.
Holder, lens, 7, 217, 228 ; needle, 167 ;

slide, 1 88.
Homogenous immersion objective, 16-

19 ; cleaning, 59 ; experiments, 58 ;

numerical aperture, 16-21.
Homogenous liquid, n ; tester for, 58,

271 ; vessel for, 199.
Horizontal camera, 230, 236-237.
Huygenian ocular, 22, 24, 32.

INDEX

293

Illuminating, cone (for condenser), aper-
ture of, 45 ; objective, 13, 238 ;
power, 21.

Illumination, for Abbe camera lucida,
129; artificial, 37, 48, 50; artificial
for photo-micrography, 229 ; center-
ing image of source of, 44 ; with air
and oil, 94, 95 ; dark ground, 49,
50 ; daylight, 35 ; of entire field, 51 ;
lamp for, 50 ; methods of, 35, 49 ; for
micro-polariscope, 151 ; for micro-
spectroscope, 144 ; oblique with air
and oil, 94, 95 ; of opaque objects,
144, 238 ; for photography, 208,
216, 229, 241 ; for photo-micrography,
23°. 233. 238 ; for projection. 250 ; for
Wollaston's camera lucida, 124.

Illuminator, 41-50; vertical, 13, 238.
See also condenser.

Image, actinic, 221 ; aerial, 30, 230 ; center-
ing i. of source of illumination, 44 ;
color, 52, 58 ; inverted, real of ob-
jective, 30 ; of flame, 45 ; geomet-
rical construction of, 6 ; and object,
size and position, 5, 9, 108 ; real, 5, 9,
30-32, 103 ; refraction, 52, 58 ; retinal,
6, 9, 32 ; swaying of, 48 ; virtual i.
and standardd'istance at which meas-
ured, 6, 109.

Image-power of objectives, 18.

Imbedding, 177, 185.

Immersion, fluid or liquid, 58, 271 ; illu-
minator, 47 ; objective, n, 58-59.

Incandescence or line spectra, 137.

Incident light, 35.

Index, of refraction, 53 ; of medium in
front of objective, 16-19.

Indicator ocular, 247.

Infiltration, collodion, 177 ; paraffin, 184;
paraffin dish for, 279.

Ink for labels and catalogs, 195 ; for
drawing, 274.

Interpretation of appearances under the
microscope, 90-102.

Inventory card, 281.

Iris diaphragm, 82, 157.

Irrigating with reagents, 170.

Isochromatic plates, 217.

Isolation, 173 ; with formaldehyde, 173 ;
nitric- acid, 175.

Isotropic, 152.

J-K

Japanese filter or tissue paper, 60.
Jelly, glycerin, 170-171, 201.
Jena glass, 71.

Jurisprudence^ micrometry in, 121.
Knife support, 277.

Labels and catalogs, 194-196, 203.

Labeling microscopical preparations, 194;
photographic negative, 211 ; serial
sections. 193.

Laboratory compound microscope, 64,
71-89.

Lamp, acetylene, 37, 51, 229 ; condenser,
250 ; electric arc, 37, 229, 250-255 ;
petroleum, 37, 50-51, 220, 229 ; spirit,
183.

Lantern, magic, 249 ; slides, 241.

Law of color, 138.

Lens, concave, 3 ; converging, 3 ; convex,
4 ; eye, 22 ; field, 32 ; holder, 7, 175,
217, 228 ; paper, 60 ; system, 9 ;
thick. 3.

Lenses of micro-projection apparatus,
cleaning, 265.

Letters in stairs, 93 ; for photo-engraving,
274.

Lettering oculars, 26.

Light, with Abbe illuminator, 48 ; ace-
tylene, 37, 51, 229 ; artificial, 37, 50,
229 ; axial, 36, 40, 48 ; direct, 35 ;
central, 36, 40 ; electric, 37, 229, 250 ;
incident, 35 ; with mirror, 37 ; ob-
lique, 36, 41, 48 ; petroleum, 37, 50,
220, 229 ; for photo-micrography, 229;
polarized, 150 ; reflected, 35 ; sun,
229 ; transmitted, 36 ; utilized with
different objectives, i 7; for vertical
illuminator, 239 ; wave length of,
142 ; Welsbach, 37, 229.

Lighting, 35 ; for Abbe camera lucida,
129 ; artificial, 50 ; experiments, 37 ;
with horizontal camera, 230 ; for mi-
cro-polariscope, 151 ; for micro-spec-
troscope, 144 ; with a mirror. 37, 40 ;
with daylight, 35, 229 ; for photog-
raphy, 208, 216 ; for photo-micro-
graphy, 229, 230 ; for vertical illum-
inator, 229.

Line spectrum, 137.

Liquid, currents in, 98 ; homogenous, n,
58, 271.

M

Magic lantern, 249.

