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Cornell University Library
» QM 551.G13 1896

iii’
| 3 1924 001 036 981 vet

cA a)

Cornell University

Library

The original of this book is in
the Cornell University Library.

There are no known copyright restrictions in
the United States on the use of the text.

http://archive.org/details/cu31924001036981

CORNELL UNIVERSITY.

THE
Roswell P. Flower Library
THE GIFT OF RS.

ROSWELL P. FLOWER Oo

1

FOR THE USE OF
THE N. Y. STATE VETERINARY COLLEGE.
1897

i
rs divided into

timeters.

S.
aes . pee ee eo ease ss NT Toeeths: Meter: 7zcron
WHE METER: oe | (Ly), I-toooths millimeter; the Micron is the unit in Micrometry (7 157).

LESGTIE » © « /eler, 1000 Meters; used in measuring roads and other long distances.

THE GRAM FOR ({ Afi/ligram (m.g.), 1-1oooths gram. . :
WEIGHT. . . . | Atlogram, 1000 grams, used for ordinary masses, like groceries, etc.

THE LITER FOR { Cubic Centimeter (c.c.), 1-100oths Liter. This is more common than the cor-
CAPACITY. . . | rect form, Milliliter.
Divisions of the Units areindicated by the Latin prefixes: decz, 1-10th; centz, 1-100th; Mill/7,

3-1000th.
Multiples are designated by Greek prefixes: deka, 10 times ; hecto, 100 times; &zlo, 1000 times ;

myrita, 10,000 times.

TABLE OF METRIC AND ENGLISH MEASURES.

METER = 100 centimeters, 1000 millimeters, 1,000,000 microns, 39 3704 inches.

Millimeter (11m.) = 1000 microns, 75th millimeter, 99th meter, jth inch, ap-
proximately.

MIcRON (/2) (Unit of Measure in Micrometry) = ygysth mm. zyydqq5th meter.
(0.000039 inch) z345yth inch, approximately.

Inch (in.) = 25.399772 mm. (25 4 mm., approx. )

LITER == 1000 milliliters or Icoo cubic centimeters, J quart (appre x.)

Cubie centimeter (cc. or cctm.) = zayath of a liter.

Fluid ounce (8 Fluidrachms) = 29 57% cc. (30 ce., approx. )

GRAM =15 432 grains.

Kilogram (kilo) = 2.204 avoirdupois pounds (2}th pounds, approx. )

Ounce Avoirdupois = (4374 grains) = 28.349 grams.
Ounce Troy or Apothecaries = (480 grains) = 31.103 grams. (30 grams, approx )
TEMPERATURE.

the equivalent of 10° Centigrade, C. = 10° (10° & 2) + 32 = 50°F.
To change Farenheit to Centigrade : (F. —32°) x 3=C. For example, to re-

duce 50° Farenheit to Centigrade, F. = 50°, and (50° — 32°) K 3 = 10°C. ; or — go
Farenheit to Centigrade, F. = — 40° (— 4o° — 32°) = — 72°, whence — 72° & 3
=e 4o° Cc :

Address of American Opticians: For the price of Microscopes and Microscopical supplies, the
student is advised to obtain a catalog of one or more of the opticians named below For the cat-
log of foreign opticians, see addressesin the table of tube-length, p. 15. Nearly all import for-

eign goods.

The Bausch & Lomb OpticalCo.,... 2. 2... New York City and Rochester, N. Y.
Esier BNO AMENG, 6 oe ok ee wa KOO 205-211 Third Avenue, New York City
The Franklin Educational Co.,,,.........0....0...00. Harcourt St., Boston, Mass.
Mt TMT OWS adc gy rl wae 1G, ee ee he 2) ae gh RAIA EL SS fe Ay eo ven se ae oe 7o West 39th St., New York
The Gundlach, Optcal CO. ac ws, a by eR ec a eS Ge ak ROR me Rochester, N. Y.
Wm. Krafft, (representative of Leitzin America),.........~. 411 West soth St., New York
The McIntosh Battery and OpticalCo.,,.........2.2.2.. 521-531 Wabash Ave., Chicago, I11.
Queen &.Co:, Incorporated) a. ee as pe me ee o1o Chestnut St., Philadelphia, Pa.
Richards & Co., Limited, ......... . 30 K, 18th St., New York, 108 Lake St,, Chicago, II].
Edward PennNoekys. 2. a. a4 a che ele oe AA Rak Se 3609 Woodland Ave., Philadelphia, Pa.
Spencer Lens OOg. 2 ik ew RPL Re Pe ee ns es o 4G Main St, Boftalo, N.Y.
Walmsley, Fuller 8Cog.s 2uoy a 8 pk 8 Ga ee we 8 134-136 Wabash Ave., Chicago, Ill.
Williams, Brown & Earle,. . ee ee ree toth & Chestnut Sts., Philadelphia, Pa.
G. S. Woolman, (Queen & Co., in New York),..........2.... 116 Fulton St., New York
J. Zentmayer,.......... BE Be castes udtobl at ey Ba Je 209 South 11th St., Philadelphia, Pa, rs

[Zo replace table on p. 168 of the Microscope and Microscopical Methods, 6th ed.,

by S. H. Gage.

Comstock Pub. Co., Ithaca, N. Y.]

ORDER OF PROCEDURE IN MAKING MICROSCOPICAI, PREPARATIONS BY

THE PARAFFIN METHOD. \

§ 284. It will be seen from this table and from sections 268 to 283

that it requires from 5 to 7 days to get a microscopical preparation by

the paraffin method if one starts with the fresh tissue.

Depending on

the method of fixing and hardening, the time may be much greater.
Unless much time is lost in waiting one must plan ahead in histological
work.

pee

Io.

Fixing and hardening the tissue or
organ (4% 269), 4 days or more.

. Dehydrating the object to be cut in

95% or stronger alcohol (@ 270), I
to 24 hours.

. Displacing the alcohol and clearing

tissues with cedar-wood oil. (See
%271a, below). 2 to 24 hours.

. Infiltrating the tissue with paraffin

in the paraffin oven (4 272, @ 2714),
2 to 24 hours.

. Imbedding in paraffin (% 273), 10

minutes.

Cutting the sections (4 274), Io min-
utes.

Extending the sections with warm
water. (See % 274a, below. )

| Fastening the sections to a slide

(2 2748, 275), 5 minutes to 24
hours.

| Removing the paraffin (4 276), 10

minutes to 24 hours.
Washing in 95% alcohol to remove
the benzin (@ 277), 2 minutes.

Il.
12%

13.

14.

15.

16.

T%

18.

19.

20.

21.

Washing with water, note, p. 162

Staining with an aqueous dye (@ 278-
279), z minutes to 24 hours.

Washing away the superfluous stain.
with water (%.278-279).

Staining with a general dye (4264a,
280a), 10 seconds to Io minutes.
Washing the sections with water

(% 280).

Dehydrating the stained sections in
95% alcohol (% 281), 3 minutes to
24 hours.

Clearing the sections (2 282), 2 min-
utes to 24 hours.

Mounting in balsam (% 283), 1 to 5
minutes. .
Sealing the cover-glass (@ 238), 2

minutes.

Labeling the preparation (% 291), 2
minutes.

Cataloging the preparation (% 294),
5 to Io minutes.

[ To replace or supplement the matter in % 264, 271, 280.}

§ 271a. Displacing the Alcohol and Clearing Tissues with
Thickened Cedar-Wood Oil. (Lee p. 66, Neelsen and Schiefferdeck-
er, Arch. f. Anat. u. Physiol., 1882, p. 206). Instead of using chloro-
form as directed in § 271 much time is saved by displacing the alcohol
with thickened cedar-wood oil. When the alcohol used for dehydration
is displaced by the oil the tissue will look clear and translucent. It is
then removed from the cedar-wood oil and placed in pure, melted paraf-
fin and this is then put into a paraffin oven and left from 2 to 24
hours. It is then imbedded for sectioning ($ 273). The tissues are
not harmed by remaining a long time in the cedar-wood oil. Small
pieces of tissue are cleared by it and: made ready for paraffin infiltra-
tion in two hours or less.

§ 274a. Extending the Sections with Warm Water. In section-
ing by the paraffin method the sections are very liable to be finely wrink-
led. These fine wrinkles or folds are very confusing. To remove them
the ribbons or separate sections as they are cut are placed on cold water in
a shallow basin. Then hot water#s slowly added till the sections extend
or straighten. ‘This removes all the folds. When the water is cold
again the ribbons are cut up with fine scissors and the pieces transferred
to albumenized slides and treated as described in § 275. If the tissue
was stained in toto, no albumen or collodion need be used. ‘The sec-
tions after an hour or more will cling very closely to the slide and may
be de-paraffined and mounted in balsam without the sections coming
off. (Gaskell, Quart. Jour. Micr. Science, xxxi (1890), p. 382;
Gulland, Jour. Anat. and Physiol. xxvi (1891), p. 56.) Where but
few sections are to be mounted on a slide, one may put them directly
from the knife to the slide, add enough water to float them, and heat
the slide gently. The sections will straighten ; the water may then be
drained off and the slide stood on end until the sections are dry and
adhere firmly. The sections appear more or less transparent when dry.

§ 264a, 280a. Counterstaining with Picro-Fuchsin instead of
Eosin. 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 Picro-Fuchsin. Formula : Saturated aqueous solution of picric
acid, 95 c.c., 1% aqueous solution of acid fuchsin, 5 ¢.c. Sections are
first strongly stained with hematoxylin, well washed with water and
then stained 15 seconds to 3 minutes in the picro-fuchsin. They are
then washed in water, dehydrated, cleared and mounted in acid balsam,
that is balsam that has not been neutralized. Neutral balsam soon
fades the red of the fuchsin. (See Freeborn, Trans. N. Y. Path. Soc.
1893, p. 73. Also studies from the Department of Pathology of tie
College of Physicians and Surgeons, Columbia University, N. Y.,
1894-1895. )

ORDER OF PROCEDURE IN MAKING MICROSCOPICAL PREPARATIONS BY
7 THE PARAFFIN METHOD.

§ 284. It will be seen from this table and from sections 268 to 283
that it requires from 7 to 10 days to get a microscopical preparation by
the paraffin method if one starts with the fresh tissue. Depending on
the method of fixing and hardening, the time may be much greater.
Unless much time is lost in waiting one must plan ahead in histological
work.

1. Fixing and hardening the tissue or | 11. Washing away the superfluous stain,
organ (% 269), 4 days or more. (% 278-279).
2. Dehydrating the object to be cut in) yo, Staining with a general dye (2 280),

95% or stronger alcohol (2 270), 5 1o seconds to 10 minutes.
to 24 hours.

3. Saturating the tissue with chloroform
(% 271), 4 to 24 hours.

4. Infiltrating the tissue with chloroform
paraffin (% 272), 3 to 10 days.

13. Washing the sections with water or
alcohol (% 280), 3-5 minutes.

14. Dehydrating the stained sections in
95% alcohol (% 281), 5 minutes to

5. Imbedding in pure paraffin (4 273), 24 OnE,

qovmiinntes: : 15. Clearing the sections (3 282), 5 min-
6. Cutting the sections (% 274), 10 min- utes to 24 hours.

utes. 16. Mounting in balsam (2% 283), 2 to 5
7. Fastening the sections to a slide minutes. ,

(@ 275), 5 minutes. 17. Sealing the cover-glass (3 238), 2 —
8. Removing the paraffin (% 276), Io anintest

niinutes to 24 hours.
g. Washing in 95% alcohol to remove
the benzin (¢ 277), 2 minutes. : :
ro. Staining with an alcoholic or aque- | 19. Cataloging the preparation (¢ 294),
ous dye (2 278-279), 2 minutes to 24 5 to Io minutes,
hours. |

18. Labeling the preparation (¢ 291), 2
minutes.

§ 293. Cataloging Preparations.—lIt is believed from personal ex-
perience, and from the experience of others, that each preparation
(each slide or each series) should be accompanied by a catalog contain-
ing at least the information suggested in the following formula :

A Catalog Card Written According to

% 294. General Formula for Catalog- }
| this Formula :

ing Microscopical Preparations : i

1. The general name and _ source.
Thickness of cover glass and of sections.

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 object
from which the preparation is derived.
Condition of rest or activity ; fasting
or full fed at the time of death.

5. The chemical treatment, —the
method of fixing, hardening, dissociating
etc., and the time required.

6. The mechanical treatment, —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.

g. The objectives and other accesso-
ries (micro-spectroscope, polarizer, etc. )
for studying the preparation.

10. Remarks, including references to
original papers, or to good figures and
descriptions in books.

Muscular Fibers. Cat.
C15.
Fibers 20 to 4o yu thick..

2. No. 475. (Drr. 1X) Oct. 1, 1891. §. |
H. G., Preparator.

3. Tendinous and intra-muscular ter-
minations of striated muscular fibers
from the Sartorius of the cat (Felis do-
muestica.)

4. Cat eight months old, healthy and
well nourished. Fasting and quiet for
12 hours.

5. Muscle pinned on cork with vas-
elined pins and placed in 20 per cent.
nitric acid immediately after death by
chloroform. Left 36 honrs in the acid ;
temperature 20°C. In alum water (%
sat. aq. sol.) 1 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.

9g. Use 18 mm. for the general appear-
ance of the fibers, then 2 or 3 mm. ob-
jective for the details of structure. Try
the micro-polariscope (% 209).

io, 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.

LABELING PREPARATIONS.

(1) The number of the preparations, the
thickness of the cover-glass and
of the sections under it,

(2) The name and source of the prepara-
tion.

(3) The date of the specimen (2 of
catalog. )

CQ. 15

AiGe ATs Secs. Su

Striated Muscle; transection of the

Sartorius of the Cat.

October 15, 1894. ‘

ORDER OF PROCEDURE IN MAKING MICROSCOPICAL PREPARATIONS BY

THE COLLODION METHOD.

S$ 265. It will be seen from this table, and sections 252-266, that

it requires about five days to get a microscopical preparation if one com-

mences with the fresh tissue.
quire as many months.
foresight in histology or much time

12.

. Imbedding the tissue (2 257),

. Cutting the sections (2 259),

. Fixing and hardening the tissues

(@ 252), 4 days or more,

. Dehydrating the object to be cut in

95% or stronger alcohol (¢ 253), 2-
24 hours.

. Saturating the tissue in ether-alcohol

(@ 254), 2-24 hours.

. Infiltrating with thin collodion

(4 255), 2 hours to 2 days.

. Infiltrating in thick collodion (4 256),

5 hours to several days.
15 to
20 minutes. :

. Hardening the collodion with chlo-

roform (4 258), 5-24 hours.

. Clarifying and further hardening the

collodion with castor-xylene (@ 258),
10-36 hours.

Io min-
utes to 2 hours.

. Transferring the sections to a slide

with paper (% 260), 1 minute.

. Fastening the sections to the slide

with ether-alcohol (% 261), 1 or 2

minutes.

Removing the oil from the sections
with benzin and alcohol (2 262), 3-5
minutes, or 24 hours.

Other methods of hardening might re-
It is evident, therefore, that one must exercise

will be wasted.

13.

14.

15.

16.

17.

18.

19.

20.

2I.

22.

23;

24.

Staining the sections with an alco-
holic dye (4263-264), 2 minutes to
24 hours.

Staining the sections with an aque-
ous dye (% 264), 2-10 minutes.

Removing the superfluous dye by
washing in water or alcohol (@ 263-
264), 2-5 minutes.

Staining with a general dye (@ 264),
15-30 seconds.

Washing with water or alcohol
(% 263-264), I to 2 minutes.

Dehydrating the sections in 95% al-
cohol (% 266), 5 min. to 24 hours.

Clearing the sections (¢ 266), 5 min.

to 24 hours.
Draining the sections, 1-2 minutes.

Mounting in Canada balsam (2 266),
I-2 minutes.

Sealing the cover-glass (¢ 238), 2
minutes.

Labeling the preparation (? 291), 2
minutes.

Cataloging the preparation (¢ 294),
5-Io minutes.

REE

10 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-tooth Meter; Millimeter (m.m.), t-1000th Meter: Micron
Ba ces FOR (ft), I-1000th Millimeter ; the Micron is the unit in’Micrometry (2 157).
"+ * + (kvrlometer, 100 Meters ; used in measuring roads and other long distances.
THE GRAM FOR { Milligram (m.g.), I-loooth gram.
WEIGHT. . . . | Avlogram, 1000 grams, used for ordinary masses, like groceries, etc.

THE LITER FOR { Cubic Centimeter (c.c.), I-1oooth Liter. This is more common than the correct
CAPACITY. . . form, Milliliter.

ober iia of the Unzts are indicated by the Latin prefixes: decz, 1-10th; centi, 1-100th; Millz,
I-100oth.

Multiples are designated by Greek prefixes: deka, 10 times; Aecto, too times; &#lo, 1000 times;
myria, 10,000 times.

[This card is the size used for library catalogs. ]
From Gage, the Micr pe and Micr ical Methods,

p

6th edition. Comstock Pub. Co., Ithaca N. Y.

RS)

a

a

on

. Compensation ocular + 12;

THE MICROSCOPE

it isa positive
ocular.

Draw-tube, by which the tube is lengthened
or shortened.

Main tube or body, tothe lower end of which
the objective or revolving nose-piece is
attached.

Society screw in the lower end of the
draw-tube.

. Society screw in the lower end of the

tube.

. Objective in position.

7. Stage, under which is the substage with the

substage condenser.

. Spring clip for holding the specimen.

IN SECTION.

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

. Iris diaphragm outside the principal focus

of the condenser for use in centering
(4 77)-

. Mirror with plane and coneave faces.
- Horse-shoe hase,
. Rack and pinion for the substage conden-

Ser,
Flexible pillar.

. Part of pillar with spiral spring of fine

adjustment.

. Serew of fine adjustment.
» Milled head of coarse adjustment.

Li

[xy 4

na

THE © LIBRA,

MICROSCOPE

AND

MICROSCOPICAL METHODS,

BY

SIMON HENRY GAGE,

Professor of Microscopy, Histology and Embryology in
Cornell University and the New York State Veterinary College,
Ithaca, N. Y., U.S. A.

SIXTH EDITION, REWRITTEN, GREATLY ENLARGED, AND
ILLUSTRATED BY 165 FIGURES IN THE TEXT.

ITHACA, N. Y.
COMSTOCK PUBLISHING Co.
1896.

i

Copyright, 1896,
By SIMON HENRY GAGE.
All Rights Reserved.

Sve So nf LF

Printed by
Andrus & Church,
Ithaca, N.Y.

PREFACE ‘TO THE SIXTH EDITION.

HE 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, con-
venient 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 fundamental
principles of the microscope, and gain a working acquaintance with it, 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.

This edition has been largely rewritten. Many new figures and about ninety
pages of new matter have been added, and it is hoped that the student will find it
a real help in his efforts to become master of the modern microscope.

SIMON HENRY GAGE,

CORNELL UNIVERSITY.
October 31, 1896.

PREFACE TO THE FIFTH EDITION.

HIS edition has been enlarged nearly one-half by the elaboration of the mat-
ter in the previous edition, and by the addition of a wholly new chapter on
photo-micrography and on photographing natural history objects in a hori-

zontal position with a vertical camera. The figures have been distributed in the
text, and many new ones added.

It is hoped that the book as it now appears may, white remaining strictly ele-
mentary, still more fully meet the needs of those who wish to use the microscope
for serious study and investigation. The aim has been to produce a book for be-
ginners in microscopy, such as the author himself felt sorely the need of when he
began the study. This purpose has been strengthened and furthered by noting the
difficulties of the various classes that have used the work and aided in its evolution
during the last fifteen years.

The author wishes to acknowledge the aid rendered by the various Optical Com-
panies for information freely given, and for the loan of cuts and instruments
(Bausch & Lomb Optical Co., Gundlach Optical Co., Queen & Co., and all the op-
ticians mentioned in the table of tube-length, p. 10). I feel under special obliga-
tion to iny various classes for the enthusiasm and earnestness with which they have
followed the instructions in the book, to my colleagues, Professor Wilder and In-
structors Hopkins and Fish for suggestion , to Mrs. Gage for criticising the manu-
script, reading proof, preparing the index and the original figures, to Dr. A. C.
Mercer for aid in preparing the chapter on photo-micrography, to Dr. M. D. Ewell
for information and for the loan of apparatus, and finally, to many other friends
who have used the previous editions, and have made suggestions whereby it is
hoped the present edition is greatly improved.

I would like to repeat a part of the preface to the third and to the fourth editions,
and to call especial attention to the address of the Hon. J. D. Cox at the recent
meeting of the American Microscopical Society: ‘‘A plea for systematic instruc-
tion in the technique of the microscope at the university,’ in the Proceedings for
1893.

Extract from the preface of the fourth edition :

‘The author would feel grateful to any person who uses this book if he would
point out any errors of statement that may be discovered, and also suggest modifi-
cations which would tend to increase the intelligibility, especially to beginners.”

From the third edition ;

“tt is thoroughly believed by the writer that simply reading a work on the mi-
croscope, and looking a few times into an instrument completely adjusted by an-
other, is of very little value in giving real knowledge. In order that the knowl-
edge shall be made alive, it must be made 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
jiave been made an integral part of the work.

‘“‘In considering the real greatness of the microscope, and the truly splendid

vi PREFACE,

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 this
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 appreciating brain
behind the eye. The microscope simply aids the eye in furnishing raw material,
so to speak, for the brain to work upon.

“The necessity for doing a vast deal of drudgery, or ‘dead work,’ as it has been
happily styled by Professor Leslie, before one has the training necessary for the
appreciation and the production of original results, has been well stated by Beale :

“«The number of original observers emanating from our schools will vary as
practical work is favored or discouraged. It is certain that they who are most
fully conversant with elementary details and most clever at demonstration, will be
most successful in the consideration of the higher and more abstruse problems,
and will feel a real love for their work which no mere superficial inquirer will ex-
perience. It is only by being thoroughly grounded in first principles, and well
practiced in mechanical operations, that any one can hope to achieve real success
in the higher branches of scientific enquiry, or to detect the fallacy of certain so-
called experiments.’ ”’

SIMON HENRY GAGE,
CORNELL UNIVERSITY,
February 12, 189]. IrHaca, New York, U.S. A.

CONTENTS.

CHAPTER I.
. ‘ : . PAGE.
@ I- 55—The Microscope and its Parts—Demonstration of the Function
ofeach Part. Figures of Laboratory Microscopes,. ... . I- 32
CHAPTER II.

@ 56-119—Lighting and Focusing, Manipulation of Dry, Adjustable, and
Immersion Objectives; Care of the Microscope and of the
STEVES pro cays sa ok! Siete any. ie eae sh tee eet Fee ces cee Oo ea dhe Ge a res Gee Cea 33- 79

CHAPTER III.
@ 120-144—Interpretation of the Appearances under the Microscope,. . . 80- 91

CHAPTER IV.

2 145-167—Magnification of the Microscope; Micrometry, ....... 92-108
CHAPTER V.
2 168-178—Drawing with the Microscope,.......-...-.-+04- Iog-I1g

CHAPTER VI.

¢ 179-218—Micro-spectroscope and Micro-polariscope ; Use and Applica-
{10 3) 5 “aa we Boe ee Rae Be ee ee ee E eee ee 120-139

CHAPTER VII.

2 219-322 —Slides and Cover-glasses ; Mounting; Isolation ; Sectioning by
the Collodion and Paraffin Methods; Labeling and Storing
Microscopical Preparations; Preparation of Reagents; Ex-
periments in Micro-chemistry,.......-........ 140-182

CHAPTER VIII.
3 322-351—Photo-micrography and the Photography of Natural History
Specimens in a Horizontal Position with a Vertical Camera, - 183-209
APPENDIX.

2 352-370—The use of Abbe’s Test-Plate and Apertometer, ....... 210
Testing Homogeneous Liquids; Experimental Determination
of the Equivalent Focus of Objectives and Oculars; Prepara-
tion of Diagrams ; Preparation of Drawings for Photo-engrav-

TPR der ited es PG ere ae ee en a A a a ee 213-219

BOOKS AND PERIODICALS ................4. » 4 4 220-225
INDEX jo. ce boat el a yah ie 20 a al axes Be eye Mia win Ce a - Ge ks Peet ha 227-237

LIST OF ILLUSTRATIONS.

The author extends grateful acknowledgments to the opticians and others who
have loaned cuts for this edition. The source of each figure is given when bor-
rowed. The other figures were drawn expressly for this work by Mrs. Gage. The
frontispiece was drawn by Mr. Gutsell, of the University Art Department.

FIG, PAGE,
MTOM tS PEGE fea ke eee, ae sa a He le eee ae ha de ae Bl tee oe Sees SS
1-9. The principal axis and center of various lenses... .........4. 2
Jo-II. Principal focus with converging and diverging lenses .. 1... be Gs 3
12, Chromatic-aberration . 2.2 aoa Ge ye ee RE Ag eR we 4
13. Spherical aberration... 44 244 ease Fe eee we eS ee ee, 04
14-15. Real and virtual image with convex lenses... . 2... ....048.- 5
16. Simple microscope and eye of observer... 2... 2... et eee 6
17; Tmpod magnifier 4 6 sac we Be ea we a HP op te BES 7
18. Achromatic triplet The Bausch & Lomb Opt.Co.). 2... ...... 7
19. Lens-holder (The Bausch & Lomb Opt. Co.). 2... 1. ee ee 8
20. Dissecting microscope (The Bausch & Lomb Opt. Co.). ........ 9
21. Principle of the compound microscope. . 2... 1 ee ee 10
225 Dry objective <4... 5-2 2 ew me Bees poe a a ee et gt a es oa II
23, Immersion objective . 2% «6 2 is eee ERR Ree 12
24. Tubéslenpth, 4 2.4 ¢ eae Se See ae e eS Swe eS we RG 15
25. Tube-length when nose piece and ocular micrometer are used (Zeiss’ cat-
IOS INO; 530) shen as ae ad) aap ae Gas OE AO Se AL er bg er Gh oe GG 16
26. Angitlarsapertare 6... 2 6 ai ele eA ey Sea BE RS, A es 17
27-29. Dry and immersion objectives (Ellenberger).. 2... ......0.4. 18
30. Section of Huygenian ocular for eye-point. ...... ae a! Gy tal eet: eam 22
31. Compensation oculars (Zeiss’ catalog, No. 30)... 6. eee ee 24
32. Projection oculars (Zeiss’ catalog, No. 30). . 2... ee ee ee 25
33-34. Ocular micrometer with movable scale (Bausch & Lomb ‘Opt. Co.) . 25
35. Ocular screw micrometer (Zeiss’ catalog, No. 30)... . 2... 2205 26
36. Triple nose-piece or revolver (Queen & Co.) 2... 2. ee ee ion OF
37. Size of field with various objectives and oculars....... is end) Go 29
38. Principle of the simple microscope (Fig. 16 repeated)... 2... .. wou SBE
39-40. Dry and immersion objectives (Figs. 22-23 repeated)... 2. .... 34
41. Achromatic condenser (Zeiss’ catalog, No. 30) .. 2... . a ee a OE
42-43. Image of diaphragm in centering... ...... 2... .0.00.4. 42
44-45. Centering the source of illumination on the object... ....... 43
46-47, Aperture of condenser (from Nelson)... ........-..... oe AS
48-51. Abbe condenser, central, oblique and dark ground illumination... . 47
52. Lamp atid bull’s'eyecondenser' s,s sya 4 4 ee eb we Re Ee YS 49
53-55. Refraction diagrams (from Carpenter-Dallinger). .. 2... 0.0.0.2. 50

56. Aberration produced by the cover-glass (Ross)... .......6... 52

x LIST OF ILLUSTRATIONS.