Magnification of compensating oculars,
24 ; effect of adjusting objective, 118 ;
determination of, 103-109 ; expressed
in diameters, 103 ; initial or indepen-
dent, 273 ; of microscope, 103 ; in
micro-projection, table, 262 ; of mi-
croscope with Abbe camera lucida,
131 ; of microscope, compound, 105-;
of microscope, simple, 104 ; of photo-

294

INDEX

micrographs, determination of, 233 ;
real images, 103 ; table of, with ocu-
lar micrometer, in : with projection
microscope, 262 ; varying with com-
pound microscope, 109 ; and velocity,
98.

Magnifier, tripod, 7, 104, 209.

Marker for preparations, 65-66.

Marking objects, 65-66,248 ; negatives,
211, 214 ; objectives, 71.

Masks for preparations, 264.

Material and apparatus, i, 34, 90, 103,
123, I34,_ 156, 198, 205, 223, 243.

Measure, unit of, in micrometry, 112 ; of
wave length, 143.

Measurer, cover-glass, 164-165.

Measuring the thickness of cover-glass,
163.

Mechanical parts of compound micro-
scope, 64 ; Frontispiece, 8 ; of micro-
scope, care of, 59 ; testing, 63.

Mechanical stage, 65, 67-70, 258, 259.

Megascope, 267.

Metallic surfaces, photography of, 235-
240 ; preparation of, 239.

Metallography, microscope in, 159.

Metals, examination of, 159, 235.

Met-hemaglobin, spectrum of, 136, 147.

Methods, collodion, 176-183 ; paraffin,
183-191.

Metric measures and equivalents, cover
ist p., 133.

Micro-chemistry, 155-157 ; slides for, 161.

Micro-metallography, objects for, 238.

Micrometer, 103 ; calipers, 164 ; cob-web,
117; filar m. ocular, 117, nS; filling
lines of, 106 ; lines, arrangement of
ocular and stage, 120 ; lines, finding,
106 ; net, 128 ; object or objective,
105; ocular or eye-piece, 114-120;
ocular, micrometry with, 116 ; ocular,
ratio, 119; ocular, valuation of, in,
114; ocular, varying valuation of,
118 ; for photo-micrography, 233 ;
screw ocular, 117; stage, 105, 106 ;
table of magnification, in.

Micrometry, definition, 112, 114; with
adjustable objectives, 118; compari-
son of methods, 119-120 ; with com-
pound microscope, 112; and juris-
prudence, 121 ; limit of accuracy in,
120; with ocular micrometer, 116 ;
with simple microscope, 112 ; remarks
on, 118 ; unit of measure in, 112.

Micro-millimeter, 113.

Micron, 113 ; for measuring wave length
of light, 143.

Micro-photograph, 220.

Micro-photography, distinguished from
photo-micrography, 220.

Micro-planar objective, 212, 260.

Micro-polariscope, 100, 150-155.

Micro-polarizer, 150.

Micro-projection, 249-267 ; apparatus,
256-257 ; carbons for, 250-254 ; cir-
culation of blood with, 257, 264 ; con-
denser, 254, 256 ; current, 254 ; dem-
onstration with, 265 ; magnification,
262 ; masks for specimens, 264 ; me-
chanical stages, 258-259 ; microscope
for, 249 ; objectives and oculars, 259 ;
pointer for, 265 ; preparations, 263 ;
screen and screen distance, 261 ;
specimen cooler, 257 ; stage, 258 ;
stains for, 263 ; water-bath, 255.

Microscope, definition, i ; amplification
of, 103 ; clinical, 243 ; demonstration,
243 ; dissecting, 8, 33, 175, 176 228 ;
care of, 59; eye and, i, 6, 9; field of, 28,
29 ; focusing, 34 ; magnification, 103;
for metallography, 159-160 ; for mi-
crochemical analysis, 153; for opaque
objects, 235-237 ; for photo-micro-
graphy, 225, 231, 237 ; polarizing, 150;
preparation, with erecting prism 176;
projection, 249-250 ; price of, 64, 71 ;
putting an object under, 27 ; sun or
solar, 249 ; screen, 59 ; stand for
large, transparent objects, 215, 216 ;
stand, for embryos, 212 ; traveling,
245-246.

Microscope compound, definition, 8 ;
drawing with, 122 ; figures, frontis-
piece, 9, 71-89, I02» J53> 160, 237,
244-246 ; focusing, 38-39 ; for High
schools, 64, 71 ; for laboratory, 64,
71-89; lamp for, 50; magnification
or magnifying power, 105 ; magnifi-
cation and size of drawing with
Abbe camera lucida, 131 ; mechani-
cal parts, 64 ; micrometry with, 112 ;
optic axis of, 9, 10 ; optical parts of,
9, 64 ; varying magnification, 109 ;
working distance of, 39 ; testing, 63.

Microscope, simple, definition, i ; exper-
iments with, 6 ; figures, 6-8, 33, 104,
175, 209, 228, 243 ; focusing with, 34;
magnification of, 104 ; micrometry
with, 112; working distance of, 34;

Microscopic, objective, 9 ;obj ective low,
attached to camera, 214 ; objects,
drawing, 122 ; ocular, 22 ; slides or
slips, 161 ; tube-length, 13, 14.

Microscopical preparations, cabinet for,
197 ; cataloging, 194 ; labeling, 194 ;
mounting, 166-193.

Microtome, hand, 274-275 ; Minot's 275-
.276 ; razor support for, 276-277 ; slid-
ing, 278.

INDEX

295

Micro-spectroscope, 134-150; adjusting,
139 ; experiments, 145 ; focusing,
144 ; focusing the slit, 139 ; lighting
for, 144 ; objectives to use with, 144 ;
reversal, apparent, of colors in, 134 ;
slit, mechanism of, 135, 139.
Micrum, 113.
Mikron, 113.

Milk globules, to overcome pedesis
of, loo

Minerals, absorption spectra of, 149.

Minot's microtomes, 275-276.

Minute objects, arrangement of 204.

Mirror, 9-11 ; for Abbe illuminator, 48;
of camera lucida, arrangement for
drawing, 126 ; concave, use of, 37 ;
dark ground illumination, 50 ; light
with, central and oblique, 40, 41 ;
lighting with, 37 ; plane, use of, 37.

Mixture, clearing, 200.

Models, wax, 274.

Moist, chamber, 171.

Molecular movement, 99.

Monazite sand, spectrum of, 149.

Mounting cells, preparation of, 168 ; me-
dia and preparation of, 198 203 ; ob- j
jects for polariscope, 151 ; perma- j
nent, 167 ; temporary, 166.

Mounting objects, dry in air, order of
procedure, 167-168; in glycerin, or- i
der of procedure, 170 ; in glycerin \
jelly, order of procedure, 170; in
media miscible with water, 169 ; mi-
nute objects, 204 ; opaque objects,
239 ; permanent, 167 ; in resinous !
media, by drying or desiccation, or- j
der of procedure, 172 ; in resinous
media, by successive displacements,
order of procedure, 172 ; temporary,"
1 66.

Movement, Brownian, or molecular, 99.

Muscae volitantes, 100.

Muscular fibers, isolation of, 175.

N

Natural balsam, 199.

Needle-holder, 167.

Negatives, labeling, 211, 214; oculars,
22 ; rack for drying, 240 ; record of,
214 ; storing, 211, 214.

Net micrometer, 128. •

Neutral balsam, 199.

Nicol prism, 150.

Nitric acid, dissociator, 203.

Nomenclature of objectives, 10.

Non-achromatic condenser, 46 ; object-
ives, ri.

Non-adjustable objectives, 12; thickness
of cover glass for, table, 14.

Normal salt solution, 203.

Nose-piece, 27, 38, 80; marking object-
ives on, 71.

Numerical aperture, of condenser, 44 ; of
objectives, 16, 270 ; table of, 19.

Object, determination of form, 93 ; hav-
ing plane or irregular outlines, rela-
tive position in a microscopical prep-
ration, 92 ; and image, size of, 10,
108 ; marking parts of, 65-66 ; mark-
ing position of, 248 ; micrometer, 105;
mounting, 166 ; putting under micro-
scope, 27 ; shading, 59 ; suitable for
photo-micrography, 227 ; transparent
with curved outlines, relative posi-
tion in microscopic preparations, 94.

Objective, 9-13, 121 ; achromatic, n ;
adjustable, n, 12, 54; adjustable,
micrometry with, 118; adjustable,
photo-micrography with, 234 ; adjust-
ment for, 54 ; aerial image of, 30 ;
anastigmat, 208 ; aperture of, 15-22,
270 ; aplanatic, 1 1 ; apochromatic,
12, 224 ; back combination of, 10, n ;
carrier, 259 ; cleaning back lens of,
61 ; collar, graduated for adjustment,
56 ; cloudiness or dust, how to deter-
mine, 92; designation of. 10; dry,
10, 17-19, 121 ; equivalent focus of,
10, 25, 29, 272 ; field of, 28-29 J focus-
ing for micro-spectroscope, 144 ; front
combination of, 10, u ; function of,
29"3T'» glass for, 11-13, 71 '•> high,
focusing with, 38 ; homogeneous im-
mersion, 17-19, 121 ; homogeneous
immersion, cleaning, 59; homogene-
ous immersion, experiments, 58 ; il-
luminating, 13, 238 ; image, power
of, 18 ; immersion, ir, 121 ; index of
refraction of medium in front of, 17,
19 ; initial or independent magnifica-
tion of, 273 ; inverted, real image of,
30 ; for laboratory microscope, 64 ;
lettering, 10 ; light utilized with, 17 ;
low, focusing with. 38 ; magnifica-
tion of, 272 ; marking, by Krauss'
method, 71 ; for micrometallography,
13, 159; micro-planar, 212, 260; to
use with micro-polariscope, 151 ; mi-
croscopic, 9 ; to use with micro-
spectroscope, 144 ; for micro- spectro-
scope, focusing, 144 ; nomenclature
of, 10 ; non-achromatic, n ; non-
adjustable, 12; non-adjustable, thick-
ness of cover-glass for, table, 14 ;
with nose piece, 38 ; numbering, 10 ;
numerical aperture, 16-22, 270 ; oil

296

INDEX

immersion, n ; panto-chromatic, 13 ;
para-chromatic, 13 ; for photography,
208, 212, 214, 224; for photo-microg-
raphy, 224; projection, 13, 212, 214,
259 ; putting in position and remov-
ing, 26 ; semi-apochromatic, 13; table
of field, 29 ; terminology of, 10 ; un-
adjustable, 12; variable, 13; visual
and actinic foci of, in photo-microg-
raphy, 226 : water immersion, 17-19,
56 ; working distance of, 39-40.