57. Cover correction by changing tube-length .......... wade satay 154
58. Screen for face and microscope. ... 6 6 1 6 eee et ee 56
59. Ward’s eye shade (Cut loaned hy Gaaen & COs) ie ew ee eS aw 59
60. Double eye-shade.. 2. 2 01 we ee ee ee 59
61-63. Marker, sectional view (Proc. Amer. Micr. Soc., 1894)... .....- 64
64-66. Specimens showing the use of the marker... .. 2... 72 eee 64
67. Krauss’ method of marking objectives on a nose-piece (from Dr. Krauss,

see Proc. Amer. Micr. Soc., 1895) . 2 6 6 0 ee ee ee 65
68. Removable mechanical stage (Leitz catalog). . °°... 1... ee ee 65
69. Removable mechanical stage (Bausch & Lomb Opt. Co.)... ..... 65
70. Zeiss’ Microscope Ia with mechanical stage (Zeiss’ catalog, No. 30) . . . 66

71. Watson & Sons, Edinburgh, student’s microscope (Watson & Sons catalog) 67
72. Nachet et Fils microscope No. 4 with movable stage (cut loaned by the

PranklincBewecationaliCos) co seg ae are tee a a ow Re 68
73. BB Microscope of the Bausch & Lomb Optical Co. (B&L)....... 69
74. Reichert’s microscope IIIb (cut loaned by Richards & Co.).. 2.2... 70
75. Queen & Co.’s microscope II of the continental pattern (Q. & Co.). . . 71
76. Leitz’ microscope Ib (cut loaned by Wm. Krafft)... .......0..- 72
77. Ross eclipse microscope (cut from Walmsley, Fuller & Co.).. 2.2... 73
78. AA. Microscope of the Bausch & Lomb Optical Co. (B.& L.). 2... .. 74
79. Beck’s star microscope (cut loaned by Williams, Brown & Earle)... . 75
80. Zentmayer’s clinical microscope (Zentmayer).........-.-2.22. 76
81. Zentmayer’s microscope, No. V (Zentmayer). . 2... 22. ee eee 76
82. Leitz’ demonstration microscope (from Wm. Krafft, N.Y.) ....... vit
83. Leitz’ microscope IV (from Wm. Krafft, N.V.)............8. 77
84. Queen & Co.’s acme microscope, No. IV (Q. & Co.).. 2. ......2. 78
85. McIntosh’s scientific microscope, No. 2 (McIntosh Battery Co.). . 2... 79
86. Letters mounted in stairs to show order of coming into focus ...... 82
87. Putting on acover-glass............ Wise OP Aw On od eh 84
88. Oil-and air bubbles, eas e-4 4 @ Aum ate ee oe Be Ae hw win OF
89. Glass rods in opticalsection. ..........0.. be Men aa 2k aa dah 86
go. Double contours ss oa! aye. aa a ae ew. a BB oe Pw 87
gt. Micrometer with ring to facilitate finding the lines... 2... ..-.. 94
92: Wollaston’s:camera lucida... 4.43 465% 2 8424S eee ew 95
93. Geometrical diagram showing size of object andimage. ........ 96
94. Image and object with differing tube-length ..... 2... ...... 96

g5. Standard distance for magnification with Wollaston’s camera lucida. . . 98
96. Standard distance for magnification with the Abbe camera lucida... . 98

97. Preparation of blood corpuscles with ring around a group... ..... IOI’
98-99. Ocular micrometer (Figs 33-34 repeated) . 2... 2... eee 103
Ioo. Ocular screw micrometer (Fig. 35 repeated). . 2... 2.2... 2. eee 104
tor. Lines of stage and ocular micrometer in getting the valuation of the ocu-

Jae MTCORVERER 5) fe}. a. ah Sona, GBS ey Qa go he Bw ale ne Ba 107
102. Abbe camera lucida with 45° mirror... . 2... 2. ee ee IIo
103. Geometrical figure going with Fig. 102. ........... 0004 IIo
104. Ocular showing eye-point (Fig. gorepeated) .. 2... 110
105. Wollaston’s camera lucida Lig. G2 TEPEALEG): so we ae aye Beal my ee I11
106, Abbe camera lucida with 35° mirror... 2... . ee ee II4
107. Geometrical figure going with Fig. 106... ... 2... ee 114

108, Upper view of the prism of the Abbe camera cilia. 3 pM! fe ea ae gas LD

109,
ITO.
IIL.

La,
113.

Ir;
II5.
116.
117.
118.
119.
120.
Yr.
122.
123.
124.
125.
126,
127.
F268,
129.

130.
13
132:
133.
134.

135.

136.
137s
138.
139.
140.
I4l,
142.
143.

144.

145.
146.
147.
148.
149.

150.
151.

152.

153.

154.

LIST OF ILLUSTRATIONS.

Quadrant attached to the mirror of the Abbe camera lucida

Inclined microscope with the Abbe camera lucida . : ei

Drawing board for the Abbe camera (The Bausch & Lomb Opt. Ce, ) a

Micrometer lines indicating the scale of adrawing.. . 0... 2...

Longisection of the Abbe micro-spectroscope (cut loawed by the Bausch
& Lomb Opt. Co.) . : aa KE

Slit mechanism of the micro- 6-Spaclneseope ‘(ive Bz & ee ) Sled avant

VATIOUS SPECIOUS a4 4 au SRS Ree Re 4 ele we Be

Absorption spectrum of iemieatobia, oe tangs & McMunn) .

Section of the micro-spectroscope-. 2... 2.2... eee ees

Prism showing apparent reversal of colors . .

Section of a micro-polariscope . .. 2... 0.2.04. Saw eGo R8

Micrometer calipers (Brown & Sharp) .. . , yt eas laine ss

Cover-glass measurer (The Bausch & Lomb Opt. Co. 7 fey ed ode te Ws

Zeiss cover-glass measurer (from Zeiss’ catalog). . . 2...

Putting on a cover-glass (Fig. 87 repeated) . .

Needle holder (Queen & Coz) ss

Turn table (Queen & Co. eg

Centering card. rae

Anchoring a cover- seolads story ar

Irrigation, staining, etc., under thie COVED 4g 2 oe ee Be we ee eG

Moist chamber for Abri, blood corpuscles, etc. (ron Proc. Amer. Micr.
SOCs ISOL) ec aa a

Adjustable lens- holder (Leis, “oul ‘fsen Win; Krafft) .

Adjustable lens-holder (The Bausch & Lomb Opt. on eo

Preparation vials (Proc. Amer. Micr. Soc., 1895) .

Pipette for stains, etc. (Whitall, Tatum & Co.) .

Waste bowl with rack and funnel (cut loaned by Wm. ‘Wood & i. =

Round aquarium for waste bowl, rinsing jar, etc. (Whitall, Tatum & Co.)

Glass box for cleaning slides and covers (Whitall, Tatum & Co.).

Balsam bottle. . . oe ee ee

Serial section slide, strowites order ot 3 arranging seebloms. deste aor vi? ks He,

Writing diamond (Queen & Co.) . ity bs aap eo Me Son v8

Drawer of cabinet for slides (Proc. Reiner: Micr. Boe, , 1883) ol Ae ae

Cabinet for microscopical specimens (Proc. Amer. Mice, Soc., 1883)...

Czapski’s iris diaphragm ocular ( Zeiss’ catalog, No. 30) a eS

Walmisley’s large photo-micrographic camera (from Mr. Walmsley). . .

Leitz’ vertical photo -micrographic camera (from Wm. Krafft)... . 2.

Projection oculars: (Fig. 32 repeated)... 2... 1... 2... ee,

Walmsleys autograph camera in a vertivs! position Cres Mr. oie

Same in horizontal position . .

Vertical photo-micrographic camera (the Reuseli & Lamb Opt. te my

Zeiss’ 70 millimeter projection objective (from Zeiss’ photo- dnjesontaplate

catalog) .
FOCUSIIIG SCHOEN 4: <a on a. ge ay ee a Be eee a pe a ee
Perigraphic dhotoaraahie oective (The Gandtach Opt. Co)... 0...
Zeiss auastigmatic photographic objective ae the Bausch and Lomb
Opt: (COs )i nm ee Sd eo op ee A

Focusing gids Strom the Gundidct Opt. Go, i, CeO Me aaeeer
The tripod asa focusing glass .. 2... ee ee ee

xii LIST OF ILLUSTRATIONS.

155. Engraving glass (The Bausch & Lomb Opt. Co.) . 2... 0.004. 193
156. Bausch & Lomb’s chain lens-holder for use with a dissecting lens, hold-

ing an engraving glass, ete. (B.&L.).. 2... 2... 199
157. Bull’s eye and lamp (Fig. 52 repeated) ........-.....0.2..-.5 200
158. Zeiss’ vertical photo-micrographic camera (Zeiss’ catalog)... .... 202
159. Rack for drying negatives (Rochester Opt. Co.). 2... .....20. 203
160-161. Sections of the head and brain of Tienycr tis (Mrs. Gage, from the

Wilder Quarter Century Book) .. 2... ........2.2.0048. 203
162, Vertical photographic camera for picturing brains and other preparations

it a Norizontal POsiOts, a .<. ¢ddare we sos RG we Alas we a a Be 207
TO4)Abbé's testsplaters (e.% gain! See Gels we is ae ge a ee we 211

165. Abbe’s apertometer (Zeiss’ catalog). . 2... ee ee 212

TELE MICROSCOPE

AND

NIChOSOOPICAL METHODS:

CHAPTER 1,

THE MICROSCOPE AND ITS PARTS.

APPARATUS AND MATERIAL FOR THIS CHAPTER.

A simple microscope (@ 2, 9); A compound microscope with nose-piece (Figs.
68-89), eye-shade (Figs. 59-60), achromatic (¢ 18), apochromatic (% 20), dry (2 15),
immersion (% 16), unadjustable and adjustable objectives (¢ 21, 22), Huygenian or
negative (% 35), positive ‘% 34) and compensation oculars (% 36), stage microme-
ter, homogeneous immersion liquid (¢ 16, Ch. IV), benzin and distilled water (% 103-
108). Mounted letters or figures (¢ 49) ; ground-glass and lens paper (@ 49).

A MICROSCOPE.

21. 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 ob-
ject were brought very close to the eye without affecting the distinctness of vision.
Whenever the microscope is used for observation, the eye of the observer forms an
integral part of the optical combination (Figs. 16, 21).

42. 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 (39). The
simple microscope may be held in the hand or it may be mounted in some way to
facilitate its use (Figs. 17-20).

2 MICROSCOPE AND ACCESSORIES. (CH. I.

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

Axis. The Principal Optic Axis. c-c’. Centers of curvature of the two surfaces
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. ¢.

23. Principal Optic Axis.—In spherical lenses, 7. é., lenses whose surfaces are
spherical, the Axis is the part of the line joining the centers of curvature and
traversing the lens; it is the unbroken part of the line cc’ in all 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 con-
tinuous 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.

24. 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 incident
ray. It is determined geometrically by drawing parallel radii of the curved sur-
faces, r-v/ 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 radii. In Figs. 4-5 it is

CH -£.) MICROSCOPE AND ACCESSORIES. 3

within the lens; in 6-7 it is at the curved surface, and in the meniscus (8, 9) it is
wholly outside 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 par-
allel 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, (7”) 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 paral-
lel 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.

@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
(1). See also Fig. 57.

Fics. 10, 11.—Sectional views of a con-
cave or diverging and a convex or con-
verging lens to show that in the concave
lens the principal focus ts virtual as indi-
cated 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. The principal focal dis-
tance ts the distance along the axis, from
the optical center to the principal focus
(Ff).

Io Ir

26. Principal Focus.—This is the point where parallel rays 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 projected back-
ward. Such a focus is said to be virtual, as it it has no real existence. In Fig. rz
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; and as 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.

4 MICROSCOPE AND ACCESSORIES. (CH. I.

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

The ray of white light (w) ts represented as dividing into the short waved, blue

(6) and the long waved, red (r) light. The blue (6) ray comes to a focus nearer
the lens and the red ray (rv) farther from the lens than the principal focus (f).
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 f/ and f’.

27. Chromatic Aberration.—This is due to the fact that ordinary light consists
of waves of varying length, and as the effect of a lens is to change the direction 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).

Fic. 13. Double Convex Lens, showing
Spherical Aberration.

Fic. 13. The ray (0) near the
edge of the lens is broughtioa
focus nearer the lens than the
ray (7). Both are brought to
a focus sooner than rays very
near theaxts. (f) Princt-
pal focus for rays very near
the axis; (f’) Focus for the
ray (t), and (f’) Focus for
the ray (0). Intermediate
rays would cross the axis all
the way from (f’’ tof).

§ 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 of parallel rays very near the axis
is at f, rays (o 7) nearer the edge would come to a focus nearer the
lens, the focus of the ray nearest the edge being nearest the lens.
Every simple lens has the defect of both chromatic and spherical aber-
ration, and to overcome this, kinds of glass of different refractive power

CH. I.) MICROSCOPE AND ACCESSORIES. 5

and different dispersive power are combined, concave lenses neutraliz-
ing the defects of convex lenses. If the concave lens is not sufficiently
strong to neutralize the aberration of the convex lens, the combination
is said to be wxder-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.

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 I5. 14. Convex lens
showing the position of the object(A-B) pt At
outside the principal focus (F), and
the course of the rays in the formation
of real images. To avoid confusion
the ravs are drawn from only one point.

A B. Object outside the principal fo-
cus. B’ A’. Real, enlarged image on
the opposite side of the lens.

Ax.
xis

Axis. Principal optic axis. 1, 2, 3. a a\ Ng
Rays after traversing the lens. They '
are converging, and consequently form ‘

“ We

arealimage. The dotted line and the
line (2) give the direction of the rays
as tf unaffected by the lens. (F). The
principal focus.

Fic. 15. Convex lens showing the po-
sition of the object (A B) within the AZINS oAesccel esandeen
principal focus and the course of rays : -
in the formation of a virtual image. tas 15:

AB. The object placed between the lens and its focus; A’ B’ virtual image
Sormed by tracing the rays backward. It appears on the same side of the lens as
the object, and is erect (4 9).

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

1, 2,3. Rays from the point B of the object. They are diverging after travers-
ing the lens, but not so divergent as if no lens were present, as ts shown by the
dotted lines. Ray (1) traverses the center of the lens, and ts therefore not deviated.
Lt is a secondary axis (3 5).

2 8a. 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, 36), and the optical.
center (Figs. 1-9) of the lens. Then a secondary axis, (2) in Fig. 14, (1) 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..

6 MICROSCOPE AND ACCESSORIES. (CH. TI.

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.

§ 9. Employ a tripod or other simple microscope, and for object a
printed page. Hold the eye about two centimeters from the upper sur-
face 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 may be 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 mi-

croscope (§ 10, 49).

Fic. 16. 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' 2B. The object within the principal focus. A®
B. The virtual image on the same side of the lens
as the object. Itts indicated with dotted lines, as it
has no actual existence.

B A*. Retinal image of the object(A' B'). The
virtual image is simply a projection of the retinal
image in the field of vision.

Axis. The principal optic axts of the microscope
and of the eye. Cr. Corneaof the eye. L. Crystal-
line lens of the eye. R. Ideal refracting surface at
which all the refractions of the eye may be assumed
to take place.

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 on the paper is obtained. This is the burning point
or focus, and as the rays of the sun are nearly parallel, the burning

CAH. I.) MICROSCOPE AND ACCESSORIES. 7

point represents approximately the principal focus (Figs. 10,11). With-
out changing the position of the paper or the magnifier, look into the
magnifier and note that the letters are very in-
distinct or invisible. Move the magnifier a cen-
timeter or two farther from the paper and no
image can be seen. Now move the magnifier
closer to the paper, that is, so that it is less than
the focal distance from the paper, and the letters
will appear distinct. This shows that in order
to see a distinct image with a simple microscope,
the object must always be nearer to it than its
principal focal point. Or, in other words, the ee

object must be within the principal focus. Com- — 7yiod Magnifier.
pare (§ 49.).

After getting as clear an image as possible with a simple microscope,
do not change the position of the microscope but move the eye nearer
and farther from it, and note that when the eye is in one position, the
largest field may be seen. ‘This position corresponds to the eye-point
(Fig. 30) of an ocular, and is the point at which the largest number of
rays from the microscope enter the eye. Note that the image appears
on the same side of the magnifier as the object. ,

Simple microscopes are
very convenient when
only a small magnifica-
tion (Ch. IV) is desired,
as for dissecting. Achro-
matic triplets are excel-
lent and convenient for

Fic. 18. Achromatic Triplet for the pocket. As shown in the left hand figure it
zs composed of three lenses, one of crown and two of flint glass. The whole is
protected by a metal covering when notin use. (Bausch & Lomb Opt. Co.)

the pocket (Fig. 18). For use in conjunction with a compound micro-
scope, the tripod magnifier (Fig. 17) is one of the best forms. For
many purposes a special mechanical mounting like that of Figs. 19, 20,
is to be preferred.

COMPOUND MICROSCOPE.

210. A Compound Microscope.—This enables one to see an enlarged, inverted
image. It always consists of two optical parts—an odjective, to produce an en-
larged, 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

8 MICROSCOPE AND ACCESSORIES. (CAL.

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).

‘TT TTD
JUTT

Fic. 19. Lens Folder with adjustments for Socusing and for turning the lens in
any direction. This is especially useful in dissecting the minute parts of animals
too large for the regular dissecting microscope (Fig. 20). (Bausch & Lomb Opt. Co.)

Fic. 20. Dissecting Microscope with hand rests and nose-piece for several lenses of
different power. (Bausch & Lomb Optical Co.).

10 MICROSCOPE AND ACCESSORIES. [CA. I.

MECHANICAL PARTS.

211. The Mechanical Parts of a laboratory, compound microscope are shown in
the frontispiece, and are described in the explanation of that figure. The student
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.

212. Microscopic Objective.—This
consists of aconverging lens or of one
or more converging lens-systems, which
give an enlarged, inverted, real image of
the object (Figs. 14, 21). Andas forthe
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 229,
49, Figs. 16, 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 geueral,
composed of a convex and a concave
lens. The combined action of the sys-
tems serves to produce an image free

; from color and from spherical distor-
tion. In the ordinary achromatic ob-
jectives the convex Jenses are of crown
and the concave lenses of flint glass
(Figs. 22, 23).

Fic. 21. Diagram showing the prin-
ciple 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? B*), and the projection of the retinal
image into the field of vision as the
zirtual tmage (B® A*),

AB. The object. A? BY. The retinal
tinage oftheinvertedrealimage,(BA!),
formed ty the objective. B A*. The in-
verted virtual image, a projection of
the retinal image.

CH, I.) MICROSCOPE AND ACCESSORIES. II

«lis. The principal optic axis of the microscope and of the eye.

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

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

Mirror. The mirror reflecting parallel rays to the object. The light 1s central.
See Ch. Ll.

Fos. 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.

§ 13. Equivalent Focus.—In America, England, and sometimes also on the Con-
tinent, 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 ;, in. or 2 mm., indicates that the objective produces a
teal image of the same size as is produced by a simple converging lens whose prin-
cipal focal distance is 7; inch or 2 millimeters (Fig. 11). An objective marked 3
in. or 75 mm., produces approximately the same sized real image as a simple con-
verging lens of 3 inches or 75 millimeters focal length. And in accordance with
the law ¢hat the relative size of object and image vary directly as their distance
Jrom 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 objec-
tive, the greater is the size of the real image, as the tube-length remains constant
and the image in all cases is found at about 160 or 250 mm. from the objective.

214 Numbering or Lettering Objectives.—Instead of designating objectives by
their equivalent focus, many Contiuental 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 niagnification of objectives). This method is entirely arbitrary and
does not, like the one above, give direct information concerning the objective.

@ 15. Dry Objectives,—These are objectives in which the space between the front
of the objective and the object or cover-glass is filled with air (Fig. 22). Most ob-
jectives of low and medium power (7. é., 4th in. or 3 mm. and lower powers) are
dry.

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

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

BC. Back Combination, composed of a
plano-concave lens of flint glass (f°), and a
double convex lens of crown glass (Cc). A aA

FC. Front Combination. a

C, O, sl. The cover-glass, object and slide.

Mirror. The mirror ts 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.

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

SS

Biage

12 MICROSCOPE AND ACCESSORIES. (CH. I.

216. 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 cedarwood oil or of glycerin containing
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. 56) 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.

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

Axts. The principal optic axis of the objective.

BC, MC, FC. The back, middle and front
combination of the objective. In this case the
Sront ts not a combination, but a single plano con-
vex-lens.

A,B. Parallel rays reflected by the mirror axi-
ally or centrally upon the object.

C. Ray reflected to the object obliquely.

I. Immersion fluid between the front of the ob-
jective 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.

217. Non-Achromatic Objectives.—These are objectives in which the cliro-
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. (22 7, 8, Figs. 12, 13).

218. Achromatic Objectives.—In these the chromatic and the spherical aberra-
tion are both largely eliminated by combining concave and convex lenses of differ-
ent kinds of glass ‘‘so disposed that their opposite aberrations shall correct each
other.’”’ All the better forms of objectives are achromatic and also aplanatic (2 19).
That is the various spectral colors come to the same focus.

219. Aplanatic Objectives, etc.—These are objectives or other pieces of optical
apparatus (oculars, illuminators, etc. ), in which the spherical distortion is 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 appa-
ratus are usually achromatic also. (27, 8).

2 20. 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

CH. £.) MICROSCOPE AND ACCESSORIES. 13

fluoride, Fluorite, or Fluor spar). The name, Apochromiatic, 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
objectives. At the present time (1896) several opticians make apochromiatic ob-
jectives without using the fluorite. Some of the apochromiatics deteriorate rather
quickly in hot, moist climates.

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 /wo 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 ove color, the correction being made for the central part of the
spectrum, the objective remaining wader corrected spherically for the red rays and
over-corrected for the blue rays (¢@ 8).

(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 apochromatic objectives
give as clear images as the same power water immersion objectives of the old form.

221. Non-Adjustable or Unaijustable Objectives.—Oljectives 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
homogeneous immersion are mostly non-adjustable. For beginners and those un-
skilled in manipulating adjustable (? 22) objectives, 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 Fig. 24).

Z 22. 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 ¢ 24. 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 @ 96).

3 23. Parachromatic, Pantachromatic and Semi-apochromatic Objectives.—These
are trade names for objectives, most of them containing one or more lenses of the
new Jena glass. They are said to approximate much more closely to the apo-
chromatics than to the ordinary objectives.

%24. Variable Objective.—This is a low power objective of 36 to 26 mm. equi-
valent focus, depending upon the position of the combinations. By means of a

14 MICROSCOPE AND ACCESSORIES. (CAE dT.

screw collar the combinations may be separated, diminishing the power or ap-
proximated and thereby increasing it.

225. Projection Objectives.—These are designed especially for projecting an
image on a screen and for photo-inicrography. They are characterized by having a
flat, sharp field brilliantly lighted. In power they vary, the lowest being of 75
mm. and the highest of 6 mm. equivalent focus (see Ch. IV).

226. Illuminating or Vertical Illuminating Objectives.—These are designed for
the study of opaque objects with good reflecting surfaces, like the rulings on metal
bars. 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.

¢ 27. 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
niicroscopist 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), ex-
cept with homogeneous immersion objectives, has.a marked effect on the light
passing from the object (Fig. 56). To compensate for this the relative positions of
the systems composing the objective are different from what 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.

CH. 1

MICROSCOPE AND ACCESSORIES.

15

Length in Millimeters and Parts included in “ Tube-Length” by

Various Opticians.*
Pts. included

in ‘‘ Tube- “ Tube-length ” in
i { a: | length.” Millimeters.
3 ~~-b See Diagram.
3 | ie Grunow, New York : 203 mm.
: OW, 3,
E. Leitz, Wetzlar . .. . 170mm.

Nachet et Fils, Paris .

a-d { Powell and Lealand, London .
C. Reichert, Vienna . ?
Spencer Lens Co., Buffalo

LW. Wales, New York

Bausch & Lomb Opt. Co., Rochester. .

. 146 or 200 mm.

254 mm.

. 160 to 180 mm.
, 235 or 160 mm.
. 254 mim.

216 or 160 mm.

Bézu, Hausser et Cie, Paris. . ... 220 mm. ;
b-d Klonne und Miller, Berlin. . . . . 160-1800r 254mm,
. 190 mm.

W. & H. Seibert, Wetzlar. . . . . ,
| Swift & Son, London... ... .
LC. Zeiss, Jena...

. 165 to 228% mm.
. 160 or 250 mm.

. Gundlach Optical Co., Rochester. . . 254 mm.
. R. Winkel, Gottingen... 2... . 220 mm,
2 . Ross & Co., London... . 2... 254 mm.
3 . R. & J. Beck, London . . . 254 mm.
& . J. Green, Brooklyn... . . 254 mm,
° . 160-180 mm.

Hartnack, Potsdam
Vérick (Stiassnie) Paris . .
Watson & Sons, London. .

. 160-200 nim,
. 160-250 mm.

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

Various Opticians.

Grunow, Brooklyn.

a Green, Brooklyn.
1
|

75mm Powell and Deslant. London.
Spencer Lens Co., Buffalo.
|W. Wales, New York.
?f;mm. Watson & Sons, London.
iésmm. Klonne und Miiller, Berlin.
y HE. Leitz, Wetzlar.
Toomm. LR Winkel, Gottingen, Germany.
16,25mm Ross & Co., London.
qo’smm. Bausch & Lomb Optical Co., Rochester.
1353°mm C. Zeiss, Jena.
135}2mm Cc. Reichert, Vienna.
Guudlach Optical Co., Rocleeien:
qesmm {* & H. Seibert, Wetzlar.
R. & J. Beck, London.
135}4mm J. Zentmayer, Philadelphia.
10.15 Nachet et Fils, Paris.
“roo wm Bézu, Hausser et Cie, Paris.
5mm Swift and Son, London.

*The information contained in these tables was very kindly furnished by the
opticians named, or by consulting catalogs. In most of the later catalogs the in-
formation 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
(@ 29) and the tube-length for which the objective is corrected. This is in accord-
ance 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.

16 MICROSCOPE AND ACCESSORIES. [CH. J.

§ 28. Aperture of Objectives.—The angular aperture or angle of
aperture of an objective is the angle ‘‘ contained, in each case, between
the most diverging of the rays issuing from the axial point of an object
[¢. e., a point in the object situated on the extended optic axis of the
microscope], that can enter the objective and take part in the formation
of an image.’’ (Carpenter).

43). Ifthe table in this edition is compared with the original table or with that in
the previous edition of this book some differences will he noted, the changes being
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,
Pp. 250, are as follows :

‘Believing in the desirability of a uniform tube-length for microscopes, we
unanimously recommend: 1. 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 defined in section 1—Be, for the short
or continental tube, 160 mm., or 6.3 inch-
es, and 216 mm., or 81% inches, for the
long tube, and that the draw tube of the
microscope possess two special marks in-
dicating 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 objectives
of 6 mm. (4% inch) and shorter focus
should be such as to bring the optical
center of the objective 114 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 7%; to 13°
mm. (z+, to 745 inch) in thickness.

These recommendations give a distance
Fig. 25. The tube of a microscope with of io inches (254 mm.) between the par-

ocular micrometer and nose piece 7 focal plane of the ocular and the optical

position to show that in measuring center of the chbjective for the long tube,
tube-length one must measure from and are essentially in accord with the
the eye lens to the place where the 0b- actyal practice of opticians.

jective is attached. (Zetss’ Catalog, At the request of the committee, a joint

No. 30). conference was held with the opticians
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. Gace,
A. CLIFFORD MERCER,
CHARLES E. BARR.

Tube length.

CH. £:] MICROSCOPE AND ACCESSORIES. 17

According to some other authors the angle of aperture is the angle
between the extreme rays from the focal point which can be transmit-
ted through the entire objective. This would give a somewhat greater
angle than by the first method as the focal point of the objective is
nearer to it than the axial point of the object (Figs. 10, 11).