Oblique light, 36, 41 ; with Abbe illumi-
nator, 48 ; with a mirror, 41, 50.

Ocular, various forms, 22-25 \ cloudiness,
how to determine and remove, 60, 92;
equivalent focus of, 25, 29, 273 ; eye-
point of, 32 ; field-lens, 32 ; filar or
screw micrometer, 26, 117; focus,
equivalent of, 25, 273 ; function of,
31-33 ; indicator, 67, 247 ; iris dia-
phragm for, 157 ; lettering and num-
bering, 26 ; micrometer, micrometry
with, 114-121 ; parfocal, 24, 38; for
photo-micrography, 224, 232, 235 ;
pointer, 247 ; projection, 260 ; spec-
troscopic,i34 ; standard size for, 26;
table, effect on field, 29.

Oil, and air, appearances and distinguish-
ing optically, 95 ; removal, 61 ; re-
moval from sections, 179.

Oil-globules, with central and oblique
illuminations, 95.

Oil immersion objectives, ri.

Opaque objects, lighting, 144, 238 ; pho-
tography of, with microscope, 235-
240 ; projection of, 267.

Opera glasses, 262.

Optic axis, 2, ; of condenser or illumi-
nator, 47 ; of microscope, 10.

Optical, bench, 237 ; center, 2 ; focus, 12 ;
parts of compound microscope, 9, 64;
parts of microscope, care of, and test-
ing, 60, 63 ; section, 98.

Order of procedure in mounting ob-
jects dry or in air, 167 ; in glycerin,
170; in glycerin jelly, 170: in resin-
nous media by desiccation, 172 ; in
resinous media by successive dis-
placement, 172.

Ordinary ray, with polarizer, 150.

Orthochromatic plates, 217.

Orthoscopic ocular, field with, 28.

Outline distinctness of, 96.

Oven paraffin, 279.

Over-correction, 5.

Oxy-hemoglobin, spectrum of, 138, 147.

Paper, bibulous, filter, lens, or Japanese
for cleaning oculars and objectives,
60, i So.

Paraffin, 185, 203 ; filtering, 185 ; infil-
trating with, 185 ; dish for infiltrat-
ing, 279; imbedding in, 185, 186 ;
method, 183 ; oven, 279 ; pail for
melting, 184 ; removal from lenses,
61 ; removing from sections, 188.

Parfocal oculars, 24, 38.

Parts, optical and mechanical of micro-
scope, 8, 64 ; testing, 63.

Pedesis, 99 ; compared with currents, 99 ;
to overcome, 100 ; with polarizing
microscope, 100 ; proof of reality of,

100.

Penetrating power, 21.

Penetration of objective, 21.

Permanent mounting, 167 ; preparations
of isolated cells, 175.

Permanganate of potash, absorption spec-
trum of, 136, 146.

Petri dish, photographing bacterial cul-
tures in, 241.

Petroleum light, 37, 229 ; as color screen,
220.

Pharmacological products, examination
of, 158.

Photo-engraving, drawing for, 273 ; let-
tering for, 274.

Photographic, camera, 207 ; negatives,
labeling, 211, 214 ; objectives, 208 ;
prints, 211.

Photography, back -ground for, 209 ; of
bacterial cultures, 241-242 ; color-
correct, 217 ; of colored objects, 218 ;
compared with photo-micrography,
222; of embryos, 211-214; focusing
and exposure, 206, 213 ; indebtedness
to photo-micrography, 220; of large
transparent objects, 214-216; lighting
for, 208, 216; metallic objects, 235-
240 ; objectives for, 208, 212, 214 ; of
objects in alcohol or water, 206 ;
opaque objects, 235-240 ; plates for,
217; stage for, 211 ; with vertical
camera, 205-209, 225.

Photo-micrograph, 220; determination
of magnification for, 233 ; at 5-20 di-
ameters, 212 ; 20-50 diameters, 230 ;
100-2500 diameters, 233 ; of metallic
surfaces, 235-240 ; objects suitable
for, 227 ; of opaque objects, 235-240 ;
prints of, 211 ; plates for, 217 ; repro-
ductions of, 232 ; with and without
1 an ocular, 230-234.

Photo-micrographic, camera, 222, 225,
236 ; outfit, 236-237 ; stand, 231.