In general, the angle increases with the size of the lenses forming the
objective and the shortness of the equivalent focal distance ($13). If
all objectives were dry or all water or all homogeneous immersion a com-
parison of the angular aperture would give one a good idea of the rela-

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

Axts, the principal optic axis of the objective. 4
BA, BC, the most divergent rays that can enter the objective, =
they mark the angular aperture. AB Dor CBD half the
angular aperture. This ts designated by u in making Nu- 8

merical Aperture computations. See the table, 3 30.

tive number of image forming rays transmitted by different objectives ;
but as some are dry, others water and still others homogeneous immer-
sion, one can see at a glance that, other things being equal, the dry ob-
jective (Fig. 27) receives less light than the water immersion, and the
water immersion (Fig. 28) less than the homogeneous immersion (Fig.
29). Inorder to render comparison accurate between different kinds of
objectives, Professor Abbe takes into consideration the rays actually
passing from the back combination of the objective to form the real im-
age; he thus takes into account the medium in front of the objective as
well as the angular aperture. The term ‘‘ Mumerical Aperture,’’ (N.
1.) was introduced by Abbe to indicate the capacity of an optical in-
strument ‘‘ for receiving rays from the object and transmitting them to
the image, and the aperture of a microscopic objective is therefore de-
termined by the ratio between its focal length and the diameter of the
emergent pencil at the point of its emergence, that is the utilized diam-
eter of a single-lens objective or of the back lens of a compound objec-
tive.”’

§ 29. Numerical Aperture (abbreviated N. A.) is then the ratio
of the diameter of the emergent pencil to the focal length of the lens,
or as usually expressed, the factors being more readily obtainable, it is
the index of refraction of the medium in front of the objective (z. ¢., air
for dry, and water or homogeneous fluid for immersion objectives) mul-
tiplied by the sine of half the angle of aperture. The usual formula

2

18 MICROSCOPE AND ACCESSORIES. [CH. I.

is N. A. =z sin «; N. A. representing numerical aperture, 7 the in-
dex of refraction of the substance in front of the objective, and w the
semi-angle of aperture.

For example, take three objectives each of 3 min. equivalent focus,
one being a dry, one a water immersion, and one a homogeneous im-
mersion. Suppose that the dry objective has an angular aperture of
106°, the water immersion of 94° and the homogeneous immersion of
go°. Simply conipared as to their angular aperture, without regard to
the medium in front of the objective, it would look as if the dry objec-
tive 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

FIGS. 27-29 are somewhat modified from

a Lllenberger, and are introduced to illustrate
| om the relativeamount of utilized light, with dry,
water immersion and homogeneous tinmier-

: iS 27 ston objectives of the same equivalent focus.

| NU Cover | The point from which the rays emenate ts in
air in each case. If Canada balsam were in

place of the air beneath the cover glass there

would be practically no refraction of the
rays on entering the cover glass (% 16).

Fic. 27. Showing the course of the rays

passing through a cover glass from an axial

28 point of the object, and the number that final-
ly enter the front of a dry objective.

Fic. 28. Rays from the avial point of the
object traversing a cover of the same thick-
ness asin Fig. 26, and entering the front lens
of a water immersion objective.

i Fic. 29. Rays from an axial point of the
eX \Asla LE LY § 29 object traversing a cover-glass and entering
es AAU ZC @ ree | oat ob
the front of a homogeneous tinmersion objec-
a all tive.

pale

is considered, that is to say, if the numerical instead of the angular ap-
ertures are compared, the results would be as follows : Numerical Aper-
ture of a dry objective of 106°, N. A.=~zsinz. In the case of dry ob-
jectives the medium in front of the objective being air, the index of
refraction is unity, whence x = 1. Half the angular aperture is 196° —
53°. By consulting a table of natural sines it will be found that the
sine of 53° is 0.799, whence N. A. = 2 or 1 X sin z or 0.799 = 0.799.
With the water immersion objective in the same way N. A. = » sin w.
In this case the medium in front of the objective is water, and its index
of refraction is 1.33, whence x = 1.33. Half the angular aperture is
§jh° = 47°, and by consulting a table of natural sines, the sine of 47° is

CA. L.) MICROSCOPE AND ACCESSORIES. 19

found to be 0.731, @. ¢., sin “= 0.731, whence N. A. = 2 or 1.33 X sin
OF 0.731 = 0.972.

With the oil immersion in the same way N. A. = 2 sin ~; 2 or the
index of refraction of the homogeneous fluid in front of the objective is
1.52, and the semi-angle of aperture is 4°° = 45°. The sine of 45° is
0.707, whence N. A. = z or 1.52 X sin w or 0.707 = 1.074.

By comparing these numerical apertures: Dry 0.799, water 0.972,
homogeneous immersion 1.074, the 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 an-
gular aperture is readily determined from the formula ; and one can de-
termine the equivalent angles of objectives used in different media (Zz. e.,
dry or immersion). For example, suppose each of three objectives has
a numerical aperture (N. A.) of 0.80, what is the angular aperture of
each. Using the formula of N. A. = 2 sin z one has N. A. = 0.80 for
all objectives.

For the dry objective 2 = 1 (Refractive index of air).
‘* water immersion objective 7 = 1.33 (Refractive index of water).
‘‘ homogeneous immersion objective 2 = 1.52 (Refractive index
of homogeneous liquid). And 2 z is to be found in each case.

For the dry objective, substituting the known values the formula
becomes 0.80 = I sin z, or sin «= 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 w, or sin“ = © 80 0.6015.

1.33
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’
2 = 73° 58% ‘

For the homogeneous immersion objective, substituting the known
values, the formula becomes: 0.80 = 1.52 sin wz whence sin w=
oa = 0.5263. And by consulting the table of sines it will be found
that this is the sine of 31° 4514’ whence 2 z or the entire angle (balsam
or oil angle) is 63° 31’.

That is, three objectives of equal resolving powers, each with a nu-
merical 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.

20 MICROSCOPE AND ACCESSORIES. (CAH. I.

§ 30. Table of a Group of Objectives with the Numerical Aper-
ture (N. A.) and the method of obtaining it. Half the angular aper-
ture 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 andn = 1, for water
immersions n = 1.33, and for homogeneous tmmersions n = 1.52. (For
a table of natural sines, see third page of cover).

2% Index of
% 3—| Natural SINE Refraction | NUMERICAL, APERTURE
OBJECTIVE. | 3 § 5 of half the angular of the poe

bh OW aperture. um in fron es;

3 a isin 2). of the oe (N.A.) = sin uw.
25Jnmt 20° Sin 7° —0.1736 m=1 |N.A.= 1 X0.1736=0.173
(Dry.) 2 .

25 1m. 4o° Sin 4° — 0.3420 n=1 |N.A= 1X03420—0.342
(Dry.) 2
ou 42° sin® = 0.3583 m=1 |N.A.= 1 X0.3583 = 0.358
oh too? | Sin <2 =0.7660 n=1 |N.A.= 1 X0.7660 = 0.766
Seca © | sin 25 — 0.6087 =: NA= 6087 = 0.608
(Dry.) 75 = n=l .A.= 1 X0.6087 = 0.60
ee 136° Sin 13° — 0.9272 n—=1 |N.A.= 10.9272 =0.927
epry.) I15° Sin “1S =0.8434 | n=1 |N.A.= 1 X0.8434==0.843
epry.) 163° Sin = =0.98909 | m=1 |N.A.= 1 X0.9890 = 0.989
2mm. 96° 12
; Water. 96° 12/ | Sin ae eee 7443| m2 =1.33/N.A. = 1.33 X 0.7443 = 0.99
mumiersion.
2mm. T1869 48”
Homogeneous |110° 38’| Sin “03° = 0.8223 m = 1.52|N.A, = 1.52 X0.8223 = 1.25
Immersion.
2mm. Ta 16! d
Homogeneous |134° 10’| Sin 134" 10" 9.9210 m= 1.52/N.A. = 1.52 X0.9210 = 1.40
Immersion 2

§ 31. Significance of Aperture.—As to the real significance of
aperture in microscopic objectives, itis now an accepted doctrine that—
the corrections in spherical and chromatic aberration being the same—
(1) 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 N. A. would define or resolve only half as many lines to

CH. f5) MICROSCOPE AND ACCESSORIES. 2r

the millimeter or inch as a similar objective of 1.00 N. A. Soalso 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.

(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.)’. 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 0.207: 0.40°= 0.0400: 0.1600 or 1: 4. That is the
objective of 0.20 N. A. would have but 1th the illuminating power of
the one of 0.40 N. A.

In considering illuminating power the equivalent focal length must
also be considered. If the N. A. were the same in a 3mm. anda 6
mm. objective their illuminating power would vary directly with the
square of the foci. Thus the illuminating power of the 6 and the 3
mm. objectives would be as 6’: 3” or 36 to 9 or 4:1, that is 4 times as
great in the 6 mm. as in the 3 mm. objective. As the magnification of
an objective varies indirectly as the equivalent focus, it follows also
that the illuminating power will vary indirectly as the square of the
magnification of the objective. The magnification of the 6 mm. is 42
and of the 3 mm. 84, whence the illuminating power of the two objec-
tives are as 427: 84° or 1764: 7056 or 1: 4. As the ratio is inverse in
this case the result is the same as before, that is 4 times as great for the
6 mm. as for the 3 mm. objective.

(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 ne

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

In comparing the penetrating power of objectives of different foci,
the numerical aperture being the same, it is found that the penetrating
power increases directly as the square of the focus. For example, two
objectives of the same N. A., one of 4 mm. and the other of 2 mm.
focus, the penetrating power would be as 4’: 2’ or 16:4 0rq4:1. That
is, the numerical aperture remaining the same, the greater the equiva-
lent focus the greater the penetration.

To briefly summarize: The numerical aperture is concerned with
resolution and the resolving power varies directly as the numerical
aperture, thus if the N. A. is doubled, twice as many lines to the mil-
limeter or inch can be resolved.

22 MICROSCOPE AND ACCESSORIES. (CH. I.

With illuminating and penetrating power the equivalent focus of the
objective must be considered as well as the numerical aperture. With
objectives of the same equivalent focus, to double the N. A. is to in-
crease the illuminating power 4-fold but the penetrating power is halved.

The numerical aperture remaining constant, the illuminating and
penetrating power vary directly as the square of the equivalent focus ;
thus a 4 mm. objective would give four times the illuminating and
penetrating power of a 2 mm. objective.

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

For a consideration of the aperture question, its history and signifi-
cance, 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. Carpenter-Dallinger, Chapters II and V.

THE OCULAR.

232 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 mi-
croscope, to magnify the real image formed by the objective.

Fic. 30. Sectional view of a Huygenian ocular (Hg. ocu-
/ lar), to show the formation of the E-ye-Point.
Axis. Optic axis of the ocular. D. Diaphragm of the
ocular, EF. L. Evye-Lens. F.L. Field-Lens.
£.P. Eye-point. As seen in section, it appears something
like an hour-glass. When seen as in looking into the ocu-
lar, 1. €., 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.

4 33. Negative Oculars are those in which the real, in-
verted 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
N absent (Fig. 21). As the field-lens of the ocular aids in the
S=E=-\ formation of the real image it is considered by some to form
i \) @ part of the objective rather than of the ocular. The upper

ae or eye-lens of the ocular magnifies the real image.

@ 34. Positive Oculars are those in which the real, inverted image of the objec-
tive 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

He. Ocwla?

CH. I.) MICROSCOPE AND ACCESSORIES. 23

eye-lens toward the object and looking into the field-lens an image may be seen,
however. ‘

Special sames have also been applied to oculars, depending upon the designer,
the construction, or the special use to which the ocular is to be applied. The fol-
lowing are most used.*

*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
seemed 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 3.19). 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
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 composé ; Ger. zusammengesetztes Okular. An ocu-
lar of two or more lenses, ¢. g., the Huygenian (see Fig. 30). Continental Ocular.
An ocular mounted in atube of uniform diameter as in Fig. 31. Jeep Ocular,
see high ocular. Lyrecting 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. /tlar micrometer Ocular; Screw m. 0, Cobweb m. o.
Ger. Okular-Schraubenmikrometer. A modification of Ramsden’s Telescopic Cob-
web micrometer ocular. Goniometer Ocular; Fr. Oculaire 4 goniométre ; Ger.
Goniometer-Okular. An ocular with goniometer for measuring the angles of mi-
nute crystals. Afigh Ocular, sometimes called a deep ocular. One that magnifies
the real image considerably, 7. 2., 10 to 20 fold. Auygenian Ocular, Huygens’ O.,
Campani's O., Airy's O.; Fr. Oculaire d’Huygens, 0. de Campani; Ger. Huy-
gens’sches Okular, Campaniches Okular, see 7 35. /udex 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 satisfacto-
rily for micrometry. Aed/ner’s Ocular, see orthoscopic ocular. Low Ocular, also
called shallow ocular. An ocular which magnifies the real image only moderate-
ly, 2.@., 2to8 fold. Micrometer or micrometric Ocular; Fr. Oculaire microme-
trique ou a micrométre; Ger. Mikrometer-Okular, Mess Okular, Bénéches O.,
Jackson m. o., see 4 38. A/icroscopic Ocular ; Fr. Oculaire microscopic ; Ger. mi-
kroskopisches Okular. An ocular for the microscope instead of one for a telescope.
Negative Ocular, see 7 33. Nelson’s screw-micrometer ocular. A modification of
the Ramsden’s screw or cob-web micrometer in which positive compensating ocu-
lars may he used. Orthoscopic Oculars ; also called Kellner’s Ocular; Fr. OQcu-
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) anda
double convex field lens. The field-lens is in the focus of the eye-lens and there

24 MICROSCOPE AND ACCESSORIES. [Gaz

235. Huygenian Ocular.—A negative ocular designed by Huygens for the tele-
scope, but adapted also to the microscope. It is the one now most commonly em-
ployed. 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 flo. 2 4 6 8 12 18

Fic. 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, g and 6 are negative,and the higher ones, 8, 12
18, ave positive. The numbers 2, 4, 6, 8,12, 18, indicate the magnification of the
ocular. (From Zeiss’ Catalog No. 30).

is no diaphragm present. The field is large and flat. /%a7-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). Pertscopic 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 % 34. Projection Ocular ; Fr. Ocu-
laire de projection ; Ger. Projections-Okular, see 437. 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 2 36.
Shallow Ocular, see low ocular. Solid Ocular, holosteric O.; Fr. Oculaire holo-
stére ; 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 or
spectroscopic Ocular ; Fr. Oculaire spectroscopique ; Ger. Spectral-Okular, see Mi-
crospectroscope, Ch. VI. Stauroscopic 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. Arheits Okular,
see 4 36.

GET MICROSCOPE AND ACCESSORIES. 25

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 im-
age achromatic.

%@ 36. 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 ocular
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).

237. Projection Oculars.—These are oculars especially designed for projecting
a microscopic image on the screen for class demonstrations, or for photographing
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.

Fic. 32. Projection Oculars with section re-
moved to show the construction. Below are
Shown the upper ends with graduated circle to in-
dicate the amount of rotation found necessary to
Socus 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. ( Zeiss’ Catalog,
No. 30].

% 38. Micrometer Ocular.—Tlis is an ocular
connected with an ocular micrometer. The
micrometer may be removable, or it may be per-
manently in connection with the ocular, and ar-
ranged with a spring and screw, by which it may
be moved back and forth across the field. (See
Ch. IV).

FIG. 33 FIG. 34.

Fics. 33-34 Ocular Micrometer with movable scale. Fig. 33 is a side view of
the ocular while Fig. 34 giv2s au sectional end view, and shows the ocular microme-
ter in position. In both the screw which moves the micrometer ts shown at the
left. (From Bausch & Lomb Opt. Co.)

26 MICROSCOPE AND ACCESSORIES. (CH. J.

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 clampiug screw to
fasten the ocular to the upper part of the mt1-
croscope tube. Below ts 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 microme-
ter very smallobjects frequently fill but a part
of an interval of the micrometer, but with this
the movable cross lines traverse the object (or
rather its realimage) regardless of the minute-
ness of the object. ( Zeiss’ Catalog, No. 30).

§ 39. Spectral or Spectroscopic Ocular.—(See Micro-Spectroscope, Ch. VI).

DESIGNATION OF OCULARS.

2 40. Equivalent Focus.—As with objectives, sone opticians designate the ocu-
lars by their equivalent focus (2713). With this method the power of the ocular,
as wit! objectives, other lenses or lens systems, 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 advocated 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).

241. 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.

442. 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 multiplied 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,

< 2, X 4; and for the long or 250 mm. tube, they are X 3 and X 6. 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. VIIT.

COMPOUND MICROSCOPE.
EXPERIMENTS.

$ 43. Putting an Objective in Position and Removing it.—Ele-
vate the tube of the microscope by means of the coarse adjustment
(frontispiece) so that there may be plenty of room between its lower

(Gah Jk] MICROSCOPE AND ACCESSORIES. 27

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 in 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.

S 44. Putting an Ocular in Position and Removing it.—Elevate
the body of the microscope with the coarse adjustment so that the ob-
jective 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 insert-
ing them of forcing the tube of the microscope downward and the ob-
jective upon the object.

§ 45. Putting an Object under the Microscope.—This is so plac-
ing an object under the simple microscope, or on the stage of the com-
pound microscope, that it will be in the field of view when the micro-
scope is in focus (§ 46).

With low powers, it is not difficult to get an object under the micro-
scope. The difficulty increases, however,
with the power of the microscope and the
smallness of the object. It is usually neces-
sary to move the object in various directions
while looking into the microscope, in order
to get it into the field. Time is usually
saved by getting the object in the center of
the field with a low objective before putting
the high objective in position. This is
greatly facilitated by using a nose-piece, or
revolver. (See also § 118, Figs. 61-66).

& 46. Field or Field of View of a Microscope.—This is the area
visible through a microscope when it isin 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, different parts are
brought successively into the field of view.

FMA, AHP
Fis. 36. Triple nose-piece
or revolver for quickly chang-
ing objectives (Queen & Co.).

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.

28 MICROSCOPE AND ACCESSORIES. (CH. J.

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.

$47. Table showing the actual size in millimeters of the field of a group
of commonly used objectives and oculars. Compare with the graphic rep-

resentation tn Fig. 37. See also § 46,

|
| Equivalent | Diameter |
' Focus and

NA. of of Field OCULAR.
Objective. in mm.
15.4 3714 inm.
85 mm... . 106 25 Be Huygenian.
8.3 ious **
. 7.0 374 mm |
‘45 mm.. 5.0 25 A Huygenian.
4.0 rete ss !
i | 3.0 37/2 mm. i
| 17 mm.. 2.0 25 “| Huygenian.
1.6 v2,“ 4
57 180 mm
| N.A. = 0.25 on oe « Compensation.
0.97 10 -

i5mm.... | 0 371 25 “ Huygenian.

N.A.=0 92 | res 2 “« Compensation,
| es. || a
j O 270 apt,
0.147 taj

|
2mm... 5 | 0.186 25 ee Huygenian.
|

Compensation.

CA. J.) MICROSCOPE AND ACCESSORIES. 29

2mm
\
@ 2

17 mm

45 mm

85 mm

Fic. 37. Figures showing approximately the actual size of the field with ob-
jectives of 85 mm., 5 mm., 17 mm., 5 mm., and 2 mm., equivalent focus, and
ocular of 37% mm., equivalent focus in each case. This figure shows graphically
what ts also very clearly indicated in the table (4 47).

S$ 48. The size of the field of the microscope as projected into the
field of vision of the normal human eye (7. 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).

FUNCTION OF AN OBJECTIVE.

§ 49. Put a 2-in. (50 mm.) objective on the microscope or screw off
the front combination of a 16 mm., (33-in.), and put the back-combina-
tion 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.*

Lower the tube of the microscope by means of the coarse adjustment
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, 7. e., if the top of the microscope 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.

* Ground glass may be very easily prepared by placing some fine emery between
two pieces of glass, wetting it with water and then rubbing the glasses together
fora 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 7.

To demonstrate that the object must be outside the principal focus
‘vith 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 dis-
tance (Figs. 10, 11). 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 § 9).

§ 50. 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
eyes for the right distance. If the paper is absent the eyes adjust
themselves for the light circle at the back of the objective, and the
aerial image appears low in the tube. Furthermore, it is more difficult
to see the aerial image in space than to see the image on the ground-
glass or paper, for the eye must be held in the right position to receive
the rays projected from the real image, while the granular surface of
the glass and the delicate fibers 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.

$51. 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 ob-
ject (Fig. 21).

FUNCTION OF AN OCULAR.

§ 52. Using the same objective as for § 49, 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 18) as if the image were an object.

CH. 1.) MICROSCOPE AND ACCESSORIES. 31

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 nakedeye ($9). Remove the
screen and observe the aerial image with the tripod.

Put a 50 mm., (A, No. 1, or 2 in.) ocular (7. e., an ocular of low
magnification) in position (§ 44). Hold the eye about to 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 mi-
croscope with the coarse adjustment.

$53. 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

Fic. 38. 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' B'. The object within the principal focus. <1*
B. The virtual image on the same side of the lens
as the object. It 7s indicated with dotted lines, as
it has no actual existence.

B’ A’, Retinal image of the object (A' B'). The
virtual image is simply a projection of the retinal
image in the field of vision. s AL

Axis. The principal optic axis of the microscope
and of the eye. Cr. Cornea of the eye. L. Crystal-
line lens of the eye. R. Ideal refracting surface at fi qt BN ‘ i
which all the refractions of the eye may be assumed :

'
'
to take place. ts ya
bat | a
certain definite lines and not in all directions ; Wy XQ se
ly :
: : rata 3 e mm
hence, in order to see this aerial image with pf. ____ mace ica) 3

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

§ 54. 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).

32 MICROSCOPE AND ACCESSORIES. (CH, SL.

Demonstrate this by screwing off the field-lens and using the eye-lens
alone as in the ocular, refocusing if necessary. Note also that the let-
ters or other 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.

§ 55. 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 ($49). Light the object brightly, focus the micrc-
scope, shade the ocular, then hold some ground-glass or a piece of the
lens paper above the ocular and slowly raise aud lower it until the
smallest circle of light is found. By using different oculars it will be
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.

Iu the appendix will be giveu 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 treatinent.
The most satisfactory work in English is Carpenter-Dallinger. For the history of
the microscope, Mayall’s Cantor Lectures on the microscope are very satisfactory.
See also Bzale, E Bausch, Belerens, Kossel and Schiefferdecker, Dippel, Frey,
Harting, Hogg, Nageli and Schwendener, Robin, Van Heurck, Clark, Cross and
Cole, Stokes.

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, 1110; same, 1890, p. 480; Zeit. f. In-
strumentenk., 1890, pp. 1-6; Micr. Bullt., 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., 1881, pp. 303, 348, 365, 388 ; 1882, pp. 300, 460 ; 1883,
p. 790; 1884, p. 20.

CHAPTER IL

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 con-
densers, dry, adjustable and immersion objectives, oculars, tripple nose-piece.
Microscope lamp and movable condenser (bull's eye or other form (Fig. 52),
Homogeneous immersion liquid; Benzin, alcohol, distilled water ; Mounted
preparation of fly’s wing (7 68); Mounted preparation of Pleurasigma. Stage or
ocular micrometer with lines filled with graphite (% 73, 74); Glass slides and
cover-glasses (Ch. VII) ; 10 per ct. solution of salicylic acid in 95 per ct. alcohol
(2 88) ; Preparation of stained microbes (% 101) ; Vial of equal parts olive or cot-
ton seed oil or liquid vaselin and benzin (% 105); Ward’s and double eye-shade
(Figs. 59, 60) ; Screen for whole microscope (Fig. 58).

FOCUSING.

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

With a simple microscope (4 9) 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 tbe 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 object and objective be brought.

#57. Working Distance.—By this is meant the space between the simple micro-
scope and the object, or between the front lens of the compound microscope and
the object, when the microscope is in focus. This working distance is always con-
siderably less than the equivalent focal length of the objective. For example,
the front-lens of a %thin., or 6 mm. objective would not be {th inch, or 6 milli-
' meters 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

3

34 LIGHTING AND FOCUSING. (CH. LZ.

high objectives, it would be necessary to use very thin cover-glasses over the ob-
ject. (See 3 22, 27). 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 ex-
amination with high powers it is always better to use thin covers.

LIGHTING WITH DAYLIGHT.

258. 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., itis 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. 58) 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 micro-
scope 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 symmetrically lighted, or lighted
more on one side than the other, light used in microscopy is designated as re-
flected and transmitted, axial and oblique.

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

4 59 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 micro-
scope. By this kind of light objects are ordinarily seen by the unaided eye, and

CH. LL) LIGHTING AND FOCUSING. 35

the objects are mostly opaque. In Vertebrate Histology, reflected light is but
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 ob-
ject, or is reflected or condensed upon it with a mirror or condensing lens, answers
very well. For high powers aud for special purposes, special illuminating appa-
ratus has been devised (2 26). (See also Carpenter-Dallinger, p. 278).

260. Transmitted Light.—By this is meant light which passes through an ob-
ject 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 (4 75), and thence transmitted to the object from below (Figs. 48-51).

¢ 61. 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 parallel with
the optic axis of the microscope. In either case the object is symmetrically illu-
minated.

262. Oblique Light.—This is light in which parallel rays from a plane mirror
form an angle with the optic axis of the microscope (Fig. 4o). 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. 49).

DIAPHRAGMS.

2 63. 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 somie cases an iris diaphragm is used, and then the same
one is capable of giving a large range of openings. The object of a diaphragm, in
geueral, 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.

264. 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 used. 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 diaphragin 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. Ifthe diaphragm is a considerable
distance below the object, (1) it allows considerable adventitious light to reach the
object and thus injures the distinctness of the microscopic image ; (2) it prevents
the use of very oblique light uuless 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.

With an illuminator or condenser (Figs. 41, 48), the diaphragm serves to narrow

36 LIGHTING AND FOCUSING. (CH. /7.

the pencil to be transmitted through the condenser, and thus to limit the aperture
‘or for any special purpose to be served (see % 80). 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

(988; Fig. 51).
ARTIFICIAL ILLUMINATION.

265. For evening work and for certain special purposes, artificial illumination
is employed. A good petroleum (kerosene) lamp with flat wick has been found
very satisfactory, but for brilliancy and for the actinic power necessary for photo-
micrography (see Ch. VIII) the new acetylene light seems to be all that could be
desired. Whatever source of artificial light is employed, the light should be bril-
liant and steady.

LIGHTING AND FOCUSING : EXPERIMENTS.

§ 66. Lighting with a Mirror.—Place a mounted fly’s wing under
the microscope, put the 16 mm. (34 in.) or other low objective in posi-
tion, also a low ocular. With the coarse adjustment, lower the tube of
the microscope to within about 1 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 (§ 46) 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
($57). 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 suniight.

§ 67. Use of the Plane and of the Concave Mirror.—The mirror
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 when a moderate amount is needed or when it is necessary to have
parallel rays or to know the direction of the rays.

§ 68. Focusing with Low Objectives.— Place a mounted fly’s
wing under the microscope ; put the 16 mm. (23 in.) objective in posi-
tion, and also the lowest ocular. Select the proper opening in the dia-
phragm and light the object well with transmitted light (§ 60, 64).

CH AT, | LIGHTING AND FOCUSING. 37

Hold the head at about the level of the stage, look toward the win-
dow, and between the object and the front of the objective ; with the
coarse adjustment lower the tube until the objective is within about
half acm. 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 turning 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. Puta high ocu-
lar in place of the low one (§ go). If the oculars are not par-focal it
will be necessary to lower the tube somewhat to get the image in focus.*

Pull out the draw-tube 4-6 cm., thus lengthening the body of the
microscope, and it will be found necessary to lower the tube of the mi-
croscope somewhat. (For reason, see Fig. 57).

§ 69. 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.

§ 70. Focusing with High Objectives.—Employ the same object
as before, elevate the tube of the microscope and remove the 16 mm.
(23 in.) objective as indicated. Put the 3 mm. (% in.) or a higher ob-
jective in place, and use a low ocular.

Light well, and employ the proper opening in the diaphragm, ete.
(§ 64). Look between the front of the objective and the object as be-
fore (§ 68), and lower the tube with the coarse adjustment till the ob-
jective 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 neces-
sary, 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

* Par-focal oculars are so constructed, or so mounted, that those of different pow-
ers nay be interchanged without the microscopic image becoming wholly out of
focus (Fig. 31, note, p. 23). 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.

38 LIGHTING AND FOCUSING. (CH. 17.

to the focal point either from the shadow or the color, if the object is
colored. With high powers and scattered objects there might be no ob-
ject in the small field (see § 46, 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. 61-65 § 118).

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 oculars are
not par-focal) or lengthening the tube of the microscope it will be found
necesssary to bring the objective still nearer the object, as with the low
objective. (For reason see Fig. 57).

§$ 71. 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 of view (§ 46). It may be neces-
sary 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
adjustment constantly, and the focus is continually varied.

72. Determination of Working Distance.—As stated in § 57
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 equivalent
focal length of the objective.

Make a wooden wedge to 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 useacover-glass. Focus the
dust carefully first with the low then with the high objective. When
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 work-
ing distance. For measuring the thickness of the wedge at the point of
contact for the high objective use a steel scale ruled in 4ths mm. and
the tripod to see the divisions. Or one may usea cover-glass measurer,
for determining the thickness of the wedge (Ch. VIII).

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-

CH, JT.) LIGHTING AND FOCUSING. 39

til the front of the objective just touches the cover-glass. Note the po-
sition of the micrometer screw and slowly focus up with the fine ad-
justment until the object isin 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 a cover-glass was raised by the fine adjustment a distance repre-
sented by 16 of the divisions on the head of the micrometer screw.
Each division represented .or 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.

CENTRAL AND OBLIQUE LIGHT WITH A MIRROR.

§ 73. Axial or Central Light (§ 61).—Remove the condenser or
any diaphragm from the substage, then place a preparation containing
minute air bubbles under the microscope. The preparation may be
easily made by beating a drop of mucilage on a slide and covering it.
(See Ch. III). Use a3 mm. (% in.) or No. 7 objective and a medi-
um ocular. Focus the microscope and select a very small bubble, one
whose image appears about 1 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 Pleurastgma angulatum or some other
finely marked diatom. Study the appearance very carefully.

§ 74. Oblique Light, (§ 62).—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 different appearance with that of cen-
tral light. The effect of oblique light is not so striking with histologi-
cal preparations as with diatoms.

It should be especially noted in §$ 73, 74, that one cannot deter-
mine the exact direction of the rays by the position of the mirror. This
is especially true for axial light (§ 73). To be certain that the light is
axial some such test as that given in § 73 should be applied. (See
also Ch. III, under Air-bubbles).

CONDENSERS OR ILLUMINATORS.*

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

*No one has stated more clearly or appreciated more truly the value of correct
illumination and the methods of obtaining it than Sir David Brewster, 1820, 1831.

40 LIGHTING AND FOCUSING. (CH. I.

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

It is of the greatest advantage to have the sub-stage condenser
mounted with rack and pinion 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 (§ 88) and with blue and neutral tint glasses to soften the
glare when artificial light is used (§ 85, 89).

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

§ 76. Achromatic Condenser.—lIt is still believed by all expert mi-
croscopists that the contention of Brewster was right, and the condenser
to give the greatest aid in elucidating microscopic structure must ap-
proach in excellence the best objectives. That is, it should be as free as
possible from spherical and chromatic aberration, and therefore would
transmit to the object a very large aplanatic cone of light. Such con-
densers 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 expen-
sive, and student microscopes as well as many others are mostly 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 (Car-

Iie 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 illu-
mination 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 chro-
matic and spherical aberration, and the greatest care be taken to exclude all ex-
traneous 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, aud the Edin-
burgh Journal of Science, new series, No. 11 (1831), p. $3.

CH ATT: LIGHTING AND FOCUSING. 41

penter-Dallinger, pp. 254, 263). ‘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 objec-
tive is put into position, the front lens being next the object. As will
be seen below (§ 79-80), the full aperture of an objective can rarely be
used, and for histological preparations perhaps never, so that an objec-
tive of greater equivalent focus, 7. ¢., lower power is used than the one
on the microscope. It is much more convenient, 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 aper-
ture of 1.00, and an aplanatic aperture of N. A. 0.65.

Fic. 41. Zetss’ Achromatic Conden-
Ser. cs cS. Centering screws for
changing the position of the condenser
and making its axis continuous with
that of the microscope. A segment of
the condenser ts cut away to show the
combinations of lenses. For very low
powers the upper lens is sometimes
screwed off. There ts an iris dia-
phragm between the middle and lower
combinations. (Zeiss’ Catalog, No.

3°)

§ 77. Centering the Condenser.—To get the best possible illumina-
tion for bringing out in the clearest manner the minute details of a mi-
croscopic object two conditions are necessary, viz.: The principal 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,
Zz. €., 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 : The object is placed
on the stage and lighted with the edge or face of the flame and then a
very small diaphragm is put below the condenser. (If the Zeiss achro-
matic 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 exact-
ly central. This is best accomplished by putting the paper disc over
the iris or metal diaphragm and then making the hole in the center of

42 LIGHTING AND FOCUSING. (CH. I.

the small circle uncovered by the metal diaphragm. For the holea fine
needle is best). 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 found accu-
rately 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 mi-
croscope be used to focus the diaphragm exactly as in ordinary work.

Fic. 42. Shows that the optic axis of
the condenser does not coincide with
that of the microscope. (D). Dia-
phragm of the condenser shown at one
side af the field of the microscope.

Fic. 43 Shows the diaphragm (D)
in the center of the field of the micro-
scope, and thus the coincidence of the
axis of the condenser with that of the
microscope.

FIG. 42.

§ 78. Centering the Image of the Source of Mlumination.—For
the best results it is not only necessary that the condenser be properly
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.
The condenser is then racked up toward the objective 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 posi-
tion of the lamp or of the bull’s eye (Figs. 34, 52).

CH. IT.) LIGHTING AND FOCUSING. 43

§$ 79. 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 prac-
tice that for diatoms, etc., the best images are obtained when the object
is lighted with a cone which shall fill 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 can be utilized.

Fic. 44 Shows the image of the
Hlame (Fl.) in the center (C) of the
field of the microscope and illuminat.-
ing the object.

Fic. 45. Shows the image of the
flame (Fl.) at one side of the center
(Lxc.) and not properly illuminating
the object.

FIG. 44. FIG. 45.

To determine this in any case, focus the object carefully, 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 advanta-
geous to use the entire aperture, for others, less than three-fourths.
Experience will teach the best lighting for special cases.

Lilum

Fic. 46. Fic. 47.

Fics. 46-47. Figures showing the dependence of the objective upon the illumt-
nating cone of the condenser. (Nelson.)

Fic. 46 (dA). The illuminating cone from the condenser ({llum). 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.

Fic. 47. (4). Ln this figure the illuminating cone from the condenser ( [llum.)
ts seen to be insufficient to fill the objective (Ob7.).

(B). The back lens of the objective only partly filled with light, due to the re-
stricted aperture of the illuminator.

44 LIGHTING AND FOCUSING. (CH. I.

§ 80. 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 (§ 63) ; for there the diaphragm
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 16mm. 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 diaphragin 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 ac-
complished 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
light is spread over a greater area (Figs. 48-51).

§ 81. Non-Achromatic Condenser.—Of the non-achromatic con-
densers or illuminators, the Abbe condenser or illuminator is the one
most generally used. It is also much more commonly used than the
achromatic condenser from its cheapness. 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 trans-
mitted 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 con-
denser like the Abbe are much more satisfactory than with no condenser.
The highest results cannot be attained with it, however. (Carpenter-
Dallinger, p. 256).

CH. IT.) LIGHTING AND FOCUSING. 45

§ 82. 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
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.

§ 83. Centering the Condenser.—The illuminator should be cen-
tered 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 cen-
tered. Centering is approximated as follows : Puta pin-hole diaphragm
over the end of the condenser (Fig. 48)—that is, a diaphragm with a
small central hole—the central opening should appear to be in the mid-
dle 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 smalldrop 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 micro-
scope and illminator axes nay not be entirely coincident even when the
center of the upper lens appears in the center 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 arraugements 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 mount-
ing for this the most commonly used condenser whereby it may be cen-
tered as described for the achromatic condenser instead of by the crude
methods described above. If the condenser mounting regularly pos-
sessed centering screws as in the microscope of Watson & Sons (Fig.
71), and there was a centering diaphragm in the proper position so that
its image 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 still
retain all their efficiency.

46 LIGHTING AND FOCUSING. (CAH. Ll.

§ 84. Mirror and Light for the Abbe Condenser.—lIt 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 ob-
ject ; 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 condenser is
close to the object (Fig. 48).

§ 85. Artificial Light.—If one uses lamplight, it is recommended
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 con-
cave mirror may be used to render the rays less divergent. It may be
necessary to lower the illuminator somewhat in order to illuminate the
object in its focus.

ABBE CONDENSER: EXPERIMENTS.

§ 86. 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 up-
per 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 Pleurasigma as object. Study carefully the appearance of the
diatom with this central light, then make the diaphragm eccentric so as
to light with oblique light. The differences in appearance will probably
be even more striking than with the mirror alone (§ 74).

§ 87. Lateral Swaying of the Image.—Frequently in studying 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 ec-
centric 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 with reference
to the illuminating ray? What in focusing up? If one understands
this experiment it may sometimes save a great deal of confusion. (See
under testing the microscope for swaying with central light § roq4).

CGHaTT.| LIGHTING AND FOCUSING. 47

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

48. 49. 50. Si.

Fics. 48-51. Sectional views of the Abbe Llluminator of 1.20 N. A. showing
various methods of illumination (% 84). 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 illumina-
tor ts centered, that ts its optic axis is a prolongation of the optic axis of the
microscope.

S. Arts. Secondary axis. In oblique light the central ray passes along a sec-
ondary axts of the illuminator, and ts therefore oblique to the principal axis.

DD. Diaphragms. These are placed in sectional and in face views. The dia-
phragm ts placed between the mirror and the illuminator. In Fig. 49 the opening
ts eccentric for oblique light, and in Fig. 51 the opening is a narrow ring, the
central part being stopped out, and thus giving rise to dark-ground illumination
(4 88).

Ob7. Obj. The front of the objective.

posed 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 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 ob-
ject will appear like a self-luminous one on a dark back-ground. This

48 LIGHTING AND FOCUSING. (CH. IT.

form of illumination is only successful with low powers and objectives
of small aperture. It is well to make the illuminator immersion for
this experiment, see § 98.

(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 graphite, use a 16 mm. (% in.) objective,
and when the light is sufficiently oblique the lines will appear some-
thing like streaks of silver on a black back-ground. A specimen like
that described below in (B) may also be used.

(B) TW7th the Abbe Condenser.—Have the illuminator so that the
light would be focused on the object (see § 82) 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 and allow it to dry and 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 dia-
phragm eccentric. The same specimen may also be tried with a mir-
ror 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 adding a
very small drop of the solution to the dried preparation, putting it under
the microscope very quickly, lighting for dark-ground illumination and
then watching the crystallization.

ARTIFICIAL ILLUMINATION.

§ 89. For evening work and for regions where daylight is not suf-
ficiently brilliant, artificial illumination must be employed. Further-
more, for the most critical investigation of bodies with fine markings
like diatoms, artificial light has been found superior to daylight.

A petroleum (kerosene) lamp with flat wick givesa satisfactory light.
It is recommended that instead of the ordinary glass chimney one made
of metal with a slit-like opening covered with an oblong cover-glass is
more satisfactory, as the source of light is more restricted. Very ex-
cellent results may be obtained, however, with the ordinary bed-room
lamp furnished with the usual glass chimney.

The new acetylene light promises to be the most perfect of all the
artificial lights for microscopic observation and for photo-micrography.
(See under Photo-micrography).

CHET.) LIGHTING AND FOCUSING. 49

Fic. 52. 1. Lamp with slit-opening in metal chimney. 2. Bull's eye on separate
stand. 3. Screen showing image of flame.

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

§ 90. 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. 52), and only by trial can this position be
determined. In some cases it is found advantageous to discard the mir-
ror and allow the light from the bull’s eye to pass directly into the conden-
ser. This method is especially excellent in photomicrography (see Ch.
VIII).

§$ 91. 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 condenser
may be lowered also. (See also § 80).

REFRACTION AND COLOR IMAGES.

792. 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 3 79).

4

5° LIGHTING AND FOCUSING. (CAH. I.

If the color and refractive index of the object were exactly like the mounting
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 isa
great advantage. This combination is generally taken advantage of in histology.
Fig. 89 is an example of a purely refractive image.

53. NG 54. N’. 55. N’%

Fics. 53-55.—Diagrams illustrating refraction in different media and at plane
and curved surfaces. In each case the denser medium ts 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 <1 C.

293. 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. 53, light passing from air into water
does not continue in a straight course but is bent éoward the normal N-N’, 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 in-
stead of to C’.

Conversely, if the ray of light is passing from water into air, on reaching the air
it is bent from the normal, the ray C B passing to A and not in a straight line to
Cc’... By comparing Figs. 54, 55, iu which the denser medium is crown glass instead
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. This relationship is expressed thus: Sine of the angle of incidence di-
vided by the sine of the angle of refraction equals the zadex of refraction. In

Sin ABN

the figures, Sin CBN? index of refraction. Worked out completely in Fig. 39,

A BN= 40°, CBN = 28° 54’ and Sin fT a NS 1.33, 2. é., the index of
Sin 28° 54’ 0.48327

refraction from air to water is 1.33. (See 430). In Figs. 54-55, illustrating refrac-

tion 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 getting the correct angle where the exact angle corresponding to the sine
cannot be found in the table it is necessary to proceed as follows: Find thesine in

CH. IT) LIGHTING AND FOCUSING. 51

2 94. Color Images.—These are images of objects which are strongly colored
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 specially applicable to the study of
stained microbes. (See below # ror).

ADJUSTABLE, WATER AND HOMOGENEOUS OBJECTIVES.
EXPERIMENTS.

$95. Adjustment for Objectives.—As stated above (§ 22), the ab-
erration produced by the cover-glass (Fig. 56), is compensated for by
giving the combinations 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 unad-
justable objectives, one can secure the best results of which the object-
ive is capable by selecting covers of the thickness for which the object-
ive was corrected. (See table in § 27). Adjustment may be made

e

also by zzcreasing the tube-length for covers ¢izzzer than the standard,

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 deter-
mined, 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 in the particular region. 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 greater the sine is than the next
lower one whose angle is known. Add this number of minutes to the angle of the
next lower sine and the sum will represeut 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 the last section 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 0.48481, the sine of 29°. Evidently then the angle sought must lie between
28° 45’ and 29°. If the difference between 0.48481 and 0.48099 be obtained, 0.48481
— 0 48099 = 0.00382, and this increase for 15’ be divided by 15 it will give the in-
crease for I minute; 0.00382 +15==0.000254. Now the difference between the
sine whose angle is to be found and the next lower sine is 0.48327 — 0.48099 =
0.00228. If this difference is divided by the amount found necessary for 1 minute
it will give the total minutes above 28° 45’ ; 0.00228 + 0.000254 =9. That is the
angle sought is 9 minutes greater than 28° 45’ = 28° 54’. If now it is found how
much less the sine is than the next higher sine it will be seen that 0.48327 and
0.48481 differ by 0.00154. If this is divided by 0.000254 the change in sine for 1
minute in this region of the arc, the result is 0.00154 + 0.000254—=6, That is the
sine of 29° is too great for the sine whose angle is to be found by 6 minutes, hence
29° less 6’ = 28° 54’, the angle sought.

52 LIGHTING AND FOCUSING. (CH. I.

and by shortening the tube-length for covers ¢iicker than the standard
(Fig. 57).

Fic. 56.—Lffect of the cover-glass on
the rays from the object to the objective
(Leoss).

Axis. The projection of the optic
axis of the microscope

F. Focus or axial point of the objec-
tive.

F’and F’’, Points on the axis where
rays 2 and 3 appear to originate if
traced backward after emerging from
the upper side of the cover-glass (Cover).

itis for the correction of this disturbance, or so-called ‘‘ negative aberration,”
produced by the cover-glass that objectives are fixed in their mounting for a given
thickness of cover, or that the combinations making up the objective are made ad-
justable; that is, so that the back combinations may be brought nearer the front
lens or combination as the cover thickens, and separated with a thinner cover or
for uncovered objects. The thicker the cover, the nearer the front and back combt-
nations » the thinner the cover the farther apart are front and back combinations
separated (% 96).

§ 96. Adjustable Objectives.—The proper adjustment of object-
ives, that is, the adjustment which gives the truest image, requires both
insight and experience ; for the structure of an object does not appear
the same with different adjustments of the objective. And as the opin-
ion of different observers on the structure of objects varies, they adjust
the objectives differently, and try to obtain the adjustment which will
show a structure in accordance with their opinion. Eyes also differ, and
two observers might find it necessary to adjust the same objective dif-
ferently to produce an identical appearance for each of them.

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, shading the stage and adjusting the objective, until the
proper appearance is obtained. The adjustment is made by turning a
ring or collar which acts on a screw and increases or diminishes the dis-
tance between the systems of lenses, usually the front and the back sys-
tems (Fig. go). In adjustable objectives the back systems should be mov-
able, the front one remaining fixed so that there will be no danger of
bringing the objective down upon the object. If the front system is
movable, the body of the microscope should be raised slightly every
time the adjustment is altered.

General Directions.—(A) The thinner the cover-glass the further
must the systems of lenses be separated, ¢. ¢., the adjusting collar is

CH. IT.) LIGHTING AND FOCUSING. 53

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, 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 wé¢hout the focus, or farthest from the ob-
jective [7. ¢., in focusing up], the lenses must be placed further asunder,
or toward the mark uncovered [?.e¢., the adjusting collar is turned
toward the zero mark as the cover-glass is too thin for the present ad-
justment]. Ifthe greater expansion is when the object is within the
focus, or nearest the objective, [7. ¢., in focusing down], the lenses must
be brought closer together, or toward the mark covered, [7. ¢., the ad-
justing collar should be turned away from the zero mark, the cover-
glass being too thick for the present adjustment].’’ Jl most objectives
the collar ts graduated arbitrarily, the zero (O) mark representing the
position for uncovered objects. Other objectives have the collar graduated
to correspond to the various thickness of cover-glasses for which the object-
ive may be adjusted. This seems to be an admirable plan , then tf one
knows the thickness of the cover-glass on the preparation (Ch. VILL) the
adjusting collar may be set at a corresponding mark, and one will feel
confident that the adjustment will be approximately correct. It ts 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 systems of the objective must be
brought closer together, z. 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.

54 LIGHTING AND FOCUSING. (CH. IT.

Object-b

Object-a

Fic. 57.—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. 56) tt 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. «1s shortening the tube serves to re-
move the object, it neutralizes the effect of the thick cover, and tf the cover ts so
thin that it does not elevate the object enough for the corrections of the objective,
then an increase in the tube-length will correct the defect.

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

CH. I.) LIGHTING AND FOCUSING. 55

engraving on each objective the ‘‘tube-length’’ for which it is correct-
ed, 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 appendix).

$97. Water Immersion Objectives.—Put a water immersion ob-
jective in position (§ 43) and the fly’s wing for object under the micro-
scope. 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 the
other till the image is clear. Water immersions are exceedingly con-
venient in studying the circulation of the blood, and for many other
purposes where aqueous liquids are liable to get on the cover-glass. If
the objective is adjustable, follow the directions given in § 95.

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 would 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.

§ 98. 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.

§ 99. Tester for Homogeneous Liquid.—In order that full ad-
vantage 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 appendix).

§ 100. Refraction Images.—Put a 2 mm. (sth 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 § 70. 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

56 LIGHTING AND FOCUSING, (CH. I.

medium will also increase the sharpness of the outline. If an air bub-
ble preparation (§ 73) were used, one would get pure, refraction images.

$ 101. Color Images.—Use some stained microbes, as Baczllus tuber-
culosis for object. Put a drop of the immersion liquid on the cover-
glass or the front lens of the homogeneous objective. Remove the dia-
phragms 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 microbes in focus. They will stand
out as clearly defined colored objects on a bright field.

Fic. 58.—Screen for shading the microscope and the
i Suce of the observer. This ts very readily constructed
as shown in the figure by supporting a wire in a disc
of lead, iron, or heavy wood. The screen ts then com-
pleted by hanging over the bent wire, cloth or manilla
paper 30. zocm. The lower edge of the screen should
be a little below the stage of the microscope and the
upper edge high enough to screen the eyes of the ob-
server.

gO cm
soem

go cm §$ 102. 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 ob-
jective will serve the purpose. Often the proper effect may be obtained
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 im-
mersion objectives. The screen (Fig. 58) is the most satisfactory
means for this purpose, as the entire microscope above the illuminating
apparatus is shaded.

§$ 103. 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 (§ 107),
then if the fluid is cedar oil, wet one corner of a fresh piece in benzin
and wipe the front lens with it. Immediately afterward wipe with a
dry part of the paper. The cover-glass of the preparation can be
cleaned inthe same way. If the homogeneous liquid is a glycerin mixt-
ure proceed as above, but use water instead of benzin to remove the
last traces of glycerin.

CH, 17.) LIGHTING AND FOCUSING. 57

CARE OF THE MICROSCOPE.

$ 104. The microscope should be handled carefully and kept perfect-
ly 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, especial-
ly from acids, alkalies, alcohol, benzin, turpentine and chloroform.

S$ 105. Care of the Mechanical Parts.—To clean the mechanical
parts put a small quantity of some fine oil (olive oil or liquid vaselin
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 cha-
mois or paper wipe off most of the oil. If the mechanical parts are
kept clean in this way a lubricator is rarely needed. Where opposed
brass surfaces ‘‘cut,’’ z. e., when from the introduction of some gritty
material, minute grooves are worn in the opposing surfaces, giving a
harsh movement, the opposing parts should be separated, carefully
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.

§$ 106. 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 b2 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 cloths 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.

§$ 107. 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 ten years for cleaning 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 and sand are not present ; and from its bibulous character it is very
efficient in removing liquid or semi-liquid substances.

58 LIGHTING AND FOCUSING. [CH. IZ.

§ 108. 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 removed
by unscrewing them and wiping as directed above. A high objective
should never be taken apart by an inexperienced person.

Tf the cloudiness cannot be removed as directed above, moisten one cor-
ner 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 distilled
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
to the glass, it may be removed more readily by adding a small quan-
tity 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 chlo-
roform, etc., may be 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 combination
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.

§ 109. Keep both eyes open, using the eye-screen if necessary (Fig.
59, 60) ; and divide the labor between the two eyes, 7. ¢.. use one eye
for observing the image awhile and then the other. In the beginning

CH. I.) LIGHTING AND FOCUSING. 59

it is not advisable to look into the microscope continuously for more
; than half an hour at a time. One
Q never should work with the micro-
} scope after the eyes feel fatigued.
After one becomes accustomed to mi-
croscopic observation he can work for
Fic. 59.—Ward’s Eye-Shade. several hours with the microscope
without fatiguing the eyes. This is due to the fact that the eyes be-
come inured to labor like the other organs of the body by judicious ex-
ercise. It is also due to the fact that but very slight accommodation is
required of the eyes, the eyes remaining nearly in a condition of rest as
for distant objects. The fatigue incident upon using the microscope at
first is due partly at least to the constant effort on the part of the ob-
server to remedy the defects of focusing of the microscope by accommo-
dation 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 suitable light—that is light which is
steady and not so bright as to dazzle the eyes nor so dim as to strain
them in determining details—microscopic work should improve rather
than injure the sight.

Fic. 60. Double Eye-Shade. This 1s Fe AD sam:
readily made by taking some thick bris- ~
tol board 7 x 14 centimeters and making
an oblong opening with rounded ends

(o—o) and of such a diameter that it goes fo A cae
readily over the tube of the microscope. — ;
This is then covered on both sides with “eee - eee eee e

velveteen and a central slit (s) made in
the cloth. This admits the tube of the
microscope and holds the screen in post-
tron. It may readily be pulled from side to side and thus serves for either eye, or
Sor the use of the eyes alternately.

§ 110. Position and Character of the Work-Table.—The work-
table should be very firm and large (122 x 72 cm. ; 28 x 48 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 com-
fortable. An adjustable stool, something like a piano stool is conven-
ient, then one may,vary the height corresponding to the necessities of
special cases. It is a great advantage to sit facing the window if day-
light is used, then the hands do not constantly interfere with the illu-
mination. To avoid the discomfort of facing the light a screen like that
shown in Fig. 58 is very useful (see also under lighting, § 58).

60 LIGHTING AND FOCUSING. (CH. ll.

TESTING THE MICROSCOPE.

2111. Testing the Microscope.—To be of real value this must be accomplished
by a person with both theoretical and practical knowledge, and also with an un-
prejudiced mind. Such a person is not common, and when found, does not
» show an 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 tbat conform to the ‘‘fad”’ of the day.
From the writer’s experience it seems safe to say that the inexperienced can do no
better than to 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 iustruments 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 ari artist's pride in their work, they

therefore have a stimulus to excellence greater than business prudence alone could
give.

@ 112, Mechanical Parts.—All of the parts should be firm, and not too easily
shaken, Bearings should work smoothly. The mirror should remain in any posi-
tion 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
moderate mechanical instinct will be able to tighten the proper screws, etc.

Lhe Fine Adjustment is more difficult to deal with. From the nature of its
purpose, unless it is approximately perfect, it would better be off the microscope
entirely.

It should work smoothly and be so balanced that one cannot tell by the feeling
when using it whether the screw is going up or down. Then there should be ab-
solutely 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 aud when using the fine adjustment. There should be no swaying of
the image with either if the light is central (2 73).

2113. 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. Inno case is the condemna-
tion of a microscope to be made 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 appendix).

CH. II.) LABORATORY MICROSCOPES. 61

LABORATORY COMPOUND MICROSCOPES.

% 114. 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 universities and medical 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 out-
fit should comprise, (a) dry objectives of 50 mm. (2 in.), 16-18 mm, (3¢ in.) and 3
mun. (1s in.) equivalent focus. There should be present also a 2 mm. (J; in.) or
1.5 mm. (;; in.) homogeneous immersion objective. Of oculars there should be
several of different power. Au illuminator or substage condenser, and au Abbe
camera lucida are also uecessities, 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 achro-
matic objectives approach them very closely.

2115. Mechanical Parts or Stand.—The stand should be low enough 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 horizontal.
The adjus:ments for focusing should be two,—a coarse adjustment or rapid move-
ment with rack and pinion, anda fine adjustment by means of a micrometer 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, and the draw-tube should be 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 exami-
nation of slides with serial sections. If there is no mechanical stage (@ 116), it is
also of considerable advantage to have the stage with a circular, revolving top,
and two centering screws with milled heads. In this way a mechanical stage with
limited motion is secured, and this is of the highest advantage in using powerful
objectives. The sub-stage fittings should be so arranged as to enable one to dis-
pense entirely with diaphragms, to use ordinary diaphragms, or to use the conden-
ser. The condenser mounting should allow up and down motion, preferably by
rack and pinion. The base should be sufficiently heavy and so arranged that the
microscope will be steady in all positions, and interfere the least possible amount
with the manipulation of the mirror and other sub-stage accessories.

2116, Mechanical Stage.—There should also be present some form of mechani-
cal stage. That 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 niotion.
It is a modification of the mechanical stage of Tolles. The modification consists

62 LABORATORY MICROSCOPES. [CH I.

in removing the thin plate and instead, having a clamp to catch the ends of the
glass slide. The slide is then moved on the face of the stage proper. This modi-
fication 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. in America.—
(Jour. Roy. Micr, Soc., 1885, p. 122. See also Zeit. wiss. Mikroskopie, (II), 1885,
pp. 289-295 ; 1887, (I1V,, pp. 25-30).