INDEX

297

Photo-micrography, 220-240 ; cover-glass
correction, 234 ; apparatus for, 223 ; i
compared with ordinary photogra-
phy, 222 ; condenser for, 42, 226, 227; |
distinguished from micro-photogra-
phy, 220 ; experiments, 229 ; expos-
ure for, 213, 220, 232, 235, 240 ; focus-
ing for, 213, 216 ; focusing screen for,
209 ; lighting, 229, 230, 233, 239 ; mi-
crometer for, 233 ; objectives and oc-
ulars for, 13, 224, 232,235 ; vertical
camera with, 211, 222, 225 ; actinic
foci in, 226 ; with and without ocular,
230,232, 234 ; record table for, 219.

Physiological histology, 196.

Picric-alcohol, 203.

Picro-fuchsin, 190.

Pillar of microscope, Frontispiece.

Pin-hole diaphragm, 47.

Pippett, 179; egg, 280.

Plane mirror, use of, 37.

Plates, color-correct, 217 ; exposure of,
213, 220, 232, 235, 240 ; isochromatic,
or orthochromatic, 217 ; size of, 224.

Pleochroism, 152.

Pleurosigma angulatum, 41.

Point, axial, 15 ; burning, 7.

Pointer ocular, 247.

Pol ari scope, 140, 150.

Polarized light, extraordinary and ordi-
nary ray of, 150.

Polarizer and analyzer, 140, 151.

Polarizing microscope, pedesis with, 100.

Position of objects or parts of same ob-
ject, 92 ; marking p., 248.

Positive oculars, 10, 22.

Power, of microscope, 103 ; illuminating,
penetrating, resolving, of objective,
19-21 ; of ocular, 25.

Preparation of Canada balsam, Farrant's
solution, glycerin, glycerin jelly, etc.,
1 98-204.

Preparation, of clearing mixture, liquid
gelatin and shellac cement, 198-204 ;
of ground, glass, 29 ; of metallic sur-
faces, 239 ; vials, 174.

Preparations, cataloging, 194-196 ; cabi-
net for, 196-197 ; labeling, 194 ; for
microprojection, 263 ; permanent, of
isolated cells, 175.

Price of American and foreign micro-
scope, 71.

Principal, focus, 3, 5 ; focal distances, 3,
30 ; optic axis, 2, 5.

Prism of Abbe camera lucida, 124, 127;
Amici, 140 ; comparison, 141 ; dis-
persing, 141 ; erecting, 176 ; Nicol,
150 ; and slit of micro-spectroscope,
mutual arrangement, 139 ; of Wollas-
ton's camera lucida, 125.

Prints, photographic, 211.

Projection, apparatus, 249, 263-297 ; mi-
croscope, 249-266 ; see micro-projec-
tion ; objective, 13, 214, 259 ; ocular.
24, 25, 232, 260 ; opaque objects, 267 ;
in photo-micrography, 232, 234.

Putting, on cover-glass, "167 ; an object
under microscope, 27 ; an objective
and ocular in position, 26, 27.

Pyroxylin, 200.

Q-R

Quadrant for camera lucida, 127, 128.

Radiant, centering, 255.

Ratio, ocular micrometer, 119.

Razor and support, 276-277.

Reagent, bottle, 179 ; for fixing, 198; irri-
gation with, 170 ; for mounting, 198.

Real image, 5, 8, 9, 30; magnification, 103,
262.

Record, of negatives, 214; table for photo-
micrography, 219.

Reflected light. 35.

Reflection, total, 54.

Refraction, 52 ; images, 52, 58 ; index of,
53 ; of medium in front of objective,

19-

Refractive, doubly, 152 ; highly, 97 ;
singly, 152.

Relative position of objects. 92.

Resinous media, mounting objects in,
order of procedure, by drying or
desiccation, 172 ; by a series of dis-
placements, 172.

Resolution and numerical aperature, 20.

Resolving power, 20.

Retinal image, 6, 9.

Revolving nose-piece, marking objectives
on, 71.

Ribbon sections, 186-187 ; trav f°r» l87-

Sagittal sections, 192.

Salicylic acid, crystallization, 50.

Salt solution, normal, 203.

Scale, of drawing, 131 ; of sizes for pho-
tographing, 206 ; of wave lengths,
142.

Screen, color, 218-219; focusing s. for
photo-micrography, 209 ; of ground
glass, 29; for micro-projection, 261 ;
for microscope, 59.

Screw, society, 64 ; micrometer, 26, 117.

Sealing cover-glass, 169, 170.

Searching ocular, 24.

Secondary axis, 3.

Section, lifter, 181-182 ; optical, 98.

Sections, arrangement of tissue for, 191 ;
clearing, 190; cutting, 178, 186 ; de-

298

INDEX

hydration of, 190 ; extending with
water, 186 ; fastening to slide, 179,
187 ; frontal, 192 ; mounting, 183,
190 ; removing benzine, oil and para-
ffin from, 179, 188 ; ribbon, 186 ;
sagittal, 192 ; serial, 191-193 ; stain-
ing, 1 80, 189; transferring, 179;
thickness for micro-projection, 263.