2 117. Society Screw.—Owing to the lack of uniformity in screws for microscopic
objectives, the Royal Microscopical Society of London, in 1857, made an earnest
effort to introduce a standard size. The specifications of this standard are as fol-
lows: ‘‘ Whitworth thread, 7 ¢., a V shaped thread, sides of thread inclined to an
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, o 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 inicroscope], not less than 0 125 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, o 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 per-
fect there soon came to be consid: rable 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 Micro-
scopical 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 considered at
the time by the Royal Microscopical Society, the mechanical difficulties were so
great that the improvements were abandoned.

Fortunately, however, during the present year (1896) that society has again
taken hold of the matter in earnest, and we are now promised a new ‘‘ Society
Screw’ which shall be accurate ; and facilities for obtaining the standard will be
so good that there is a reasonable certainty that the universal screw for micro-
scopic objectives may be realized. It is indeed astonishing to see how widely
spread the ‘Society Screw’’ has become. 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 Standard, 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., 0. 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, Aug., 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.

CH. IT.) LABORATORY MICROSCOPES. 63

FIGURES OF LABORATORY MICROSCOPES AND
ACCESSORY APPARATUS.

It was deemed advisable in this new edition to figure some of the
most common of the laboratory microscopes and this has been rendered
possible mostly by the courtesy of the makers and importers. During
the last five years very 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 of study and research. The
production of the new kinds of glass (Jena glass), and the apochromatic
objectives have been a no less potent factor in promoting progress. It
is gratifying also to know that with the increase in the use of the mi-
croscope, not only are the optical and mechanical parts improved, and
that very greatly, but the price has decreased so that at the present time
schools cannot afford to be without one or more, and individuals are not
debarred from the possession of an instrument adequate to their needs.
The cost of a complete outfit varies from 25 to 600 dollars. The stu-
dent is advised to write to one or more of the opticians for complete
catalogs. See list, p. 2 of cover.

2118. Marker tor 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).

2119 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
purposes of demonstrating any particular structure or object in the field, a tempo-
rary 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 who devised this method.
It is certainly 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

64 LABORATORY MICROSCOPES. (CH. L7/.

have in it many other objects which are more easily seen, as the red blood corpus-
cles or particles of vegetation or dirt in the case of the blood preparation or of the

Ps. sit TLS:
ar KK ie lias
R so ite,

amoeba.

!

61. 62. 63.

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

Fic. 61. The simplest form of marker. It consists of the part SS with the
milled edge (MM). 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 ison awire(W). This wire ts 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 1s connected
with the“ Society Screw’? by the small screw (S).

Fic. 62. SS, R,and B. All paris same as with Fig. 61, except that the brush
is carried by a sliding cylinder the end view being indicated in Fig. 63.

Sees eee colle come mle a arse.

@8@08e8e
S@ ®@GOe@
$88008
64. 65

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 vine, and in fig. 66 the lines of a

Do

micrometer are ringed to facilitate finding the lines.

CHa TLE) LABORATORY MICROSCOPES. 65

Fic. 67. Avauss’ Method of Mark-
ing Objectives on a Revolving Nose-
Piece.

As seen in the figure, the equiva-
lent focus of the objective is engraved
on the diaphragm above the back lens
and may be very readily seen in ro-
tating the nose-piece. Thists of great
advantage and facilitates the chang-
ing of objectives, as one can see what
objective 1s coming into place with-
out trouble.

(g 116)

Both these mechanical stages have the great advantage of large movement in both
directions, so that a series may be studied with great certainty and facility, Both have
scales and verniers, so that the position of any particular feature of a preparation may
be readily re found The figures on the scale being different there 1s never doubt as to
the position of each from the recora. :

Fic. 68. The Tolles- Mayall mechanical stage as constructed by Leitz. It is shown in
position on the stage of the microscope: tt is fastened to the stage by a pin and screw
near the pillar.

Fic. 69. The Tolles-Mayall mechanical stage made by the Bausch & Lomb Optical
Company. It 1s separated from the microscope. It ts attached to the microscope by a
clamp surrounding the pillar. This form of connection was employed by Reichert &
Zeiss in the earlier forms devised, and ts still used by then

i)

66 LABORATORY MICROSCOPES. (CH. IT.

Fic. 70. Zeiss’ Microscope 1° with Mechanical Stage. This figure, from Zeiss’
Catalog No. 30, represents the Continental Model of Microscope in its most per-
sect 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 unscrew-
ing this the laterally moving part may be removed, leaving a plain stage.

W. Screw for moving the stage forward and backward.

CH. IT.) LABORATORY MICROSCOPES. 67

Fic. 71. Watson & Sons’ Edinburgh Students’ Microscope (Stand G). This is
a good representative of the tripod-base, English models of to-day. It is in gen-
eral like the Powell and Lealand stands which have held their position with the
foremost English microscopists for the last go years. (See Carpenter-Dallinger,
Pi t72): '

Microscopes with tripod bases something after this pattern are now being made
on the Continent.

Attention is called to the sub-stage for the condenser. It possesses centering
screws so that any apparatus used in tt may be accurately centered. It is to be
hoped that all microscopes of this grade will soon be supplied with a centering sub-
stage.

68 LABORATORY MICROSCOPES. [CH ll.

Fic. 72. Natchet & Fils’ Medium Microscope No. 4 with Movable Stage. The
French microscopes set the fashion for the Continent of Europe, and the Contt-
nental or Hartnack Model with the horse shoe base has extended to all lands, but
see Carpenter-Dallinger, p. 208.

CAL TT LABORATORY MICROSCOPES.

Fic. 73. The BB Microscope of the Bausch & Lomb Optical Co.

70

FIG.

LABORATORY MICROSCOPES. LCH. IT.

74. Reichert’s Stand LIT B, after Specifications by Richards & Co., N. Y.

CH. TT] LABORATORY MICROSCOPES. 71

i
ity}

Fic. 75. Queen & Company's Microscope of the Continental Pattern, No. II.

72

LABORATORY MICROSCOPES.

(CH. II.

a oe

Fic. 76. Leitz’ Microscope, £6

CH. I1.) LABORATORY MICROSCOPES. 73

Fic. 77. feoss Eclipse Microscope. The heavy circular base rotates to gt
Sirmness upon inclination.

74 LABORATORY MICROSCOPES. (CH. Il.

i

Fic. 78. The AA Microscope of the Bausch & Lomb Optical Co., with slid-
ing tube instead of rack and pinion. This microscope is also furnished with rack
and pinion.

CH. IT]

LABORATORY MICROSCOPES. 75

Fic. 79. Beck’s Star Microscope.

76 LABORATORY MICROSCOPES. (CH. Il.

Fic. 80. Zentmayer’s Clinical Microscope. This ts supplied with a lamp and
so mounted that it may readily be passed around a class for the purpose of demon-
Strating some microscopic structure.

il

Fic. 81. Zentmayer's Microscope, No. V.

i

CHALT A LABORATORY MICROSCOPES. Teh

Fic. 82. Leitz’ Demonstration Microscope. This is designed for class denon-
stration and is pointed toward the window, or some other source of light, by the
student. It has an arrangement for holding a sketch of the object under the
microscope.

Fic. 33. Leitz’ Aficroscope,
No. LV, with sliding tube for
coarse adjustment, and no joint
for inclination. This mtcro-
scope must always be used in the
vertical position. Microscopes
of this form are not somuch the
SJashion as they were a few years
ago. If any instrument re-
guires mechanical aids to en-
able one to attain a desired re-
sult with ease and certainty, it
is a microscope.

Most of the Leitz microscopes
are now supplied with joint and
with vack and pinion for the

coarse adjustment (Fig. 76).

78

LABORATORY MICROSCOPES.

Fic, 84. Queen & Company's Acme Microscope, No. 4.

(CH.

fT,

CH. I1.]

LABORATORY MICROSCOPES.

Fic. 85. McIntosh Scientific Microscope, No.

2,

79

CHAPTER IIT.

INTERPRETATION OF APPEARANCES.

APPARATUS AND MATERIAL FOR CHAPTER III.

A laboratory, compound microscope (% 114) ; Preparation of fly’s wing ; 50 per
cent. glycerin ; Slides and covers ; Preparation of letters in stairs (Fig. 86) ; Muci-
lage for air-bubbles and olive or clove oil for oil-globules (% 127-130). Solid glass
rod, and glass tube (% 135-137) ; Collodion (% 137); Carmine, India ink, or lamp
black (2139-141) ; Frog, castor oil and micro-polariscope (¢ 143).

INTERPRETATION OF APPEARANCES UNDER THE MICROSCOPE.

§ 120, 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 puzzled by appearances which are
constantly met with in microscopical investigation. Any one can look
into a microscope, but it is quite another matter to interpret correctly
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 un-
dertakes 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 ap-
pearances being quite differently interpreted by eminent observers.

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-

CH. IIT] INTERPRETATION OF APPEARANCES. 81

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 watchfulness
lest the appearances may be deceptive are thus admirably stated by Dal-
linger (See Carpenter-Dallinger, pp. 368-369): ‘‘ 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 azy 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 prac-
ticed observer is apt to take no note of such phenomena as his mind is
not prepared to appreciate. Errors and imperfections of this kind can
only be corrected, it is obvious, by general advance in scientific knowl-
edge; but the history of them affords a useful warning against hasty
conclusions drawn from a too cursory examination. If the history of
almost any scientific investigation were fully made known it would gen-
erally appear that the stability and completeness of the conclusions fin-
ally arrived at had been only attained after many modifications, or even
entire alterations, of doctrine. And it is therefore of such great impor-
tance 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 possi-
ble. 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
ourselvés, or may be proved fallacious at some future time, perhaps even
by our own more extended and careful researches. The suspension of
the judgment whenever there seems room for doubt is a lesson inculcated
by all those philosophers who have gained the highest repute for prac-
tical wisdom ; and it is one which the microscopist cannot too soon learn
or too constantly practice.’’

6

82 INTERPRETATION OF APPEARANCES. (CH. Ll.

For these experiments no condenser ts to be used except where specifi-
cally indicated,

§ 121. Dust or Cloudiness on the Ocular.—Employ the 16 min.
(34 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 posi-
tion ($44). Light the field well and focus sharply. The image will
be clear, but part of the field will be obscured by the irregular outline
of the particles of lint. Move the object to make sure this appearance
is not due to it.

Grasp the ocular by the milled ring, just above the tube of the micro-
scope, and rotate it. The irregular object will rotate with the ocular.
Cloudiness or particles of dust on any part of the ocular may be detect-
ed in this way.

§ 122. 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 usea 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 objective.
Dust would make a dark patch on the image that would remain station-
ary 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.

§ 123. Relative Position of Objects or parts of the same object.
The general rule is that objects highest up come into focus /asf in
focusing up, fst in focusing down.

a Fic. 86. Letters mounted in stairs to show
- the order of coming into focus.
d

‘Sine ial airs TS a,b,c¢,d. The various letters indicated

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

§$ 124. Objects having Plane or Irregular Outlines.—As object

use three printed letters in stairs mounted in Canada balsam (Fig. 86).
The ‘first letter is placed directly upon the slide, and covered with a

CH. IIT.) INTERPRETATION OF APPEARANCES. 83

small piece of glass about as thick asa 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 (§ 121).

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
soon. 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. (%thin.) objective, it is easily deter-
mined. 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 microm-
eter 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 con-
siderable practical importance.

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

§ 126. Transparent Objects having Curved Outlines.—The suc-

84 INTERPRETATION OF APPEARANCES. (CH. IIT.

cess of these experiments will depend entirely upon the care and skill
used in preparing the objects, in lighting, and in focusing.

Employ a 3mm, (% in.) or higher objective and a high ocular for all
the experiments. It may be necessary to shade the object (§ 102) to
get satisfactory results. When a diaphragm is used the opening should
be small and it should be close to the object.

§ 127. Air Bubbles.—Prepare these by placing a drop of thin muci-
lage 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.

Fic. 87. Diagram showing

3 fine J re al how to place a cover-glass upon
lass LT) an object with fine forceps.

§ 128. Air Bubbles with Central Ilumination.—Shade the ob-
ject ; and with the plane mirror, light the field with central light (Fig.
23) '

Search the preparation until an air bubble is found appearing about
1mm. 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, ad-
just 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 (§ 61).

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.

§ 129. 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. 23, C).
The bright spot will appear no longer in the center but on the side away
from the mirror (Fig. 88).

§ 130. Oil Globules.—Prepare these by beating a small drop of clove
oil with mucilage on a slide and covering as directed for air bubbles
(§ 128), or use a drop of milk.

§ 131. Oil Globules with Central Illumination.—Use the same
diaphragm and light as above (§ 128). Find an oil globule appearing
about 1 mm. in diameter. | If the light is central the bright spot will ap-

GH. fT.) INTERPRETATION OF APPEARANCES. 85

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 /as¢ in focusing up.

Fic. 88. 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.

$ 132. Oil Globules with Oblique Illumination.—Re-
move the sub-stage, etc., as above, and swing the mirror 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. 88).

§ 133. Oil and Air Together.— Make a preparation ex-
actly as described for air bubbles (§ 127), and add at one
edge a little of the mixture of oil and mucilage (§ 130) ;
cover and, examine, 2

The sub-stage need not be used in this experiment. Search the prep-
aration until an air bubble and an oil globule, each appearing about 1
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 foward it. See Fig. 88.*

§ 134. Air and Oil by Reflected Light.—Cover the diaphragm or
mirror so that no transmitted light ($ 60) can reach the preparation,
using the same preparation as in § 133. The oil and air will appear
like globules of silver on a dark ground. The part that was darkest in
each will be lightest, and the bright central spot will be somewhat
dark.+

§ 135. Distinétness 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 out-

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

+ It is possible to distinguish oil and air optically, as described above, only when
quite high powers are used and very small bubbles are selected for observation. If
a 16 mm. (% in.) is used instead of a 3 mm. (% in.) objective, the appearances
will vary considerably from that given above for the higher power. It is well to
use a low as well as a high power. Marked differences will also be seen in the ap-
pearanices with objectives of small and of large aperture.

86 INTERPRETATION OF APPEARANCES. (CH. 1/1.

line as both differ greatly in refractive power from the medium which
surrounds them, the oil being more refractive than the mucilage and
the airless. (Figs. 53-55).

Place a fragment of a cover-glass on a clean slide, and cover it (see
under mounting). The outline will be very distinct with the unaided
eye. Use it as object and employ the 16 mm. (% in.) objective and
high ocular. Light with central light. The fragment will be outlined
by a dark band. Puta 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. Puta
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 narrower than
before.

Draw a solid glass rod out to a fine thread. Mount one piece in air,
and the other in 50% glycerin. Put a cover-glass on each. Employ
the same optical arrangement as before. Examine the one in air first.
There will be seen a narrow, bright band, 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.89b). 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 remem-
bered 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. 53, 55) 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 ob-
server.

Fic. 89. Solid glass rod showing the ap-

atbeaiih i, - pearance when viewed with transmitted,

central light, and with an objective of medi-
um aperture.

a. Mounted in air. 6. 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.*

*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.

CH. Hh INTERPRETATION OF APPEARANCES. 87

A very striking and satisfactory demonstration may be made by paint-
ing 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.

$136. Highly Refractive.—This expression is often used in de-

scribing microscopic objects, (medulated 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
($ 135), 1t would be known that the refractive power of the object, and
the medium in which it was mounted must differ considerably.
§ 137. 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 (1) a dark line, (2) a light
band, and (3) a second dark line (Fig. 90).

This may be demonstrated by coating a fine glass rod (§ 135) with
one or more coats of collodion or celloidin and allowing it to dry, and
then mounting in 50% glycerin as above. Employ a 3mm. (% 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. 90). een aay

Fic. 90. Solid glass rod coated with col- 7 ;
lodion to show a double contour. Toward
one end the collodion had gatheredin a fusi-
form drop.

§ 138. Optical Section.—This is the appearance obtained in examin-
ing transparent or nearly transparent objects with a microscope when
some plane below the upper surface of the object isin 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 satisfac-
tory optical sections are obtained with high objectives having large
aperture.

Nearly all the transparent objects studied may be viewed in optical

* 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. 90).

88 INTERPRETATION OF APPEARANCES. (CH. IIT.

section. A striking example will be found in studying mammalian red
blood-copuscles on edge. ‘The experiments with the solid glass rods
(Fig. 89) furnish excellent and striking examples of optical sections.

§ 139. Currents in Liquids.—Employ the 16 mm. (% in.) object-
ive, 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 pro-
duced 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.

§ 140. 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 ap-
parent 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 ap-
pear 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 quiteslow. One should keep this in mind in studying the circulation
of the blood. The truth of what has just been said can be easily dem-
onstrated in studying the circulation in the gills of Necturus, or in the
frog’s foot, by using first a low power in which the field is actually of
considerable diameter (Fig. 37, Table, § 47) and then using a high
power. With the high power the apparent motion will appear much
more rapid. For the form of motion, spiral, serpentine, etc., see Car-
penter-Dallinger, p. 375.

§ 141. Pedesis or Brownian Movement.—Employ the same ob-
ject as above, but a 3mm. ( in.) or higher objective in place of the 16
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'sts or Brownian movement, Also, but improperly, molecu-
lar movement, from the smallness of the particles.

The motion is increased by adding a little gum arabic solution or a

CH. III, INTERPRETATION OF APPEARANCES. 89

slight amount of silicate of soda or of 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 pow-
dered pumice-stone in water has for many years been a favorite object.

Pedesis is exhibited by all solid matter if it is finely enough divided
and in a suitable liquid. In the minds of most, no adequate explana-
tion has yet besn offered. See Carpenter-Dallinger, p. 373; 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. NNNIII, pp. 163-167, entitled ‘‘The Secret of the Brownian
Movement.’' This paper is suggestive if not wholly satisfactory.

For the original account of this see Robert Brown, ‘‘ Botanical appen-
dix to Captain King’s voyage to Australia,’’ Vol. II, p. 534. (1826).

See also Dr. C. Aug. Sigm. Schultze, ‘‘ Mikroskopische Untersuch-
ungen tiber des Herren Robert Brown Entdeckung lebender, selbst im
Feuer unzerst6rbarer Theilchen in allen Korpern.’’ From ‘‘ Die Gesell-
schaft fiir Beforderung der Naturwissenschaften zu Freiburg.’’ 1828.

Compare the pedetic motion with that of a current by slightly inclin-
ing the tube of the microscope. The small particles will continue their
independent leaping movements while they are carried along by the
current.

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

Open the abdomen of a dead frog (an alcoholic specimen will do if it
is soaked in water for some time, but a fresh specimen is more satisfac-
tory). 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. Adda 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 ap-
pearance. 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 will be seen
multitudes of crystals of carbonate of lime; the larger crystals are mo-
tionless but the smallest ones exhibit marked pedetic movement.

Use the micro-polariscope, light with great care and exclude all ad-

g9 INTERPRETATION OF APPEARANCES. [CH 1.

ventitious light from the microscope by shading the object (§ 102) 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, motionless
crystals will shine continuously and a part will remain dark, but the
small crystals between the large ones will shine for an instant, then dis-
appear, only to appear again the next instant. This demonstration is
believed to furnish absolute proof that the pedetic movement is real and
not illusory.

§ 143. 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 ob-
ject under the microscope. They may also be seen by looking at the
brightly illuminated snow or other white surface. By studying them
carefully it will be seen that they are somewhat movable and float across
the field of vision, and thus do not remain in one position as do the ob-
jects 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.

§ 144. In addition to the above experiments it is very strongly rec-
ommended that the student follow the advice of Beale, p. 248, and ex-
amine first with a low then 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 fibres accidentally present have been considered
nerve fibres, 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 erroneous 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, ete.) of the various forms of starch,
see Bulletin No. 13 of the Chemical Division of the U. S. Department
of Agriculture. For Hair and Wool, see Bulletin of the National Asso-

CH. Ill) INTERPRETATION OF APPEARANCES. gI

ciation of Wool Growers, 1875, p. 470, Proc. Amer. Micr. Soc., 1884,
pp. 65-68.

For different appearances due to the illuminator, see Nelson, in Jour.
Roy. Mier. Soc., 1891, pp. 90-105 ; and for the illusory appearances due
to diffraction phenomena, see Carpenter-Dallinger, p. 376.

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, ete., 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.

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.

CHAPTER IV;

MAGNIFICATION AND MICROMETRY.

APPARATUS AND MATERIAL FOR THIS CHAPTER.

Simple and compound microscope ; Steel scale or rule divided to millimeters and
4ths; Block for magnifier and compound microscope (§ 147, 151) ; Dividers (@ 147,
151); Stage micrometer (7150); Wollaston camera lucida (¢ 151); Ocular screw-
micrometer (Fig. 100); Micrometer ocular (Figs. 98-99). Abbe camera lucida
(Fig. 96).

S$ 145. 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 obtained
by measuring the virtual image (Figs. 21, 38). The object for deter-
mining magnification must be of known length and is designated a m7-
crometer (§ 150). In practice a virtual image is measured by the aid of
some form of camera lucida (Figs. 92, 96), or by double vision (§ 147).
As the length of the object is known, the magnification is easily deter-
mined by dividing the apparent size of the image by the actual size of
the object. For example, if the virtual image measures 4o mm. and
the object magnified, 2 mm., the amplification must be 40 + 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 microscop-
ical or histological drawing is made, the word magnification is frequent-
ly 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.

§ 146. Magnification of Real Images.—In this case the magnifi-
cation is the ratio between the size of the real image and the size of the
object, and the size of the real image can be measured directly. By
recalling the work on the function of an objective (§ 49), 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
eye or measured as if it were an actual object. For example, suppose
the object were 3 millimeters long and its image on the ground glass
measured 15 mim., then the magnification must be, 15 + 3 = 5, thatis,

CH. II] MAGNIFICATION AND MICROMETRY. 93

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 ex-
ample, if the object is 4oo millimeters long and its image on the ground
glass is 25 mm. long, the ratio is 25 + 4oo= ,y. That is, the image
is zgth 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 x 75th.

MAGNIFICATION OF A SIMPLE MICROSCOPE.

§ 147. The Magnification of a Simple Microscope is the ratio
between the object magnified (Fig. 16, A’B’), and the virtual image
(A®B*). 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 magnifier to the table is 250 millimeters.

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

Close one eye, and hold the head so that the other will be near the
upper surface of the lens, Focus if necessary to make the image clear
($9). 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 binocular or double vision in which the
microscopic image is seen with one eye and the dividers with the other,
the two images appearing to be fused in a single visual field.

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

As 4mm. cannot be seen plainly by the unaided eye, place one arm
of the dividers at a centimeter line, and then 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, A®B*), and as the size of the ob-
ject is known, the magnification is determined by dividing the size of
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

O94 MAGNIFICATION AND MICROMETRY. (CH. IV.

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 714 diame-
ters, or 7%4 times linear.

The magnification of any simple magnifier may be determined exper-
imentally in the way described for the tripod.

MAGNIFICATION OF A COMPOUND MICROSCOPE.

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

The determination of the magnification of a compound microscope may
be made as with a simple microscope (§ 147), but this is very fatiguing
and unsatisfactory.

§ 150. Stage, Object or Objective Micrometer.—For determin-
ing the magnification of a compound microscope and for the purposes
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 on glass is most convenient. The spaces
between the lines should be 5 and ;4, millimeter, and when high pow-
ers are to be used the lines should be very fine. It is of advantage to
have the coarser lines filled with graphite (plumbago), especially when
low powers are to be used. If one has
th an uncovered micrometer the lines may

be very readily filled by rubbing some of
the plumbago on the surface with the end
of acork ; the superfluous plumbago may

Fic. 91. Diagram of a stage be removed by using a clean dry cloth or
micrometer, with a ring on the 4 piece of lens paper. After the lines are
SNEED PRUNTIE ROME ERETE ST sand Hine plumbago wiped from the
surface, the slide should be examined and if it is found satisfactory, z.e.,
if the lines are black, a cover-glass on which is a drop of warm balsam
may be put over the lines to protect them.

If one desires to have a part of the micrometer uncovered and a part
covered for using homogeneous objectives, the lines may be filled with
fine graphite, as described, and a piece of oblong cover-glass placed over
a part of the band of lines.

§ 151. Determination of Magnification.—This is most readily ac-
complished by the use of some form of camera lucida (Ch. V), that of

CH. IV.) MAGNIFICATION AND MICROMETRY. 95

Wollaston being most convenient as it may be used for all powers,
and the determination of the standard distance of 250 meillimeters at
which to measure the image is very readily determined (Fig. 92, § 153).

Employ the 16 mm. (23 in.) objective and a 50 mm. (2 in., A. or
No. 1) ocular and stage micrometer as object. For this power the 75th
mim. spaces of the micrometer should be used as object. Focus sharply,
and make the tube of the microscope horizontal, by bending the flexible

Fic. 92. Wollaston's Camera Luci-
da, showing the rays from the micro-
scope and from the drawing surface,
and the position of the pupil of the eye.

alvis, Axis. Axial rays from the
microscope and from the drawing sur-
Jace (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.

al B, The lateral rays from the mi-
croscope and their projection on the
drawing surface.

CD. Rays from the drawing sur-
face to the eye.

aD, A’ D’. Overlapping portion of the two fields, where both the microscopic
image and the drawing surface, pencil, etc., may be seen. It is represented by the
shaded part in the overlapping circles at the right.

Ocular. The ocular of the microscope.

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

FIG. 92.

pillar, being careful not to bring any strain upon the fine adjustment.
(Frontispiece ).

Put a Wollaston camera lucida (Ch. V.) in position, and turn the oc-
ular around if necessary so that the broad flat surface may face directly
upward, as shown in Fig. 92. Elevate the microscope by putting a
block under the base, so that the perpendicular distance from the upper
surface of the camera lucida to the table is 250 mm. ($153). Place
some white paper on the work-table beneath the camera lucida.

Close one eye, and hold the head so that the other may be very close
to the camera lucida. Look directly down. The image will appear to
be on the table. It may be necessary to readjust the focus after the
camera lucida is in position. If there is difficulty in seeing dividers and

96 MAGNIFICATION AND MICROMETRY. (CH. IV.

image consult Ch. V. Measure the image with dividers and obtain the
power exactly as above (§ 147, 148).

Thus: Suppose two of the jth 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 92 millimeters. If now the object is #;ths
of a millimeter and the magnified image is 92 millimeters, the magnifi-
cation (which is the ratio between size of object and image) must be
9% +75 = 47. That is, the magnification is 47 diameters, or 47 times
linear. If the fractional numbers in the above example trouble the stu-

Image
E

L 2

Objectzve

Object-b

rs Object-a
Object.
FIG. 93. FIG. 9}.

Fics. 93-94. Figures showing that the size of object and image vary directly as
their distance from the center of the lens. In Fig. 94 one can also see why it is
necessary to focus down, t. é., bring the object and objective nearer together when
the tube ts lengthened.

CH. IV. MAGNIFICATION AND MICROMETRY. 97

dent, both may be reduced to the same denomination, thus: If the size
of the image is found to be 93 mm. this number may be reduced to
tenths mun., so it will be of the same denomination as the object. Ing
mm. there are go tenths, and in 2 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 there must have been a magnification
of 94 + 2 = 47 diameters in order to produce an image 94 tenths of a
millimeter long.

Put the 25 mm. (1 in. C, or No. 4) ocular in place of one of 50 mm.
focus, and then put the camera lucida in position. Measure the size
of the image with dividers anda rule as before. The power will be
considerably greater than when the low ocular was used. This is be-
cause the virtual image (Fig. 21, B’A*) seen with the high ocular is
larger than the one seen with the low one. The real image (Fig. 21,
A'B') remains nearly the same, and would be just the same if positive,
par-focal oculars (§ 34, 68, 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. 94) is formed far-
ther from the objective when the tube is lengthened, and the objective
must be brought nearer the object (Fig. 94). 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. 93 and 94.