Selenite plate for polariscope, 154.

Semi-apochromatic objective, 13.

Serial sections, 191-193 ; arranging and
labeling, 192, 193 ; stage for, 67, 69 ;
thickness of cover-glass for, 193.

Shell vials, 174.

Shellac cement, preparation of, 203 ; re-
moval from lenses, 61.

Significance of aperture, 20.

Simple microscope, see under microscope.

Sines, table of, 3d page of cover.

Slides, 161 ; box for 198 ; cleaning, 161 ;
holder for, 188-189 ; lantern, 241 ; for
micro-chemistry, 161 ; tray for, 187.

Sliding microtome, 278.

Slips, 161.

Slit mechanism of micro-spectroscope,

135, 139-

Society screw, 64.

Sodium, lines and spectrum, 136-137.

Solar spectrum or s. of sunlight, 136-137.

Soluble cotton, 200.

Solution, alum, 198; Farrants', 201.

Specimen cooler, 257.

Spectral, colors, 138 ; ocular, 134, 139.

Spectroscope, direct vision, 134, 145.

Spectroscopic, examination of color-
screens, 220 ; ocular, 134.

Spectrum, 136-150; absorption, 137;
amount of material necessary and its
proper manipulation, 145 ; analysis,
150 ; Angstrom and Stokes' law of,
138 ; banded, not given by all colored
objects, 148 ; of blood, 146 ; of carbon
monoxide hemaglobin, 147 ; of car-
mine solution, 148 ; of minerals, 149 ;
of colorless bodies, 148 ; comparison,
142 ; complementary, 139 ; continu-
ous, 137 ; double, 142 ; incandescence;
137 ; line, 137 ; met-hemaglobin, 147,
monazitesand, 149 ; oxy-hemoglobin,
138, 147; permanganate of potash, 136,
146 ; single-banded of hemaglobin,
138, 147 ; sodium, 136, 137 ; solar,
J36' T37 ; two-banded of oxy-hetna-
globin, 147.

Spherical aberration, 4, 5 ; test for, 268.

Stage, Frontispiece, 65 ; mechanical, 65,
67-70, 259 ; micrometer, 105 ; for mi-
cro-projection, 258 ; for photograph-
ing, 211.

Stain, alcoholic, 180, 189; aqueous, 180,
189 ; for micro-projection, 263.

Staining, cells, 174 ; dish, 190 ; sections,
1 80, 189.

Stand, of microscope, 65 ; photo-micro-
graphic, 231 ; special for embryos,
212 ; special for large transparent ob-
jects, 215, 216.

Standard, distance (250 mm.) at which
the virtual image is measured, 109 ;
screw, 64 ; size for condenser, 47 ;
size for oculars, 26.

Stokes and Angstrom's law of absorption
spectra, 138.

Storing negatives, 211 ; preparations, 196.

Substage, 86, Frontispiece.

Substances for crystallography, 156.

Sulphonal with polarizer, 154.

Sulphuric ether, 201.

Support for knife of microtome, 276-277.

Swaying of image 48.

Synthol, 198.

System, back, front, intermediate of
lenses, 10, u ; crystal, 156; metric,
cover ist p., 133.

Table, for immersion fluid, 272 ; of mag-
nification and valuation of ocular mi-
crometer, in ; magnification with
projection microscope, 262 ; of tube-
length and thickness of cover-glasses,
14 ; natural sines, third page of cover;
of numerical aperture, 19 ; record,
for photo-micrography, 219 ; size of
fields, 29 ; testing homogeneous liq-
uids, 272 ; of valuations of ocular
micrometer, in ; weights and meas-
ures, 2d page of cover.

Temporary mounting, 166.

Terminology of objectives, 10.

Test of chromatic and spherical aberra-
tion, 268-270.

Tester, cover-glass, 164-165 ; for homo-
geneous liquids, 58, 271.

Testing a camera, 223 ; a microscope and
its parts, 63.

Test-plate, Abbe's, method of using, 268.

Textile fibers, examination of, 101, 158.

Thickness, of cover-glass for non-adjust-
able objectives, table, 14 ; of serial
sections, 193.

Tissues, arranging for sections, 191 ; fix-
ing or hardening, 176, 183 ; washing
apparatus for, 280.

Tolles-Mayall mechanical stage, 67.

Transections, 192. .

Transferring sections, 179.

Transmitted light, 36.

INDEX

299

Tray for slides, 187.

Triplet, aplanatic, 7.

Tripod, 7, 104 ; base for microscope, 86 ;
as focusing glass, 209.

Tube of microscope, Frontispiece.

Tube-length, 13, 14,56, for cover-glass
adjustment, 56, 57 ; importance of,
56 ; microscopical, 13, 14 ; of various
opticians, table, 14 ; and optical com-
binations, 118.

Turn-table, 168.

U— V— W— X

Unadjustable objectives, 12.

Under-correction, 5.

Unit of measures, in micrometry, 112 ; of
wave length, 143.

Valuation of ocular micrometer, 114-116 ;
table, in.

Variable objective, 13.

Varying magnification of compound mi-
croscope, 109.

Varying ocular micrometer valuation,
118.

Velocity under microscope, 98.

Vertical, camera, 205-210, 225 : illumina-
tor, 13, 238-239.

Vials, preparation, 174 ; blocks for, 174.

Virtual image, 5, 6, 9, 32 ; standard dis-
tance at which measured, 109.

Visibility with objectives, 20.

Vision, double, 103, 105 ; microscopic, 21.

Washing apparatus for tissues, 280.

Waste bowl, 181.

Water immersion objective, 16-19, 56 ;
light utilized, 17 ; numerical aper-
ture, 19.

Water, bath, 235, 255 ; for immersion ob-
jectives and removal of, 56, 58.

Wave length, designation of, 143 ; scale
of, 142.

Wax models, 274.

Weights and measures, see 2d page of
cover.

Welsbach light, 37, 229.

Wollaston's camera lucida, 107, 125.

WTork-room for photo-micrography, 224.

WTork-table, position, etc., 62.

Working distance of microscope or ob-
jective, ii, 34, 39-40.

WTriting diamond, 196.

Xylene, 178, 200 ; balsam, 199 ; for re-
moving oil, 179 ; removing from
slides, 189.

Xylol, German form of xylene, 178, 200.

GENERAL LIBRARY
UNIVERSITY OF CALIFORNIA— BERKELEY

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9 1954

LD 21-100m-l,'54(1887sl6)476

TABLE OF NATURAL SINES
Compiled from Prof. G. W. Jones' Logarithmic Tables

MINUTES.

DEGREES AND

QUARTER DEGREES UP TO

90°.