$152. Varying the Magnification of a Compound Microscope.
It will be seen from the above experiments (§ 151) that independently
of the distance at which the microscopic image is measured (§ 153),
there are three ways of varying the power of a compound microscope.
These are named below in the order of desirability.

(1) 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. 94).*

§ 153. Standard Distance of 250 Millimeters at which the Vir-
tual Image is Measured.—For obtaining the magnification of both

* Amplifier.—In addition to the methods of varying the magnification given in
2 152, the magnification is sometimes increased hy the use of an amplifier, that is
a diverging lens or combination placed between the objective and ocular and serv-
ing 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.

7

98 MAGNIFICATION AND MICROMETRY. (CA. IV.

Fic. 95. Figure showing the
position of the microscope, the
camera lucida, and the eye, and
the different sizes of the image
depending upon the distance at
which it is projected from the
eve. (a) The size at 25 cm. ;
(6) at 35 cm., (§ 153).

‘28

]

the simple and the compound microscope the directions were to measure
the virtual image at a distance of 250 millimeters. This is not that the
image could not be seen and measured at any other distance, but be-

Fic. 95. Sectional view of
the Abbe Camera Lucida to
Show that in measuring the
Standard distance of 250
millimeters, onemust meas-
ure along theaxts from the
point P, at the left of the
prism, to the mirror, and
from the mirror to the
drawing surface. For a
Sull explanation of this
camera lucida, see next
chapter, (Figs. 102, 106).

Fic. 96.

cause 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. 95, where is represented graphically the fact
that the size of the virtual image depends directly on the distance at
which it is projected, and this size is directly proportional to the verti-
cal distance from the apex of the triangle, of which it forms a base.

CH, II") MAGNIFICATION AND MICROMETRY. 99

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.

Demonstrate the difference in magnification due to the distance at
which the image is projected, by raising the microscope so that the dis-
tance 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 standard. In
such a case the magnification must be determined for the special dis-
tance. (See the next chapter. )

For discussions of the magnification of the microscope, see: Beale,
Pp. 41, 355; Carpenter-Dallinger, pp. 26, 238; Nageli and Schwen-
dener, 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.

$154. Table of Magnifications and of the Valuations of the
Ocular Micrometer.—7he following table should be filled out by each
student. In using tt for Micrometry and Drawing it is necessary to keep
clearly tn mind the exact conditions under which the determinations were
made, and also the ways in which variation in magnification and the val-
uation of the ocular micrometer may be produced (§ 152, 153, 163, 166).

| OCULAR OCULAR
: 50 mm. 25 mm.
TUBE TuBE | OcuULAR MICROMETER
OBJECTIVE. oe our aver our VALUATION.
—MM. ——MM. |TUBEIN. OUT——MM.
K 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

100 MAGNIFICATION AND MICROMETRY. (CH. IV.
MICROMETRY.

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

MICROMETRY WITH THE SIMPLE MICROSCOPE.

§ 156. With a simple microscope (A), the easiest and best way is to
use dividers and then the simple microscope to see when the points of
the dividers exactly include the object. The spread of the dividers is
then obtained as above ($ 148). 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 (§ 147), measure the image at the standard
distance. If now 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 (§ 166-167) being most accurate.

§ 157. Unit of Measure in Micrometry.—As most of the objects
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 (;;55 mm. or 0.001 mm.) or
one millionth of a meter (, 554977 OF 0.000001 meter) as the unit. He
named this unit micro-millimeter and designated it mmm. In 1869,
Listing (Carl’s Repetorium fiir Experimental-Physik, Bd, X, Pp. 5)
‘favored the thousandth of a millimeter as unit and introduced the name
Mikron or micrum, In English it is most often written AZicron, plural
micra or microns, pronunciation Mic’r6n or Mic’rdn. By universal con-
sent the sign or abbreviation used to designate it isthe Greek ». Adopt-

CH. IV.) MAGNIFICATION AND MICROMETRY. IOI

ing this unit and sign, one would express five thousandths of a milli-

meter (<7 OF 0.005ths mm.) thus, 5.*

@158. 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
my 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.

§$ 159. Micrometry dy dividing the size of the image by the magnifi-
cation of the microscope.—For example, employ the 3 mm. (% in.) ob-
jective, 25mm. (1in.) ocular, and a Necturus’ red blood-corpuscle prepa-
ration 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 ob-
taining the magnification of the microscope ($151). 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 dianieters (§ 145), then the actual length of this long axis of
the corpuscle is 18 mm. + 400 = .045 mm. or 45 m (§ 157).

Fic. 97. 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, marking on
the sketch the corpuscles measured. The different corpuscles vary con-
siderably in size, so that accurate comparison of different methods of
measurement can only be made when the same corpuscles are measured
in each of the ways (Figs. 61-66).

*The term Micromillimeter ab. mmm. is very cumbersome, and besides is en-
tirely inappropriate since the adoption of definite meanings for the prefixes mzcro
and mega, meaning respectively one millionth and one million times the unit be-
fore 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,
aud 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. XX XVII (1888), p. 388.

102 MAGNIFICATION AND MICROMETRY. [CH ZT.

§ 160. Micrometry dy 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 § 159.

Remove the object, place the stage micrometer under the microscope,
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 (§ 148, 167).

OCULAR MICROMETER.

§ 161. Ocular Micrometer, Eye-Piece Micrometer.—This, as the
name implies, is a micrometer to be used with the ocular. It is a mi-
crometer on glass, and the lines are sufficiently coarse to be clearly seen
by the ocular. The lines should be equidistant and about 75th or |yth
mm. apart, and every fifth line should be longer and heavier to facili-
tate counting. If the micrometer is ruled in squares (2e¢-micrometer)
it will be very convenient for many purposes.

The ocular micrometer is placed in the ocular, no matter what the
form of the ocular (7. 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 is measuring the size of the real
image, however, and the actual size of the object can only be deter-
mined 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.

§ 162. Valuation of the Ocular Micrometer.—This is the value
of the divisions of the ocular micrometer for the purposes of microm-
etry, and is entirely relative, depending 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 specially
determined for every optical combination (7. e., objective and ocular),

CH. IV.) MAGNIFICATION AND MICROMETRY. 103

and for every change in the length of the tube of the microscope. That
is, it is necessary to determine the ocular micrometer valuation for every
condition modifying the real image of the microscope (§ 152).

Any ocular may, however, be used as a micrometer ocular by placing
the ocular micrometer at the level of the ocular diaphragm, where
the real image is formed. ‘This is very conveniently arranged for by
some opticians by a slit in the side of the ocular, and the ocular mi-
crometer is mounted in some way and simply introduced through the
opening in the side. When no side opening exists the mounting of the
ocular may be unscrewed and the ocular micrometer, if on a cover-glass,
can be laid on the upper side of the ocular diaphragm.

It is a great convenience to have a micrometer ocular with a spring
and screw to enable one to accurately place the ocular micrometer.
The screw or filar ocular micrometers are excellent and for some pur-
poses almost indispensable (Fig. 100). They are expensive, however,
and for ordinary measurement unnecessary. If one isto do much meas-
ureing it is almost a necessity to havean ocular micrometer with screw
for moving the scale, (Figs. 98-99).

Fic. 98. FIG. 99.

Fics 93-99. Ocular Micrometer with movable scale. Fig. 98 is a side view of
the ocular while Fig. 99 gives a sectional end view, and shows the ocular microme-
ter in position. Ln both the screw which moves the micrometer is shown at the
left. (From Bausch & Lomb. Opt. Co.)

§ 163. Obtaining the Ocular Micrometer Valuation.—As an ex-
ample, employ the 25 mm. (1 in.) ocular and 16 mm. (2% in.) objec-
tive. Piace the stage micrometer under the microscope for an object,
and put the ocular micrometer in position, either through a slit in the
ocular, or renove the eye-lens and place it upon the ocular diaphragm.

Light the field well, and look into the microscope. The lines on the
ocular micrometer should be very sharply defined. If they are not,

104 MAGNIFICATION AND MICROMETRY. (CH. LV.

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 mi-
croscope (§ 45, 56) for the stage micrometer. The image of the stage
micrometer will appear to be directly under or upon the ocular microm-
eter.

Fic. 100. 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.
Alt the left is shown a clamping screw to
Sasten the ocular to the upper part of the mi-
croscope tube. Below 1s a face view, showing
the graduation on the wheel. An ocular mt-
crometer like this is tn general like the cob-
web micrometer and may be used for measur-
ing objects of varying sizes very accurately.
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 rath-
erits real image) regardless of the minuteness
of the object. ( Zeiss’ Catalog, No. 30).

§$ 164. ALake the lines of the two micrometers parallel by rotating the
ocular, or changing the position of the stage micrometer, or both if nec-
essary, and then make any two lines of the stage micrometer coincide
with any two on the ocular micrometer. Todo thisit may be necessary
to pull out the draw-tube a greater or less distance. See how many
spaces are included on each of the micrometers.

Divide the value of the included space or spaces on the stage microm-
eter 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 ~jth and
zyoth 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 ;'jth millimeter on the stage micrometer, obviously one
space on the ocular micrometer would include only one-tenth as much,
or zgth mm. + 10 = 7}y mm. That is, each space on the ocular mi-
crometer would include 7}, of a millimeter on the stage micrometer,
or zyoth millimeter of length of any object under the micrcscope, the
conditions remaining the same. Or, in other words, it would require

CH. IV.) MAGNIFICATION AND MICROMETRY. 105

100 spaces on the ocular micrometer to include 1 millimeter on the stage
micrometer, then as before 1 space of the ocular micrometer would have
a valuation of >} th millimeter for the purposes of micrometry ; and the
size of any minute object may be determined by multiplying this valua-
tion 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 ;jjth mm. x 8 = ;&; mm. or 80 » ($ 157).

§$ 165. Warying 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 corre-
sponding change in the ocular micrometer valuation (§ 152, 161).

§ 166. Micrometry with the Ocular Micrometer.—Use the 3 mm.
(% in.) objective and 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 ($ 159, 160).

Count the divisions on the ocular micrometer required to enclose or
measure the long and the short axis of each of the three corpuscles,
then multiply the number of spaces in each case 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
eacli case.

The same corpuscle is, of course, of the same actual size, when meas-
ured in each of the three ways, so that if the methods are correct and
the work carefully enough done, the same results should be obtained by
each method. See general remarks on micrometry (§ 167).*

* 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 inethod given in the text, and is 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 #,ths millimeter of the
stage micrometer, then obviously one space on the ocular micrometer would in-
clude }th of ,ths mm. or jth mm.; and the size of any unknown object under
the 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, yth 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 3 = #ths, or 0.6 mm.

106 MAGNIFICATION AND MICROMETRY. (CH. IV.

4167, Remarks on Micrometry.—In using adjustable objectives (% 22, 96), the
magnification of the objective varies with the position of the adjusting collar, be-
ing greater when the aljustment 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 ex-
act size of microscopic objects. This is due to the lack of perfection and uni-

(B) By finding the number of divisions on the ocular micrometer required to in-
clude 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 #,ths mm., on the stage micrometer,
then evidently it would require 5 + 3; = 25 divisions on the ocular micrometer to
include a whole millimeter on the stage micrometer, then 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 + 25
=foro.6mm. This method is really exactly like the one in (A), for dividing by
25 isthe same as multiplying by y,th.

(C) By having the ocular micrometer ruled in millimeters and divisions of a mil-
limeter, and then getting the size of the real image in millimeters. In employing
this method a stage micrometer is used as object and the size of the image of one
or more divisious is measured by the ocular micrometer, thus: Suppose the stage
micrometer is ruled in 75th and 35th mm. and the ocular micrometer is ruled in
millimeters and jth mm. Taking ~;th mm. on the stage micrometer as object,
as in the other cases, suppose it requires 10 of the ,jth mm. spaces or I mmi. to
measure the realimage, then the real image must be magnified 18 + 2, =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 7, mm. divisions = 29mm _ or 3 mm.
to include the image of the part measured, then evidently the actual size of the
part measured would be 3 mm. +5 = $ mm., the same result asin 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 and an exact division of a millimeter or an inch are two quite different
matters.

CH. IV.) MAGNIFICATION AND MICROMETRY. 107

formity 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.

A difficulty when one is using high powers is the width of the lines of the mi-
crometer. If the micrometer is perfectly accurate half the width of each line be-
longs 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, oue 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. 101).

It is also necessary in micrometry to use an objective of sufficient power to en-
able 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. 101. The appearance of the coarse A B

stage and of the fine ocular micrometer OS TNS
lines when using a high objective.

(A). The method of measuring the spaces
by putting the fine ocular micrometer lines
opposite the center of the coarse stage mt-
crometer lines. N

o.m

(B). Method of measuring the spaces of
the stage micrometer by putting one line
of the ocular micrometer (o.m.) at the in-
side and one at the outside of the coarse
stage micrometer lines (5s.nt.). FIG. tot.

As to the limit of accuracy in micrometry, one who has justly earned the right
to speak with authority, expresses himself as follows: ‘‘Z assume that 0.2 ts 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 (158, 159, 160, 166), the one given in 3 158 is impracticable, that given in
2 159 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 ope-
rations are necessary, each operation adding to the probability of error. Theoret-
ically the method given in 2 160 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 object to be measured and so ar-
ranging the ocular micrometer that these limits are inclosed by some divisions of

108 MAGNIFICATION AND MICROMETRY. [CH IV.

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
lines. Ifthe 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-micrometer (Fig. 100) obviates this entirely as the
cross hairs 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 juris-
prudence, 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. I,
Pp. 97, 208; 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., 1880, p. 702 ;
Amer. Monthly Mier. Jour., 1880, p. 67.

If one consults the medico-legal 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 be-
ing done.

CHAPTER V.
DRAWING WITH THE MICROSCOPE.

APPARATUS AND MATERIAL FOR THIS CHAPTER.

Microscope, Abbe camera lucida, drawing board, thumb tacks, pencils, paper,
and microscope screen (Fig. 58).

DRAWING MICROSCOPIC OBJECTS.

§ 168. Microscopic objects may be drawn free-hand directly from the
microscope, but in this way a picture giving only the general appear-
ance and relations of parts is obtained. For pictures which shall have
all the parts of the object in true proportions and relations, it is neces-
sary 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 un-
der the microscope. The appliance used in obtaining outlines, etc., of
the microscopic image is known as a camera lucida.

§ 169. Camera Lucida.—This is an optical apparatus for enabling
one to see objects in greatly different situations, as if in one field of vis-
ion, 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 image
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, pen-
cils, dividers, etc., by the same eye, and in the same field of vision.
The microscopic image appears like a picture on the pit paper.
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, pencils, etc. In some of the camera luci-
das of this group (Wollaston’s, Fig. 105), the rays are reflected twice,
and the image appears as when looking directly into the microscope.
In others the rays are reflected but once, and the image has the inver-
sion produced by a plane mirror. For drawing purposes this inversion
is a great objection, as it is necessary to similarly invert all the details
added free-hand.

TIO DRAWING WITH THE MICROSCOPE. (CH. V.

(B) By a camera lucida reflecting the rays of light from the drawing
paper, etc., so that their direction when they reach the eye coincides
with the direction of the rays from the microscope (Fig. 57, 60). In
all of the camera lucidas of this group, the rays from the paper are twice
reflected and no inversion appears.

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. 105), while others are

FIG. 103. FIG. 104.

Fic. 102. Abbe Camera Lucida with the
mirror at 45°, the drawing surface hori-
zontal, and the microscope vertical.

Axis, Axis. Axial ray from the mi-
croscope and from the drawing surface.
AB. Marginal rays of the field on the
drawing surface. ab. Sectional view of
the silvered surface 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 guadrant (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 ts
placed.

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

FIG. 102.

Fic. 103. Geometrical figure showing the angles made by the avial ray with the
drawing surface and the mirror. ‘
AB. The drawing surface.

Fic. 104. 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 (% 173).

CH. V.J DRAWING WITH THE MICROSCOPE. III

designed to be used on the microscope ina vertical position. As in bio-
logical work, it is often necessary to have the microscope vertical, the
form for a vertical microscope is to be preferred ; but see Figs. 102-111.

§ 170, Avoidance of Distortion.—/n order that the picture drawn
by the aid of a camera lucida may not be distorted, it ts necessary that the
axial ray from the image on the drawing surface shall be at right angles
to the drawing surface (Figs. 102, 105, 106).

2171. Wollaston’s Camera Lucida,—This is a quadrangular prism of glass put
in the path of the rays from the microscope, and it serves to change the direction
of the axial ray 90 degrees. In using it the microscope is made horizontal, and the
rays from the microscope enter one-half of the pupil while rays from the drawing
surface enter the other half of the pupil. As seen in the figure (Fig. 105), the fields
partly overlap, and where they do so overlap, pencil or dividers and microscopic
image can be seen together.

In drawing or using the dividers with the Wollaston camera lucida it is necessary
to have the field of the microscope and the drawing surface about equally lighted.
If the drawing surface is too brilliantly lighted the pencil or dividers may be seen
very clearly, but the microscopic image will be obscure. On the other band, if the
field of the microscope has too much light the microscopic image will be very defi-
nite, but the pencil: or dividers will not be visible. It is necessary, as with the
Abbe camera lucida (2 173), 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 microscope and from the draw-
ing 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 sat-
isfactory camera to draw with, but it is a very good form to measure the vertical
distance of 250 mm. at which the drawing surface should be placed when determin-
ing magnification (¢ 153).

Fic. 105. Wollaston’s Camera Lu-
cida, showing the rays from the mt-
croscope and from the drawing sur-
face, and the position of the pupil of
the eve.

For full explanation see Fig. 92.

§ 172. Abbe Camera Luci-
da.—This consists of a cube of
glass cut into two triangular
prisms and silvered on the upper
one. A small oval hole is then
cut out of the ceuter of the sil-
vered surface and the two prisms
are cemented together, thus giv-
ing a cubical prism with a per-
forated 45 degree mirror (Fig.

112 DRAWING WITH THE MICROSCOPE. (CA. 7,

102,ab), The upper surface of the prism is covered by a perforated
metal plate (Fig. 108). This prism 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 arawing
surface is reflected by a mirror to the silvered surface of the prism and
reflected by this surface to the eye in company with the rays from the
microscope, so that the two fields appear as one, and the image is seen
as ifon the drawing surface (Figs. 102, 106). It is designed for use
with a vertical microscope, but see § 174.

§ 173. Arrangement of the Camera Lucida Prism.—In placing
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 arranging
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 (Fig. 104). 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 co-incident 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 cut off. 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 directed in § 55 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 arrangements
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 different levels,
and the prism is hinged to turn aside without disturbing the mirror.

See the latest catalogs of Zeiss, Leitz, and the Bausch & Lomb Op-
tical Co.

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 position. If one side of the field is dark, then
the prism is to one side of the center ; if the field is considerably smaller
than when the prism is turned off the ocular, it indicates that it is not
at the correct level, 7. ¢., it is above or below the eye-point.

$174. Arrangement of the Mirror and the Drawing Surface.—

CH. V,j DRAWING WITH THE MICROSCOPE. 113

The Abbe camera lucida was designed for use with a vertical microscope
(Fig. 102). 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 draw-
ing board which is horizontal, and a drawing made under these condi-
tions would 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. 106-107). 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. 107). 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
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 (et micrometer) the distortion produced by not hav-
ing 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. Witha 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

8

114 DRAWING WITH THE MICROSCOPE. [CH. V.

Fic. 108, FIG. 107.

J.
><

FIG. 106. FIG. 109.

Fics. 106-109. 4bbe Camera Lucida in position to avoid distortion.

Fic. 106. The Abbe Camera Lucida with the mirror at 35°.

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

AB. Drawing surface raised toward the microscope 20°.

Foot. The foot or base of the microscope.

Mirror with quadrant (Q). The mirror ts 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.

Fic. 107. 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 35° 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 tt, and thus fulfill the requirements necessary to avoid adis-
tortion (% 170, 174).

Fic. 108. Upper view of the prism of the camera lucida. A considerable portion
of the face of the prism ts covered, and the opening in the silvered surface appears
oval,

Fic. 109. Quadrant to be attached to the mirror of the Abbe Camera Lucida to
indicate the angle of the mirror. As the angle is nearly always at 45°, 40°, Or 35°,
only those angles are shown.

CH. V.) DRAWING WITH THE MICROSCOPE. 115

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.

§$ 175. Abbe Camera and Inclined Microscope.—It is very fatigu-
ing 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 perfectiy 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° (§ 174),
then it is raised exactly as many degrees as the microscope is inclined,
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 (Fig. 110).

Fic. 110. Arrangement of the
drawing board for using the mi-
croscope in an inclined position
with the Abbe camera lucida (de-
signed by Mrs. S. P. Gage).

A very elaborate and convenient drawing board has been devised by
Bernhard (Zeit. wiss. Mikroskopie, Vol. XI, (1894) p. 298), whereby
the proper inclination can be given the drawing board for the vertical
microscope and also for an inclined microscope. The drawing surface
as a whole can be raised or lowered to meet the needs of different ob-
jects. Fig. 111 shows an excellent drawing board after the Bernhard
form.

§ 176. 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

116 DRAWING WITH THE MICROSCOPE. [CH V.

low powers (16 mm. and lower objectives) by covering the mirror with
white paper when transparent objects are to be drawn. For high pow-
ers it is best to use a substage condenser. Often the ight may be bal-
anced 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 linage
of the microscopic object is very distinct and the pencil is invisible or
very iadistinct. If the drawing surface is too brilliantly lighted the
pencil can be seen clearly, but the microscopic image will be very ob-

scure.

Fic. 111. Drawing Board for the Abbe Camera Lucida This drawing board,
devised by the Bausch & Lomb Optical Co., is adjustable vertically, and the board
may be inclined to prevent distortion. It is also arranged for use with an inclined
microscope by having the base board hinged. Microscope and drawing surface
are then inclined together. The camera lucida has a graduated arm to bear the
mirror and a graduated quadrant at the mirror joint so that the angle of the mir-
vor may be accurately determined. (See also Fig. 105). (From the Bausch &

Lomb Optical Co.)

When opaque objects, that is objects which must be lighted with re-
flected light (§ 59), like dark colored insects, etc., are to be drawn the
light must usually be concentrated upon the object in some way. The

CH. WV] DRAWING WITH THE MICROSCOPE. 117

microscope may be placed ina 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. 52).

If the drawing surface is too brilliantly illuminated, it may be shaded
by placing a book or a ground glass screen between it and the window,
also by putting one or more smoked glasses in the path of the rays from
the mirror (Fig. 102 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 excel-
lent plan to blacken the end of the drawing pencil with carbon ink.
Sometimes it is easier to draw on a black surface, using a white pencil
or style. The carbon paper used in manifolding letters, etc., may be
used, or ordinary black paper may be lightly rubbed on one side with a
moderately soft lead pencil. Place the black paper over white paper
and trace the outlines with a pointed style of ivory or bone. A corre-
sponding dark line will appear on the white paper beneath. (Jour.
Roy. Mier. Soc., 1883, p. 423).

(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
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 obtained
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 adjustable drawing
surface like that shown in Fig. 111. The image of a few spaces of the
micrometer, will give the scale of enlargement, or the power may be
determined for the special case (§ 177-178).

(F) It is of the greatest advantage, as suggested by Heinsius (Zeit.
w. Mikr., 1889, p. 367), to have the camera lucida hinged so that the

118 DRAWING WITH THE MICROSCOPE. (CH. V.

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. Any skilled mechanic
can add the quadrant.

§ 177. Magnification of the Microscope and Size of Drawings
with the Abbe Camera Lucida.—In determining the standard dis-
tance 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. 102) 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,

ee ek ae

1
iwoth mm.

Fic. 112. 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 width.

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
micrometer were zi 5th millimeter, then the enlargement would be
25 +77 = 500. That is, the image was drawn at a magnification of
500 diameters.

If the micrometer scale is used with 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 magnification 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.

§ 178. 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

CH. V.j DRAWING WITH THE MICROSCOPE. 119

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 (§ 177).

If one has many large objects to draw at a low magnification, then
some form of embryograph is very convenient. ‘The writer has made
use of a photographic camera and different photographic objectives for
the purpose. The object is 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. Nothing 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,
233; 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., 1881,
p. S19, 1882, p. 402, 1883, pp. 283, 560, 1884, p. 115, 1886, p. 516, 1888, pp. 113, 809,
798; Zeit. wiss. Mikroskopie, 1884, pp. I-21, 1889, p. 367, 1893, pp. 289-295.
Here is described an excellent apparatus made by Winkel. See Zeiss’ catalog
No 30, and the 15th (1896) edition of the Bausch & Lomb Optical Company for
improved forms of the Abbe camera lucida and for improved drawing boards to
accompany it.

CHAPTER VI.

MICRO-SPECTROSCOPE AND POLARISCOPE.

APPARATUS AND MATERIAL REQUIRED FOR THIS CHAPTER.

Compound microscope ; Micro-spectroscope (? 179); Watch-glasses and sinall
vials, slides and covers (2 198) ; Various substances for examination (as blood and
ammonium sulphide, permanganate of potash, chlorophyll, some colored fruit,
etc., (% 199-209) ; Micro-polarizer (% 211) ; Selenite plate (% 220) ; Various doubly
refracting objects, as crystals, textile fibers, starch, section of bone, etc.

MICRO-SPECTROSCOPE.

2179. A Micro-Spectroscope, Spectroscopic or Spectral Ocular, is a direct vision
spectroscope in connection with a microscopic ocular. The one devised by Abbe
and made by Zeiss consists of a direct vision spectroscope prism of the Amici pat-
tern, 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, 7. ¢., the edges 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 aspring. 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 (¢ 192). Finally near
the top is a lateral tube with mirror for the purpose of projecting an Angetront
scale of wave lengths upon the spectrum (3 193, Figs. 113-114).

3 180. Apparent Reversal of the Position of the Colors in a Direct Vision Spec-
troscope.—In accordance with the statements in 3 179 the dispersion or separation
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, conse-
quently the colors have the position indicated in the top of Fig. 117, also above
Fig. 118. But if one looks into the direct vision spectroscope or holds the eye close
to the single prism (Fig. 118), the colors will appear reversed as if the red were
more bent. The explanation of this is shown in Fig. 118, where it can be readily
seen that if the eye is placed at E, close to the prism, the different colored rays

CH. VI.) MICRO SPECTROSCOPE AND POLARISCOPE. 121

MP ei
a
FIG. I13. Abbe's Micro spectroscope. Fic. 114.
Longitudinal Section of Slit Mechanism separately.
the whole instrument. (Plan view, Full size.)

(1% full Size.)

‘« The eye lens 1s adjustable so as to accurately focus on the slit situated between
the lenses. The mechanism for contracting and expanding the slit 1s 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 HT 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 ts inserted.
The comparison prism is provided with a lateral frame and clips to hold the ob-
ject and the illuminating mirror. All these parts together with the eye-piece are
encased in a drum.

Above the eye-piece is placed an Amici prism of great dispersion which may be
turned aside about the pivot K, so as to allow of the adjustment of the object being
controlled, the prism being retained in its axtal 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 1s
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 prisms and the
scale of wave lengths, are simultaneously illuminated.’’ (Zeiss Catalog, No. 30.)

will appear in the direction from which they. reach the eye and consequently are
crossed in being projected into the field of vision and the real position is inverted.
The sanie is true in looking into the micro-spectroscope. The actual position of
the different colors may be determined by placing some ground glass or some of
the lens-paper near the prism and observing with the eye at the distance of distinct
vision.*

* The author wishes to acknowledge the aid rendered by Professor E. L. Nichols
in giving the explanation offered in this section.

122 MICRO SPECTROSCOPE AND POLARISCOPE. [CH. VI.

J E F f :

_ a “| 50 40 <

F edt iyi! ee Lt i ! 42

Solar i ©

Shectru if 2
Sodium : Sia: é

ee

. 7 a0 7 T on a)

Perman. : Ke i | oe

fotagh. inl! a
Metvra@r. 5 5
Tha grolin i 4

Fic. 115. Various Spectrums —All except that of Sodium were obtained by dif-
fused day light with the slit of such a width as gave the most distinct Fraunhofer
lines.

Lt 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. r1z.

Sodium Spectrum.—The line Spectrum (% 182) of sodium obtained by lighting
the microscope with an alcohol lame 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
(2.193). 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 ts characterized by the presence of five
absorption bands in the middle of the spectrum and is best shown by using a 5 per
cent. solution of permanganate in water in a watch glass asin Fig. 177.

Met-hemoglobin.— The absorption Spectrum of net-hemoglobin is characterized
by a considerable darkening of the blue end of the spectrum and of four absorp-
tion bands, one in the red near the line Cand two between Dand 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 ts best obtained by making a solu-
tion of blood in water of such a concentration that the two oxy-hemoglobin bands
run together (% 202), and then adding three or four drops of a ty per cent. aqueous
solution of permanganate of potash or a few drops of hydrogen dioxid (H,0,).
Soon the bright red will change to a brownish color, when tt may be evamined.

VARIOUS KINDS OF SPECTRA.

By a spectrum is meant the colored bands appearing when light traverses a dis-
persing prism or a diffraction grating, or is affected in any way to separate the dif-
ferent 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.

CH, VI.) MICRO-SPECTROSCOPE AND POLARISCOPE. 123

2181. Continuous Spectrum.—In case a good artificial light or 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.

@ 182. Line Spectrum.—If a gas is made incandescent, the spectrum it produces
consists, not of the various rainbow colors, but of sharp, narrow, bright lines, the
color depending on the substance. All the rest of the spectrum is dark. These
line spectra are very strikingly shown by various metals heated till they are in the
form of incandescent vapor.

2183. 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 ab-
sorption 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 ( 182).
For example, if sodium is putin the flame of a spirit lamp it will vaporize and be-
come 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. 114).
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.

3.184. Absorption Spectra from Colored Substances.—While the solar spectrum
is an absorption spectrum, the term is more commonly applied to the spectra ob-
tained with light which has passed through or has been reflected from colored ob-
jects which are not self-Juminous.

It is the special purpose of the micro-spectroscope to investigate the spectra of
colored objects which are not self-luminous, as blood and other liquids, various
minerals, as monazite, etc. The spectra obtained by examining the light reflected
from these colored bodies or transmitted through them, possess, like the solar
spectrum dark lines or bands, but the bands are usually much wider and less
sharply defined. Their number and position depend on the substance or its con-
stitution (Fig. 116), 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.

2.185. Angstr6m and Stokes’ Law of Absorption Spectra,—The wave lengths of
light absorbed by a body when light is transmitted through some of its substance

124 MICRO-SPECTROSCOPE AND POLARISCOPE. (CH. VI.

Aa BC 1°) Eb
90 80 10 60

ublubild dbo! aft |
.. ye
: : é ewe

Roof a LOckngey Yeo}

Fic. 116. Absorption Spectrum of Oxy-Hemoglobin or arterial blood (1) and of
Hemoglobin or venous blood (2). (from Gamgee and McMunn.)

A,B,C D, EB, FG, H. Some of the Principal Fraunhofer lines of the solar
Spectrum (% 183).

90, .80, .70, .60, .50, .go. Wave lengths in microns, as shown in Angstrém’s
scale (3.193). It will be seen that the wave lengths increase toward the red and
decrease toward the violet end of the spectrum.

Red, Orange, Yellow, 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 spec-
trum being dark.

2 186, 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 object.
For example, a thin layer of blood under the microscope will appear yellowish
green, buta thick layer will appear pure red. If now these two layers are exam-
ined with a micro-spectroscope, the thin layer will show all the colors, but the red
end will beslightly, and the blue end considerably restricted, and some of the colors
will appear of considerably lessened intensity. Finally there may appear two
shadow-like bands, or if the layer is thick enough, two well-defined dark bands in
the green (% 202).

If the thick layer is examined in the same way, the spectrum will show only red
with a little orange light, all the rest being absorbed. Thus the spectroscope shows
which colors remain, in part or wholly, and it is the mixture of this remaining or
unabsorbed light that gives color to the object.

2 187. Complementary Spectra.—-While it is believed that Angstrém’s law (2 185)
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).

CH. VI.) MICRO-SPECTROSCOPE AND POLARISCOPE. 125

ADJUSTING THE MICRO-SPECTROSCOPE.
$ 188. 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.

$189. Adjustment of the Slit.—In place of the ordinary diaphragm
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 approach till they appear about half a millimeter apart. If now
the Amici prism is put back in place and the microscope well lighted,
one will see a spectrum by looking into the upper end of the spectro-
scope. 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 Fraun-
hofer 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.

§ 190. 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 mutu-
ally arranged, the colored bands will appear oblique, and the whole
spectrum may be greatly narrowed. If the colored bands are oblique,
grasp the prism tube and slowly rotate it to the right or to the left until
the various colored bands extend directly across the spectrum.

§ 191. Focusing the Slit.—In order that the lines or bands in the
spectrum shall be sharply defined, 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 for
the ocular micrometer ($161). If one now uses daylight there will be
seen in the spectrum the dark Fraunhofer lines (Fig. 116 E. F., etc.).

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 necessary
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

126 MICRO-SPECTROSCOPE AND POLARISCOPE. [CH. V1.

|

Ocular.

FIG. 117. Fic. 118. FIG. 119.

Fig. 117 (1). Section of the tube and stage of the microscope with the spectral
ocular or micro spectroscope in position.

Amici Prism (4 167).—The direct vision prism of Amici in which the central
shaded prism of flint glass gives the dispersion or separation into colors, while the
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

CH. VI) MICRO-SPECTROSCOPE AND POLARISCOPE. 127

prism, the red being least and the blue most bent toward the base of the flint prism
(see Fig. 118).

Hinge.—The hinge on which the prism tube turns when it is swung off the
ocular.

Ocular (% 179).—The ocular in which the slit mechanism takes the place of the

diaphragm (2189). The eye-lens is movable as in a micrometer ocular, so that
the slit may be accurately focused for the different colors (% 191).

S. Screw for setting the scale of wave lengths (% 193).

S’. Screw for regulating the width of the slit (% 189).

S’, Screw for clamping the micro-spectroscope to the tube of the microscope.

Scale Tube.—The tube near the upper end containing the Angstrém scale and
the lenses for projecting the tmage upon the upper face of the Amici prism, whence
ut is reflected upward to the eye with the different colored rays. At the right is a
special mirror for lighting the scale. For arranging and focusing the scale, (see
@ 193).

Slit, —The linear opening between the knife edges Through the slit the light
passes to the prism. Lt must be arranged parallel with the refracting edge of the
prism, and of sucha width that the Fraunhofer or Fixed Lines are very clearly
and sharply defined when the eye-lens ts properly focused (3 189-191).

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 width and length of the slit.
Fig. 118. Flint-Glass Prism showing the separation or dispersion of white light

iuto the three groups of colored rays ( Red, Yellow, Blue), the blue rays being bent
the most from the refracting edge (¢ 180).

Fig. 119. Sectional View of a Microscope with the Polariscope in Position (% 209-
B17").

Analyzer and Polarizer.— They are represented with corresponding faces paral-
lel 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 (4 212). (a) Slot in the an-
alyzer tube so that the analyzer may be raised or lowered to adjust it for difference
of level of the eye-point in different oculars (% 214).

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 (% 211).

Object.— The object whose character ts lo be investigated by polarized light.

128 MICRO-SPECTROSCOPE AND POLARISCOPE. (CH. VI.

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 on the spectrum may be due to irregularity
of the edge of the slit or to the presence of dust. They are most
troublesome with a very narrow slit.

§ 192. Comparison or Double Spectrum.—In order to compare
the spectra of two different substances it is desirable to be able to exam-
ine 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 reflecting or com-
parison prism, the colored bands and the Fraunhofer dark lines will
appear directly continuous across the two spectra. The prism for the
comparison spectrum is moyable and may bz thrown entirely 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 spectrums shall have about the same
width.

§$ 193. Scale of Wave Lengths.—In the Abbe micro-sp2ctroscope
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 previous
investigation to have that particular wave length. The point most often
selected for setting the scale is opposite the sodium lines where the wave
length is, according to Angstrom, 0.5892". In adjusting the scale, one
may focus very sharply the dark sodium line of the solar spectrum and
set the scale so that the number 0.589 is opposite the sodium or D line,
or a method that is frequently used and serves to illustrate § 171, is to
sprinkle some salt of sodium (carbonate of sodium is good) in an alco-
hol lamp flame and to examine this flame. If this is done in a dark-
ened 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

CH. VI.) MICRO-SPECTROSCOPE AND POLARISCOPE. 129

solar spectrum (Fig. 114). Now, by reflecting light into the scale-tube
the image of the scale will appear on the spectruin, and by a screw just
under the scale-tub2, but in 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. Ifso, 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 neces-
sary in adjusting the scale to be sure that the larger number, 0.70, is at
the red end of the spectrum.

The numbers on the scale should be very clearly defined. If they do
not so appear, the scale-tube must be focused by grasping the outer tube
of the scale-tube and moving it toward or from the prism-tube until the
scale is distinct. In focusing the scale, grasp the outer scale-tube with
one hand and the prism-tube with the other, and push or pull in oppo-
site directions. In this way one will be less liable to injure the spec-
troscope.

$194. Designation of Wave Length.—Wave lengths of light are
designated by the Greek letter A, followed by the number indicating the
wave length in some fraction of a meter. With the Abbe micro-spec-
troscope the micron is taken as the unit as with other microscopical
measurements (§ 157). 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 numbers. 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 p. The last would be indi-
cated thus, AD = 0.5892.

§ 195. Lighting for the Micro-spectroscope.—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. Asa 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 removed.
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 spectrums should be about equally

9

130 MICRO-SPECTROSCOPE AND POLARISCOPE, [CH VI.

illuminated. One can manage this by putting the object requiring the
greater amount of illumination on the stage of the microscope and light-
ing it with the Abbe illuminator. In lighting it is found in general
that for red or yellow objects, lamp-light gives very satisfactory results.
For the examination of blood and blood crystals, the light from a petro-
leum lamp is excellent (§ 201-203). For objects with much blue or
violet, daylight or artificial light rich in blue light is best. The new
acetylene light ought to be very satisfactory (§ 65).

Furthermore, one should be on his guard against confusing the ordin-
ary 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.

§ 196. Objectives to Use with the Micro-spectroscope.—lf the
material is of considerable bulk, a low objective (18 to 50 mm.) 15 to be
preferred. This depends on the nature of the object under exaimina-
tion, 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 entering the spec-
troscope with the light from the object might obscure the absorption
bands.

In using high objectives with the micro-spectroscope one must very
carefully regulate the light (§ 58, 102), and sometimes shade the object.

§ 197.: 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. When the slit is open, one can see objects
when the microscope is focused as with an ordinary ocular. After an
object is focused, it may be put exactly in position to fill the slit of the
spectroscope, then the knife edges are brought together till the slit is of
the right width ; if the slit is then too long it may be shortened by using
one of the mechanism screws on the side, or if that is not sufficient, by
bringing the comparison prism farther over the field. If one now
replaces the Amici prism and looks into the microscope, the spectrum is
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.

S198. Amount of Material Necessary for Absorption Spectra
and its Proper Manipulation.—The amount of material necessary to
give an absorption spectrum varies greatly with different substances,
and can be determined only by trial. If a transparent solid is under
investigation it is well to have it in the form of a wedge, then succes-

CH. VI.) MICRO-SPECTROSCOPE AND POLARISCOPE. 131

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 homceopathic 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.

§ 199. Put the micro-spectroscope in position, arrange the slit and
the Amici prism so that the spectrum will show the various spectral
colors going directly across it (§ 188-189) and carefully focus the slit.
This may be done either by swinging the prism-tube aside and proceed-
ing as for the ocular micrometer (§ 161), 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 (§ 191). With the lever move the comparison prism across
half the field so that the two spectra shall be of about equal width. For
lighting, see § 195.

§ 200. 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 istransparent. Put this solution in a watch-
glass with sloping sides, and put it under the microscope. Usea 50mm.
or 16mm. objective, and use the full opening of the illuminator. 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 com-
parison spectrum (Fig. 113). For strength of solution see § 202.

§ 201. 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
in 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-

132 MICRO-SPECTROSCOPE AND POLARISCOPE. (CH. V1.

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 be done easily by setting the scale properly as described in § 193.
Make another preparation, but use a homceopathic vial instead of a
watch-glass. Cork the vial and lay it down upon the stage of the mi-
croscope. 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 very much darker, and if the solution is thick enough
only red and alittle orange will appear. Re-insert the cork and incline
the vial so that the light traverses a very thin layer, then gradually ele-
vate 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 fol-
lowing :

Add to the vial of diluted blood a drop or two of ammonium sulphide,
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 and the color will
change tothe bright red of fresh blood. Examine it again with the spec-
troscope 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 very 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 frst experiment. Further-
more, 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 dilut-
ed blood, say that from the watch-glass, before the opening of the com-
parison 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 of oxy-hemoglobin, or arterial blood, the
single banded spectrum is 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

CH, VI) MICRO-SPECTROSCOPE AND POLARISCOPE. 133

the oxy-hemoglobin to hemoglobin. This may be repzated many times
(Fig. 114).

§ 202. Met-Hemoglobin.—The absorption spectrum of met-hemo-
globin 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 (§ 201), and then adding three or four drops of a
qa per cent. aqueous solution of permanganate of potash. Soon the
bright red will change to a brownish color, when it may be examined
(Fig. 113).

§ 203. Carbon Monoxide Hemoglobin (CO Hemoglobin).—To
obtain this one may kill an animal, after anesthetization, in illuminat-
ing gas, or one may allow illuminating gas to bubble through some
blood already taken from the body. The gas should bubble through a
minute or two. The oxygen will be displaced by carbon monoxide.
This forms quite a stable compound with hemoglobin, and is of a bright
cherry-red color. Its spectrum is nearly like that of oxy-hemoglobin,
but the bands are farther toward the blue. Add several drops of am-
monium sulphide and allow the blood to stand some time. No reduc-
tion 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.

§ 204. Carmine Solution.—Make a solution of carmine by putting
qigth 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 com-
pare the spectrum with that of oxy-hemoglobin or carbon monoxide he-
moglobin. It has two bands nearly in 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 very markedly changed, while with either oxy-hemo-
globin or CO-hemoglobin the color would be very markedly 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-hemoglo-
bin can be distinguished by the use of ammonium sulphide, the carmine
remaining practically unchanged while the blood shows the single band
of hemoglobin (§ 201). The acetic acid serves to differentiate the CO-
hemoglobin as well as the O-hemoglobin.

§ 205. Colored Bodies not giving Distinctly Banded Absorp-

134 MICRO.SPECTROSCOPE AND POLARISCOPE, [CH. VI.

tion Spectra.—Some quite brilliantly colored objects, like the skin of
a red apple, do not give a banded spectrum. Take the skin of a red
apple, mount it on a slide, put on a cover-lass and add a drop of water
at the edge of the cover. Put the preparation under the microscope
and observe the spectrum. Although no bands will appear, in some
cases at least, yet the ends of the spectrum will be restricted and vari-
ous 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 wave lengths, as occurs with other colored objects. In this
case, however, not all the light of a given wave length is absorbed,
consequently there are no clearly defined dark bands, the light is simply
less brilliant in certain regions and the red rays so predominate that
they give the prevailing color.

§ 206. Nearly Colorless Bodies with Clearly Marked Absorp-
tion Spectra.—In contradistinction to the brightly colored objects with
no distinct absorption bands are those nearly colorless bodies and solu-
tions which give as sharply defined absorption bands as could be de-
sired. The best examples of this are afforded by solutions of the rare
earths, didymium, etc. These in solutions that give hardly a trace of
color to the eye give absorption bands that almost rival the Fraunhofer
lines in sharpness.

§ 207. Absorption Spectra of Minerals.—As example take some
monazite sand ona slide and either mount it in balsam (see Ch. VII),
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 very satisfactory under such conditions. It is
frequently of great service in determining the character of unknown
mineral sands to compare their 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 § 195.

§ 208. While the study of absorption spectra gives one a great deal
of accurate information, great caution must be exercised in drawing
conclusions as to the identity or even the close relationship of bodies
giving approximately the same absorption spectra. The rule followed
by the best workers is to have a known body as control and to treat the
unknown body and the known body with the same reagents, and to

CH. VI.) MICRO-SPECTROSCOPE AND POLARISCOPE. 135

dissolve them in the same medium. If all the reactions are identical
then the presumption is very strong that the bodies are identical or
very closely related. For example, while one might be in doubt be-
tween a solution of oxy- or CO-hemoglobin and carmine, the addition
of ammonium sulphide would serve to change the double to a single
band in the O-hemoglobin, and glacial acetic acid would enable one to
distinguish between the CO-blood and the carmine, although the am-
monium sulphide 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 different bodies show absorption bands in exactly the
same position (solid nitrate of uranium and permanganate of potash in
the blue).’”’, (MacMunn). The rule given by MacMunn is a good
one: ‘‘ The recognition of a body becomes more certain if its spectrum
consists of several absorption bands, but even the coincidence of these
bands with those of another body, is not sufficient to enable us to infer
chemical identity ; what enables us to do so with certainty is the fact :
that the two solutions give bands of equal intensities in the same parts of
the spectrum which undergo analogous changes on the addition of the
same 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.

Angstrém, Recherches sur le spectre solaire, etc. Also various papers in period-
icals. See Royal Soc’s Cat’! Scientific Papers; Anthony & Brackett; Beale, p.
269; Behrens, p. 139; Kossel und Schiefferdecker, p. 63; Carpenter, p. 104; Brown-
ing, 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 Schwendener ; Proctor; Ref.
Hand-Book Med. Sciences, Vol. I, p. 577, VI, p. 516, VII, p. 426; Roscoe; Schel-
len ; 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. IT, p. 419. The larger works on Phiysi-
ology, Chemistry and Physics may also be consulted with profit.

Vogel, Spectrum analysis, also in Nature, Vol. xix, p. 495, on absorption spectra.
The bibliography in MacMunn is excellent and extended.

136 MICRO-SPECTROSCOPE AND POLARISCOPE. [CH. VI.

MICRO-POLARISCOPE.

2 209. 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.

2 210. 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 2. ¢.,
when the faces through which the oblique section passed 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 go° aud 270°, where the light is extin-
guished. In the intermediate points there is a sort of twilight.

@ 211. 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 0°. 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 0°, clamp the ring to the
microscope, then rotate the draw-tube of the microscope till the field is lightest.

@ 212. 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 ocu-
lar 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 ina proper posi-
tion 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.

q 213. Objectives to Use with the Polariscope.—Objectives of the lowest power

CH. VI.) MICRO-SPECTROSCOPE AND POLARISCOPE. 137

may be used, and also all intermediate forms up to a 2 mm. homogeneous immer-
sion. 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,—wse the power that most clearly and
satisfactorily shows the object under investigation.

@ 214. 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.

@215. Mounting Objects for the Polariscope.—So far as possible objects should
be mounted in balsam to render them very transparent. In many cases objects
mounted in water do not give satisfactory polariscopic appearances. For example,
if starch is mounted dry or in water, the appearances are not so striking as in a
balsam mount (Davis, p. 337 ; Suffolk).

2 216, 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 axzsotropic, 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 produc-
tion 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. It is also found of advantage in investigating objects with
polarized light where colors appear, to combine a polariscopic and spectroscope
(Spectro-Polariscope).

MICRO-POLARISCOPE—EXPERIMENTS.

§ 217. Arrange the polarizer and analyzer as directed above (§ 211)
and use a 16 mm. objective except when otherwise directed.

(A) Isotropic or Singly Refractive Objects.—Light the micro-
scope well and cross the Nicols, shade the stage and make the field as
dark as possible (§ 210). Asan isotropic substance, put an ordinary
glass slide under the microscope. The field will remain dark. Asan
example of a crystal belonging to the cubical system and hence iso-
tropic, make a strong solution of common salt (sodium chloride Na Cl.),
put a drop on a slide and allow it to crystallize, put it under the micro-
scope, remove the analyzer, focus the crystals and then replace the an-
alyzer and cross the Nicols. The field and the crystals will remain
dark.

(B) Anisotropic or Doubly Refracting Objects.—Make a fresh

138 MICRO-SPECTROSCOPE AND POLARISCOPE. [CH. VI.

preparation of carbonate of lime crystals like that described for pedesis
($ 142), 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 an-
alyzer 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 crys-
tals will alternately flash and disappear. By observing carefully, some
of the larger crystals will b2 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 axis,* the
crystals are isotropic like the salt crystal and remain dark. If, how-
ever, the light traverses them in any other direction the ray from the
polarizer is divided into two constituents vibrating in planes at right
angles to each other, and one of these will traverse the analyzer, hence
such crystals will appear as if self-luminous ina dark field. The experi-
ment with these crystals from the frog succeeds well with a 2 mm. ho-
mogeneous immersion.

As further illustration of anisotropic objects, mount some cotton
fibers in balsam (Ch. VII), also some of the lens paper (§ 107). These
furnish excellent examples of vegetable fibers.

Striated muscular fibers are also very well adapted for polarizing ob-
jects. ; ;

As examples of biaxial crystals, allow some borax solution to dry
and crystallize on a slide ; use the crystals as object. Asall doubly re-
fracting objects restore the light with crossed Nicols, they are some-
times called depolarizing.

(C) Pleochroism.—This is the exhibition of different tints as the an-
alvzer is rotated. An excellent subject for this will be found in blood
crystals.

(D) For the aid given by the polariscope in micro-chemistry, see
(Ch, VII).

(E) See works on petrology and mineralogy for the application of
the micro-polarizer in those subjects.

§ 218. Production of Colors.—For the production of gorgeous
colors, a plate of selenite giving blue and yellow colors is placed between

*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 2 142) are uniaxial crystals.
Borax crystals are bi-axial.

CH. VI.) MICRO-SPECTROSCOPE AND POLARISCOPE. 139

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 a selenite or piece of mica for the produc-
tion of the most glorious colors in many objects. One of the most
beautiful preparations, 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 hypnotic medi-
cine, 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 most beautiful
colors, but the black cross with perfection. (Clark).

It is very instructive and interesting to examine organic and inor-
ganic substances with a micro-polarizer. If the objects enumerated in
S$ 144 were all examined with polarized light an additional means of de-
tecting them would be found.

REFERENCES TO THE POLARISCOPE AND TO THE USE OF POLARIZED
LIGHT.

Anthony & Brackett; Behrens, 133 ; Behrens, Kossel und Schiefferdecker ; Car-
noy, 61; Carpenter-Dallinger, 262, 269, 992; Clark ; Daniell, 494; Davis; v. Ebe-
ner; Gage; Gamgee; Halliburton, 36, 272; Hogg, 133, 729; Lehmann; M’Ken-
drick ; Nageli und Schwendener, 299; Queckett ; Suffolk, 125; Valentin. Physi-
cal Review, I., p. 127. Daniell, Physics for Medical Students,

CHAPTER VII.

SLIDES AND COVER-GLASSES ; MOUNTING ; ISOLATION,
SECTIONING BY THE COLLODION AND THE PARAF-
FIN METHODS; LABELING AND STORING MICRO-
SCOPICAL PREPARATIONS; EXPERIMENTS IN MICRO-
CHEMISTRY.

APPARATUS AND MATERIAL FOR THIS CHAPTER.

Microscope, compound and simple (Ch. I) ; Micro-Spectroscope and polariscope
(Ch. VI); Slides and cover-glasses (4 219-220) ; Cleaning mixtures for glass (? 227) ;
Alcohol and distilled or filtered water (¢ 222); fine forceps for handling cover-
glasses (4 222-226) ; Old handkerchiefs or lens paper (7 107, 223). Paper boxes for
storing cover-glasses (4 223, 225) ; Cover-glass measurer (Figs. 120-122) ; Mount-
ing material,—Farrant’s solution, glycerin, glycerin-jelly and Canada balsam (4 243,
246) ; Centering card and card for serial sections (? 236) ; Material for dissociation
and for the paraffin and collodion method (2 244) ; Material for paraffin and collo-
dion sectioning (% 250) ; Net-micrometer for arranging minute objects like diatoms
(@ 317); Labels (2 309); Carbon ink for writing labels (¢ 295); Writing diamond
(4 295) ; Shellac cement (% 316); Cabinet (¢ 296); Re-agents for experiments in
micro-chemistry (2 315).

SLIDES AND COVER-GLASSES.

§ 219. 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 (1 x 3 inches). For rock sections, slides 25 x 45 mm.
or 32 x 32 mm. are used; for serial sections, slides 25 x 76 mm., 50x 75
mm. or 37 x 87mm. are used. For special purposes, slides of the nec-
essary 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.

§ 220, Cleaning Slides.—For new slides a thorough rinsing in clean
water with subsequent wiping with a soft towel, and then an old soft

CH. VII.) SLIDES AND COVER-GLASSES. 141

handkerchief, usually fits them for ordinary use. If they are not satis-
factorily 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 blot-
ting paper, and then wipe witha 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 used 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 gum-
my 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 clean-
ing mixtures for glass. If they are first soaked in xylene, benzin or tur-
pentine to dissolve the balsam, then soaked in the cleaning mixture, the
time required will be much shortened (§ 227). After all foreign mat-
ter is removed the slides should be very 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, etc., avoids this, and
greatly shortens the time necessary 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 cleaning
mixture can then act on the entire surface. It should be said, however,
that at the present price of slides and cover-glasses it is hardly worth
while to clean those that have been used in balsam mounting.

§ 221. Cover-Glasses or Covering Glasses.—These are circular
or quadrangular pieces of thin glass used for covering and protecting
microscopic objects. They should be very thin, 7% to 75 millimeter
(see table, § 27). It is better never to use a cover-glass over ~9; 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 (§ 47). Indeed, if
one wishes to employ high powers, the thicker the sections the thinner
should be the cover-glass (see § 235).

The cover-glass should always be considerably larger than the object over
which tt ts placed.

§ 222, Cleaning Cover-Glasses.—New cover-glasses should be put
into a glass dish of some kind containing one of the cleaning mixtures

142 SLIDES AND COVER-GLASSES. (CH, VII.

($ 227) and allowed to remain a day or longer. In putting them in,
push one in at a time and be sure that it 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 to injure
the slides or covers. After one day or longer, pour off the cleaning
mixture 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.

§ 223. 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 paper to let them drain. Grasp a
cover-glass by its edges, cover the thumb and index of the other hand
with a soft, clean cloth or some of the lens paper. Grasp the cover be-
tween the thumb and index and rub the surfaces. In doing this it is
necessary to keep the thumb and index well opposed on directly oppo-
site 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 seen. 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 paper box. 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. Wood-pulp
paper, from which most of the boxes are now made, constantly sheds
particles into the boxes ana thus soils the covers stored in them . This
can be largely obviated by coating the inside of the boxes with a thin
solution of shellac.

§ 224. Cleaning Large Cover-Glasses.—For serial sections and
especially large sections, large quadrangular covers are used. These
are to be put one by one into 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. A safe and good way to clean the large covers is to take two

CA. VII.) SLIDES AND COVER GLASSES. 143

perfectly flat, smooth blocks, considerably larger than the cover-glasses.
These blocks are covered with soft clean cloth, or with several thick-
nesses of the lens paper; if now the cover-glass is placed on the one
block and rubbed with the other, the cover may be cleaned as by rub-
bing its faces with the cloth-covered finger and thumb. It is especially
desirable that these large covers should be thin—not over 74° to 7o%
nim,—otherwise high objectives cannot be used in studying the prepa-
rations.