i' 0.00029 i°,

1
0.01745 16°,

0.2756431°,

0.5150446°,

0.7193461°,

0.87462 76°,

0.97

2 000058 i°,i5/

0.02181 i6°,i5x

0.27983 31°, 1 5

' 0.51877 46°, 15' 0.72236 61°, 15' 0.87673 76°,i5

'0.97

3 0.00087 1,30

0.02618 16,30

0.28402 31,30

0.5225046,30

0.72537 61,30

0.87882 76,30

o-97

4 0.00116 1,45

0.03054 16,45

0.2882031,45

0.52621 46,45

0.7283761,45

o 88089 76,45

0.97

5 0.00145! 2

0.03490! 1 7

0.2923732

o 52992 47

0.73135 62

0.88295 77

0.97

6 0.00175: 2,15

0.03926117,15

0.2965432,15

0.53361:47,15

07343262,15

0.8849977,15

o.97

7 0.00204 2,30

0.04362 17,30

0.30071 32,30

o.5373o|47,30

0.7372862,30

0.88701 77,30

0.97

8 0.00233 2,45

0.0479 N 1 7,45

o 30486 32,45

0.54097 47,45

0.74022 62,45

o 8890277,45

0.97

9 o 00262 3

0.05234 18

0.30902 33

o 54464 48

0.7431463

0.89101 78

0.97

10 0.00291

3,i5

00566918,15

0.3131633,15

0.5482948,15

0.7460663,15

0.89298178,15

o.97

ii 0.00320 3,30

0.06105118,30

0.3173033,30

0.5519448,30

0.74896:63,30

o 8949378,30

0-97

12 0.00349 3,45

0.06540 18.45

0.3214433,45

o.555574«,45

0.75184163,45

0.89687 78,45

0.98

13 0.00378 4

0.06976 19

o 32557 34

o.559J9 49

0.75471164

0.89879 79

0.98

14 000407 4,15

o.o74ii|i9,i5

0.3296934,15

0.5628049,15

0.75756:64,15

0.9007079,15

0.98

15 000436 4,30

0.07846:19,30

0.3338134,30

o 56641 49.30

0.76041 64,30

0.9025979,30

0.98

1 6 0.00465 4,45

0.08281 19,45

0.33792 34,45

0.5700049,45

0.7632364,45

0.9044679,45

0.98

17 0.00495 5

0.0871620

0.3420235

0.5735850

0.76604165

0.90631 80

o.gS

18 0.00524 5,15

0.09150 20 15

o.346i2'35,i5

05771550,15

0.7688465,15

0.9081480,15

0.98

19 0.00553 5.30

0.09585120,30

03502135,30

0.5807050,30

0.77162 65,30

0.90996:80,30

0.98

20 0.00582: 5,45

0.10019 20,45

o 35429 35,45

0.58425)50,45

0.7743965,45

0.9117680,45

0.98

21 0.00611 6

0.1045321

0.35837 36

0.5877951

0.77715 66

o.9l335pi

0.98

22 0.00640! 6,15

0.10887 21,15

036244136,15

0.5913151,15

07798866,15

0.91531 81,15

0.98

23 0.00669 6,30

0.1132021,30

o 36650 36,30

0.59482 51,30

0.78261:66 30

0.91706 81,30

0.98

24 o 00698

6,45

0.1175421,45

0.37056 36,45

0.5983251,45

0.7853266,45

0.9187981,45

0.98

25 0.00727

7

O.I2I87 22

0.37461 37

0.60182 52

o 78801 67

o 92050 82

0.99

26 0.00756
27 000785

7,i5
7,30

O.I262O 22,15

0.1305322,30

0.37865137,15
038268:37,30

0.6052952.15
0.6087652.30

0.7906967,15
o 79335 67,30

0.9222082,15
0.92388182,30

099
0.99

28 0.00814

7,45

0.1348522,45

0.38671 37,45

0.61222 52,45

o 79600 67,45

o.92554;82,45

0.99

29 0.00844

8

0 I39i7'23

o 39073 38

0.6156653

0.79864 68

0.9271883

0.99

30 0.00873

8,15

0.1434923,15

03947438,15

0.61909153,15

0.80125 68,15

0.9288183,15

0.99

31 0.00902

8.30

0.14781 23,30

0.39875 38,30

062251(53,30

0.8038668,30

0.93042 83,30

0.99

32 0.00931

8,45

o 15212 23,45

o 40275 38,45

0.62592(53,45

0.80644! 68, 45

0.93201 83.45

0.99

33 0.00960

9

o. 15643 24

o 40674(39

0.62932154

0.8090269

0.9335884

0.99

34 0.00989

9,i5

0.16074 24 15

0.41072:39,15

0.63271154,15

0.81157 69 15

0.93514184,15

0-99

35 0.01018

9.30

0.16505 24,30

0.4146939,30

0.6^608154,30

0.8141269.30

0.93667 84,30

0-99

36 o 01047

9,45

0.16935:24,45

0.41866 39.45

0.6394454,45

0.81664169,45

0.93819 84,45

0.99

37 0.01076

10

o 17365125

0.42262 40

0.6427955

0.8191570

09396985

0.99

38 0.01105

10,15

0.1779425,15

04265740,15

0.6461255,15

0.82165170,15

0.9411885,15

0.99

39 001134
40 0.01164

10,30
io,45

0.1822425,30
0.1865225,45

0.43051 40,30
0.43445 4°,45

0.6494555,30
0.6527655,45

0.82413 70,30
0.82659 70,45

0.94264185,30
0.94409 85,45

0.99
0.99

41 0.01193

ii

0.19081 26

0.4383741

0.6560656

0.82904 71

0.94552 86

0-99

42 0.01222

11,15

0.19509126,15

0.4422941,15

0.6593556,15

0.8314771,15

0.94693:86,15

0-99

43 0.01251

11,30

0.19937126,30

0.4462041,30

0.66262 56,30

0.8338971,30

0.94832 86,30

0-99

44 0.01280

n,45

0.2036426,45

0.45010 41,45

0.66588:56,45

0.83629 7i,45

0.949708645

0.99

45 0.0130912
46 0.01338 12,15

o 20791(27
0.21218 27,15

0-4539942
0.4578742,15

0.66913157
0.6723757,15

0.83867 72
0.8410472,15

0.9510687
0.9524087,15

0-99
0.99

47 0.01367 12,30

0.21644 27,30

0.4617542,30

o.67559i57-3°

0.84339 72,30

0.95372 87,30

0.99

48 0.01395 12,45

0.22070^7,45

0.46561 42,45

0.67880157,45

o 84573 72,45

0.95502 87,45

0-99

49 0.01425 13

0.22495:28

0.46947 43

o.682ooi58

0,84805 73

0.95630 88

0.99

50 0.01454 13,15

0.2292028.15

o.47332!43,i5

0.68^1858,15

0.8503573,15

o.95757|88,i5

0.99

51 0.01483

13-3°

0.2334528,30

0.47716143,30

0.6883558,30

0.8526473.3°

0.95882188.30

0.99

52 0.01513

1345

0.2376928,45

0.48099143,45

0.69151 58.45

0.85491 73,45

o 96005 88,45

0.99

53 0.01542

14

0.24192129

0.48481144

0.69466 59

0.85717 74

0.9612689

0.99

54 0.01^7!

14.15

0.24615 29,15

048862144,15

0.6977959,15

0.85941 74,15

0.9624689,15

o.99<

55 o.oi6o0

14,3°

0.25038:29,30

0.49242 44,30

0.70091:59,30

0.86163 74,3°

0.9636389,30

0-99

56 0.01629 14,45

0.254602945

0.49622 44,45

o 70401 59,45

0.8638474,45

0.9647989,45

0-99

57 0.01653115

0.2588230

0.5000045

0.70711 60

0.8660375

0.96593:9°

I. OCX

58 0.01687

15,15

0.2630330,15

0.50377145,15

0.71019,60,15

0.86820:75,15

0.96705 . .

,

59 0.01715

15,30

0.2672430,30

o.50754!45-30

0.71325160,30

o 87036175,30

0.96815 . .

.

60 o.oi745 15,45

0,2714430,45

0.5112945,45

0.71630:60,45

0.8725075,45

0.96923 . .

•