S 225. Measuring the Thickness of Cover-Glasses.—lIt is of the
greatest advantage to know the exact thickness of the cover-glass on an
object ; for, (a) One would not try to use objectives in studying the
preparation of a shorter working distance than the thickness of the cover
($57); (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 thick-
ness of cover may be selected corresponding to that for which the object-
ive was corrected (see table, § 27). Furthermore, if thefe is a varia-
tion from the standard, one may remedy it, in part at least, by length-
ening the tube if the cover is thinner, aud shortening it if the cover is
thicker than the standard (§ 96).

In the so-called No. 1 cover-glasses of the dealers in microscopical
supplies, the writer has found covers varying from ;'y’5 mm. to 333, min.
To use cover-glasses of so wide a variation in thickness without know-
ing whether one has a thick or thin one is simply to ignore the funda-
mental principles on which correct microscopic images are obtained.

Fic. 120. Micrometer Calipers (Brown and Sharpe). Pocket Calipers, gradu-
ated in inches or millimeters, and well adapled for measuring cover-glasses.

It is then strongly recommended that every preparation shall be cov-
ered with a cover-glass whose thickness is known, and that this thick-
ness should be indicated in some way on the preparation.

§ 226. Cover-Glass Measurers.— For the purpose of measuring
cover-glasses there are three very excellent pieces of apparatus. The

144 SLIDES AND COVER-GLASSES. GEA. VL,

micrometer calipers, used chiefly in the mechanic arts, is convenient, and
from its size easily carried in the pocket. The two cover-glass meas-
urers, specially designed for the purpose, are shown in Figs. 120-122.
With either of these the covers may be more rapidly measured than with
the calipers.

With all of these measurers 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.

Fic. 121. Cover-Glass Measurer (Edward Bausch).

The cover glass 1s placed in the notch between the two screws, and the drum ts
turned by the milled head at the right till the cover is in contact with the screws.
The thickness ts then indicated by the knife edge on the drum, and may be read of
directly in ph sth mm. or polyath inch. In other columns is given the proper tube-
length for various unadjustable objectives (+, 4, 4, and | in.) made by the Bausch
and Lomb Optical Company.

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

GHEE SLIDES AND COVER.GLASSES. 145

réy mm. may be put in the same box. For example, if one takes 74%
mim, as a standard, covers varying ;7> mm. on each side may be put
into the same box. In this case the box would contain covers of 74%,

4 15 6 7
toy [tv ais a , and die mim.

Fic, 122. Zeiss Cover-Glass Meas-
urer. With this the knife edge jaws
< S See are opened by means of a lever, and
HT : : the cover inserted. The thickness
t aii al may then be read off on the face as
ae ( , the pointer indicates the thickness in

= = | Se hundredths millimeter in the outer

circle and in hundredths inch on the
inner circle.

$227. Cleaning Mixtures for Glass.—The cleaning mixtures used
for cleaning slides and cover-glasses are those commonly used in chem-
ical laboratories :

(A) Dichromate of Potash and Sulphuric Acid.

Dichromate of potash (K,Cr,O,) — - - - 200 grams.
Water, distilled or ordinary e 2 2 1000 cc.
Sulphuric acid (H, SO,) - = - - 1000 cc.

Dissolve the dichromate in the water by the aid of heat. Pour the
solution into a bottle that has been warmed and surrounded by a wet
towel. Add slowly and at intervals the sulphuric acid. It is safer to
mix the ingredients in an agate-ware basin, and put into the bottle only
after the mixture is cool.

For making this mixture, ordinary water, commercial dichromate and
strong commercial sulphuric acid should be used. It is not necessary
to employ cheniically pure materials.

This is a very excellent cleaning mixture, and is practically odorless.
It is exceedingly corrosive and must be kept in glass vessels. It may
be used more than once, but when the color changes markedly from
that seen in the fresh mixture it should be thrown away.

(B) Sulphuric and Nitric Acid Mixture.
Nitric acid (H NO,) - - - - - 200 cc.
Sulphuric acid (H, SO,) S 7 - - - 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 corrosive mixture than (A), and has the undesirable
feature of giving off very stifling fumes, therefore it must be carefully

10

146 MOUNTING AND LABELING. (CH. VIT.

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.

§ 228. Mounting a Microscopical Object is so arranging it upon
some suitable support (glass slide) and in some suitable mounting me-
dium that it may be satisfactorily studied with the microscope.

The cover-glass on a permanent preparation should always be consider-
ably larger than the object; and where several objects are put under one
cover-glass it ts false economy to crowd thent too closely together.

§ 229. Temporary Mounting.—For the study of living objects, like
amoebae, white blood corpuscles, and many other objects both animal
and vegetable, their 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 inthe serum. Some-
times it is not easv 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 little commion salt (water 10> cc.,
common salt ;;ths gram) is employed. ‘This is the so-called normal
salt or saline solution. For the ciliated cells from frogs and other am-
phibia, 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. 123. After Shi
the cover is in place, if the preparation is to be studied lide
for some time, it is better to avoid currents and evapora-
tion 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. 140). ,

Fic. 123. 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.

Fic. 124. Needle Holder (Queen G& Co.). By means of the
screw clamp or chuck at one end, the needle may be quickly
changed.

CH. VIT) MOUNTING AND LABELING. 147

§ 230. Permanent Mounting.—For making permanent micro-
scopical preparations, there are three great methods. Special meth-
ods of procedure are necessary to mount objects successfully in each of
these ways. The best mounting medium and the best method of mount-
ing in a given case can only be determined by experiment. In most
cases some previous observer has already made the necessary experi-
ments and furnished the desired information. ,

The three methods are the following: (A) Dry or tz atr (§ 231) ;
(B) Ln some medium miscible with water, as glycerin or glycerin jelly
(S$ 235); (C) Lz some resinous medium like dammar or Canada balsam
(S$ 240).

§ 231. 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.

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 (§ 233).

2, The object is thoroughly dried (desiccated) either in dry air or by
the aid of gentle heat.

3. If practicable the object is mounted on the cover-glass ; if not it is
placed in the bottom of the cell.

4. The slide is warmed till the cement forming the cell wall is some-
what sticky, or a very thin coat of fresh cement is added ; the cover is
warmed and put on the cell and pressed down all around tilli a shining
ring indicates its adherence (§ 234).

5. The cover-glass is sealed (§ 234).

6. The slide is labeled (§ 292).

7. The preparation is cataloged and safely stored (§ 293, 296).

§ 232. Example of Mounting Dry, or in Air.—Prepare a shal-
iow cell and dry it (§ 233). Select a clean cover-glass slightly larger
than the cell. Pour upon the cover a drop of a 10% solution of sali-
cylic acid in 95% alcohol. Let it dry spontaneously. Warm the slide
till the cement ring or cell is somewhat sticky, then warm the cover
gently and put it on the cell, pressing down all around (§ 231). Seal
the cover, label and catalog (§ 234, 292, 293).

A preparation of mammalian red blood corpuscles may be made very
satisfactorily by spreading,a very thin layer of fresh blood on a cover

148 MOUNTING AND LABELING. [CH. VIL.

with the end of a slide. After it is dry, warm gently to remove the last
traces of moisture and mount precisely as for the crystals. One can get
the blood as directed for the Micro-spectroscopic work (§ 201).

Fic. 125. Turn-Table for sealing cover-glasses and making shallow mounting
cells. (Queen & Co.)

§ 2433. Preparation of Mounting Cells.—( A) 7/in Cells. ‘These
are most conveniently made of some of the microscopical cements.
Shellac is one of the best and most generally applicable (§ 316). To
prepare a shellac cell place the slide on a turn-table (Fig. 125) and cen-
ter 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 spontane-
ously, or heat the slide in some way. Before the slide is used for
mounting, the cement should be so dry when it is cold that it does not
dent when the finger nail is applied to it.

A cell of considerable depth may be made with the shellac by adding
successive layers as the previous one 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.

§ 234. Sealing the Cover-Glass for Dry Obje¢ts 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 remaining moisture from the ob-
ject. When the cover is put on it is pressed down all around over the

CH. VII.) MOUNTING AND LABELING. 149

cell until a shining ring appears, showing that there is an intimate con-
tact. 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 ar-
ranged so that the cover-glass and cell wall will be concentric with the
guide rings of the turn-table. Then the turn-table is whirled and a
ring of fresh cement is painted, half on the cover and half on the cell
wall (Fig. 140). If the cover-glassis 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 ina
safe place for the drying of the cement. It is advisable to add a fresh
coat of cement occasionally.

§ 235. 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 ob-
ject moist and therefore in a condition resembling the natural one.
The object is usually and properly treated with gradually increasing
strengths of glyceriu or fixed by some fixing agent before being per-
manently mounted in strong glycerin or either of the other media.

In all of these different methods, unless glycerin of increasing
strengths has been used to prepare the tissue, the fixing agent is
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 con-
siderable thickness.

Fic. 126. 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 of the slide. (From the Mi-
croscope, Dec., 1886. )

§ 236. Order of Procedure in Mounting Objects in Glycerin.
1. A cell must be prepared on the slide if the object is of considerable
thickness (§ 233, 234).

150 MOUNTING AND LABELING. (CAH. IST.

2. A suitably prepared object (§ 235) is placed on the center of a clean
slide, and if no cell is required a centering card is employed to facilitate
the centering (Fig. 126.)

3. A drop of pure glycerin is put upon the object, or ifa 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. 123). The cover
is then gently pressed down. If a cell is used, a fresh coat of cement
is added before mounting (§ 234.)

Fic. 127. Slide and cover glass showing method of anch-

C) oring a cover-glass with a glycerin preparation when no

cell ts used. A cover-glass so anchored is not liable to
move when the couzr is being sealed (% 238).

Fic. 128. Glass slide with cover-glass, a drop of reagent

and a bit of absorbent paper to show method of trriga-
: tion (% 247, 248).

5. The cover-glass is sealed (§ 234).

6. The slide is labeled (§ 292).

7. The preparation is cataloged and safely stored (§ 293, 296).

§ 237. 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.
126) 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. 123),
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 (§ 234, 248).

6. The slide is labeled (§ 292).

7. The preparation is cataloged and safely stored (§ 296).

§ 238. Sealing the Cover-Glass when no Cell is used.—(A)
for glycerin mounted specimens. The superfluous glycerin is wiped

CH: VIL)J MOUNTING AND LABELING. E51

away as carefully as possible with a moist cloth, then four minute drops
of cement are placed at the edge of the cover (Fig. 127), and allowed to
harden for half an hour or more. These will 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.

A BO C

Fic. 129. 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
ts 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 toa strip of glass of the desired length and width.

B—Two cover-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 tt 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, Farrant’s solution or a resinous me-
dium. ‘The mounting medium is first allowed to harden, then the su-
perfluous 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 Farrant’s solution or with alcohol, chloroform or turpentine, etc.,
if a resinous medium is used. ‘Then the slideis 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 seal-
ing preparations in balsam or other resinous media unless the solvent
of the cement is not a solvent 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. Shellacis an excellent cement for sealing
balsam preparations, as it never runs in, and it serves to avoid any in-
jury to the preparation when cedar oil, etc., are used for homogeneous
immersion objectives.

152 MOUNTING AND LABELING. [CH. VI.

§ 239. Example of Mounting in Glycerin Jelly.—For this select
some stained and isolated muscular fibres or other suitably prepared
objects. (See under isolation § 244). Arrange them on the middle of
a slide, using the centering card, and mount in glycerin jelly as
directed in § 223. Air bubbles are not easily removed from glycerin
jelly preparations, so care should be taken to avoid them.

§ 240. 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 trausparency to enable
one to use high enough powers for the demonstration of minute details.

By using sufficient care almost any tissue may be mounted in a resin-
ous 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 bring-
ing this about: (A) By drying or desiccation (§ 241), and (B) by
successive displacements (S$ 243).

S$ 241. Order of Procedure in Mounting Objects in Resinous
Media by Desiccation :

1. The object suitable for the purpose (fly's wings, etc.) is thorough-
ly 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. 126).

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.
123), but in no case does one breathe on the cover as when media mis-
cible with water are used.

5. The cover-glass is pressed down gently.

6. The slide is labeled (§ 292).

7. The preparation is cataloged and safely stored (§ 293, 296).

8. Although it is not absolutely necessary, it is better to seal the
cover with shellac after the medium has hardened round the edge of
the cover (§ 228 C).

S$ 242. 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. Carefully remove the fly’s wings, being especially care-
ful to keep them the dorsal side up. With a camel’s hair brush remove
any dirt that nay be clinging to them. Place a clean slide on the cen-
tering card, then with fine forceps put the two wings within one of the

CH VIT:] MOUNTING AND LABELING. 153

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. 123). 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., (§ 291-
295).

§ 243. Mounting in Resinous Media by a Series of Displace-
ments.—For examples of this see the procedure in the paraffin and in
the collodion methods (§ 265, 284). The first step in the series is De-
Ayvdration, that is, the water is displaced by some liquid which is misci-
ble 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 ina
dish of the alcohol, or alcohol from a pipette may be poured upon it.
Dehydration usually occurs in the thin objects to be mounted in balsam
in 5 to 15 minutes. Ifa dish of alcohol is used it must not be used too
many times, as it loses in strength.

The second step is clearing. ‘Yhat is, some liquid which is miscible
with the alcohol and also with the resinous medium is used. This
liquid is highly refractive in most cases, and consequently this step is
called clearing and the liquid a clearer. The clearer displaces the alco-
hol, 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 resin-
ous 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.

154 ISOLATION OF ELEMENTS. (CH. VII.

ISOLATION OF HISTOLOGICAL ELEMENTS.

§ 244. 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 oue 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. It is
better also to dilute the fixing agents with normal salt solution (§ 313)
than merely with water.

S 245. Isolation by Means of Formaldehyde.—Formaldehyde in
a zy solution in normal salt solution is one of the very best dissociat-
ing agents for brain tissue and all the forms of epithelium (§ 308). It
is prepared as follows: 5 cc. of formal, formol, formalin or formalose,
that is, a 4o% solution of formaldehyde, are mixed with 995 cc. of
normal salt solution. ‘This acts quickly and preserves delicate struct-
ures like the cilia of ordinary epithelia, and also of the endymal cells of
the brain. It is very 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 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.
See Figs. 130 and 131.

S 246. 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 form-
aldehyde dissociator. After two hours or more, up to two or three
days, excellent preparations of ciliated cells may be obtained by scrap-
ing 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 profile. 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,

CH. VIL.) ISOLATION OF ELEMENTS. 155

S$ 247. Staining the Cells.—Almost any stain may be used for the
formalin dissociated cells. As an example, one may use eosin (§ 305).
This may be drawn under the cover of the already mounted preparation
(Fig. 128), 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
stained. The stain makes the structural features somewhat plainer ; it
also accentuates some features and does not affect so markedly others.

S$ 248. Permanent Preparations of Isolated Cells.—If one de-
sires to make a permanent preparation of the 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

ran<,
eS

Fic. 130. Adjustable lens holder with universal joint. This ts especially useful
for gross dissections, and for dissecting the partly isolated elements with needles.

a piece of blotting paper, as shown in Fig. 128. 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 (§ 239, 309).

§ 249. Isolation of Muscular Fibers.—For this the formal disso-
ciator may be used (§ 245, 308), but the nitric acid method is more suc-

156 ISOLATION OF ELEMENTS. [CH., VEL.

cessful (§ 312). ‘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 very easy to separate the fascicles and fibers either with nee-
dles or by shaking in a test tube or reagent vial (Fig. 132) with water.
It takes longer for some muscles to dissociate than others, even in the
same temperature, so one must try occasionally to see if the action is
sufficient. When it is, the acid is poured off and the muscle washed

Fic. 131. Adjustable lens holder for the same purposes as Fig. 130. (The Bausch
& Lomb Optical Company).

gently with water to remove the acid. If one is ready to make the prep-
arations at once they may be isolated and mounted in water. If it is de-
sired to keep the specimen indefinitely, or several days, the water should
be poured off and a half saturated solution of alum added ($ 299). ‘The
alum solution is also very advantageous if the speciiens are to be
stained. The specimens may be mounted in glycerin, glycerin jelly or
balsam. Glycerin jelly is the most satisfactory, however.

CH. VIL)J COLLODION SECTIONING. 157

THE PREPARATION OF SECTIONS OF TISSUES AND ORGANS.

S$ 250. At the present time there are three principal methods of ob-
taining thin sections of tissues and organs for microscopic study. ‘These
methodsare: The Collodion Method, the Paraffin Method, and the Ireez-
ing \ethod. Each of these methods has its special application, although
the collodion method is perhaps 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 struct-
ure are not so important. With the paraffin method the thinnest sec-
tions may be made, and in some ways it is the most satisfactory of all.
A good microtome is of very great aid in sectioning.

§ 251. The Collodion Method.—In sectioning by this method the
tissues are first hardened properly and then entirely infiltrated with col-
lodion, and the collodion hardened. It is not removed from the tissue,
but on account of its transparency does no harn).

§ 252. 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. For the preparation of the solution see (§ 315). A
small piece of tissue or organ not 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 replaced 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.

§$ 253. Dehydration before Infiltration.—When one is ready to
imbed for sections, the tissue must first be dehydrated in plentiful 95%
or stronger alcohol. It is better to take only a small piece for this.
The smaller the piece the thinner the sections may be made. The de-
hydration will usually be completed in 2 to 24 hours. If the alcohol is
changed two or three times the dehydration will be hastened.

$ 254. Saturating with Ether-Alcohol (§ 306).—The next step is
to remove the tissue from the alcohol and place it in a vial of ether-
alcohol ($ 306) for 2 to 24 hours. The dehydration is 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 omitted, however, but one is surer of success
if the ether-alcohol is used.

§ 255. Infiltration with Thin Collodion.—The ether - alcohol is
poured off, and a mixture of thin collodion is added (§ 304). Two or

158 COLLODION SECTIONING. [CH. VIT.

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.

§ 256. Infiltration with Thick Collodion.—The thin collodion is
poured off and thick collodion (§ 304) 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 during a day or even a week is more
certain to insure a perfect infiltration.

§ 257. Imbedding.—The tissue may be imbedded in a paper box,
such as is used for paraffin imbedding, or in any of the other boxes de-
vised for paraffin. It is better, if paper is used, to put a very small
amount of oil on the paper to prevent the collodion from sticking to it.
Vaselin spread over lightly and then all removed, so far as possible,
with a cloth or with lens paper, gives the right surface. For small ob-
jects 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. 132). The vial should contain sufficient chloroform 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 ad-
vantageous 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 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.

§ 258. Hardening and Clarifying the Collodion.—After a few
hours the collodion is hardened by the chloroform, If it acts long
enough the imbedding mass is rendered entirely transparent, if no water
is present. Whenever the collodion is hard, whether it is clear or not,

CH. VTI1.J COLLODION SECTIONING. 159

the chloroform is poured off and the carbol-xylene* clarifier (§ 302)
added. Ina few hours the imbedded mass will become as transparent as
glass and the tissue will seem to have nothing around it. 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, the opacity may result. Sometimes, in objects of consid-
erable size, this may remain for a week. This is the exception, how-
ever, and if the mass seems sufficiently hard and tough, the cutting may
proczed even if the clarification is incomplete. +

Fic. 132. Preparation Vials for Histology and Embryology. These represent
the two vials, natural size, that have been found most useful. They are kept in
blocks with holes of the proper size.

In case the imbedding mass will not clarify after a few days the im-
bedded 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.

* The hydrocarbon xylene (C, H,,) is called xylolin German. In English, mem-
bers of the hydrocarbon series have the termination ‘‘ene,’’ while members of the
alcohol series terminate in ‘“‘ol.”’

+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.

160 COLLODION SECTIONING. (CH. VI/T.

$259. Cutting the Sections.—For collodion sectioning a long, draw-
ing 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
placed 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 two millime-
ters all around the tissue. This is to render the diameter of the ena to
be cut as small as possible. The smaller the object the thinner can the
sections be made. With an object two to three millimeters thick and
not over five millimeters wide, and a good sharp knife, sections 5 to
6 can be cut without difficulty. When knife and tissue are properly
arranged the tissue is well wet and the knife 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.

§ 260. 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 pa-
per* about twice the size of a slide and place it directly upon the sec-
tions. 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 any
absorbent paper, and press it down gently upon the transfer paper.
This will absorb the oil, and then the transfer paper may be lifted, with
a rolling motion, from the slide. The sections will remain on the slide.

§ 261. Fastening the Sections to the Slide.—Drop just enough

*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 ab-
sorbent. One of the first and most successful papers recommended is ‘‘ closet or
toilet paper.’’ Cigarette paper is also excellent. In my own work the silky Japa-
nese paper, called ‘‘ Usago”’ paper, has been found almost perfect for the purpose.
Ordinary lens paper or thin blotting paper for absorbing the oil is used with it.

t If one is a long time 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.

CH. VII.) COLLODION SECTIONING. 161

ether-alcohol (equal parts of sulphuric ether and 95% alcohol) 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 sur-
face 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.

Fic. 133. Pipette for adding liquids drop-
wise and for washing preparations. (Whit-
all, Tatum & Co.)

§ 262. 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.

§ 263. Staining the Sections with an Alcoholic Stain. —If an
alcoholic stain containing 50% or more alcohol (for example, hydro-
chloric 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 sec-
tions dehydrated in 95% alcohol, cleared and mounted in balsam.

§ 264. 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 amoment. ‘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, however, 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

II

162 COLLODION SECTIONING. (CH. VII.

of water. They may then be counterstained for half a minute with
some general dye, like eosin or picric acid, or mounted with but the one

stain.*

Fic 134. Waste Bowl with rack for supporting slides and a small funnel tm
which the slides stand while draining. This oulfit is easily made by any tin smith.
The rack is composed of two brass rods about 3 mim. in diameter. The bent end
pieces are sheet brass. The funnel is made of tin, copper or brass. Either copper
or brass ts preferable to tin. A glass dish like that shown in Fig. 135 1s betler than
a bowl, as tt can be more readily and thoroughly cleaned. (Cut loaned by Wm.
Wood & Co.).

Fic. 135. Round glass aquarium. This glass vessel 1s better than the bowl for
all the uses described for the bowl. (Whitall, Tatum & Co.)

Fic. 136. Glass box with cover. These boxes may be had of various sizes and
can be used advantageously for water, and for cleaning mixture for slides and
cover-g lasses (4.227). (Whitall, Tatum & Co.)

*In 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% alcoliol 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 65% 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 hy the flood of alcohol. This plan has
been submitted to the severe test of laboratory work, and has proved itself perfectly
satisfactory.

CH. VII.) COLLODION SECTIONING. 163

ORDER OF PROCEDURE IN MAKING MICROSCOPICAL PREPARATIONS BY
THE COLLODION METHOD.

§$ 265. It will be seen from this table, and sections 252-266, that
it requires about five days to get a microscopical preparation if one com-
mences with the fresh tissue. Other methods of hardening might re-
quire 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 tissues| 13. Staining the sections with an alco-
(% 252), 4 days or more. holic dye (3263-264), 2 minutes to

2. Dehydrating the object to be cut in 24 hours.

95% or stronger alcohol (% 253), 2-/ 14. Staining the sections with an aque-
24 hours. ous dye (% 264), 2-10 minutes.

3. Saturating the tissue in ether-alcohol

5 ea) ‘ 15. Removing the superfluous dye by
254), 2-24 hours.

washing in water or alcohol (¢ 263-
4. Infiltrating with thin collodion 264), 2-5 minutes.

2255), 2h to 2 days. oer :
Uh apo = eseerore a 16, Staining witk a general dye (% 264),

5- Infiltrating in thick collodion (3 256), 15-30 seconds.

5 hours to several days.
17. Washing with water or alcohol

6. Imbedding the tissue (@ 257), 15 to (2 263-264), I to 2 minutes.

20 minutes.
18. Dehydrating the sections in 95% al-

. Hardening th llodi ith chlo-
ie ee cohol (¢ 266), 5 min. to 24 hours.

roform (¢ 258), 5-24 hours.

8. Clarifying and further hardening the | 19- Clearing the sections (2266), 5 min.
collodion with castor-xylene (7 258), to 24 hours.

iorgh Biotirs. 20. Draining the sections, 1-2 minutes.

g. Cutting the sections (% 259), 10 min-

iitestoee hours: 21. Mounting in Canada balsam (3 266),

I-2 minutes,
jo. Transferring the sections to a slide :
with paper (2 260), 1 minute. 22, Sealing the cover-glass (3 238), 2

: . minutes.
11. Fastening the sections to the slide

with ether-alcohol (? 261), 1 or 2| 23. Labeling the preparation (@ 291), 2
minutes. miuutes.

12. Removing the oil from the sections} 24, Cataloging the preparation (3 294),
with benzin and alcohol (2 262), 3-5 5-Io minutes.
minutes, or 24 hours.

§ 266. 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 clear-

164 PARAFFIN SECTIONING. [CH. VII.

ing 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 (§ 303). 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. 134). Then the bal-
sam is put on the sections or spread on the cover-glass and that placed
over the sections.

For cataloging and labeling, see § 291-295.

tf |
[| Fic. 137. Small spirit lamp modified into a balsam
I| bottle, or a glycerin or glycerinyelly bottle, or a bottle
for homogeneous tinmersion liquid. For all of these
purposes tt should contain a glass rod as shown in the
figure. By adding a small brush, it answers well for
a shellac bottle also. i

2 267. The Collodion Method with Alcohol.—A good method of procedure for
making collodion sections is to proceed exactly as described including % 257, 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 (?% 263-266) are as described. One may preserve the
tissue after imbedding for a long time in the 82% alcohol before sectioning, or for
successive sections. While this method appears somewhat simpler, the results are
not so satisfactory as by the oil-method given above.

THE PARAFFIN METHOD.

§ 268. As with the collodion method, the tissues are first properly
fixed and hardened and then entirely filled with the imbedding mass,
but unlike collodion the mass must be entirely removed before the sec-
tions are finally mounted, ‘The tissue thus imbedded and infiltrated is
like a homogeneous mass and sections may be cut of extreme thinness.

§ 269. Harden perfectly fresh tissue in picric-alcohol (§ 315)

CH. VIL.) PARAFFIN SECTIONING. 165

from one to three days. (Any other good method for fixing and hard-
ening the elements may be used, only the special conditions necessary
for the reagent must be observed. The time might be longer or shorter
than for picric-alcohol. )

After the tissue is properly fixed and hardened in, pour off the
fixative and add 67% alcohol. Leave this on the tissue from 1 to 3
days.

Pour off the 67% alcohol and add 82% alcohol. Leave this on the
tissue at least 1 day. The tissue may remain indefinitely in 82% alco-
hol.

§ 270. Dehydration and Preparation for Imbedding.—From the
pieces of tissue fixed and hardened, cut pieces 5 to 10 millimeters long
and 2 to 3 millimeters in diameter and dehydrate them one day in 95%
or stronger alcohol using a preparation vial (Fig. 130). Leave the
stock tissue in the 82% alcohol. If one changes the 95% alcohol after
3 or 4 hours and adds fresh 95% the pieces of tissue of the size here
given will be sufficiently dehydrated in 5 or 6 hours. Larger pieces
require more time.

(If one is studying organs then the whole organ may need to be pre-
pared for imbedding, but for the minute structure small pieces are
preferable as thinner sections may be made. )

§ 271. Saturating with Chloroform.—After dehydration pour off
the 95% alcohol and add pure chloroform. The chloroform will re-
place the alcohol in a few hours. It will do no harm to leave the tissue
in chloroform a day or more. Small pieces are usually sufficiently pene-
trated in 4 to 6 hours.

$ 272. Infiltrating with Chloroform Paraffin. ee the tissue
from the chloroform into chloroform paraffin (§ 301) using a tin dish
(pattie dish). Place the dish into the paraffin oven where the paraffin
will remain melted. The tissue being full of chloroform will allow the
penetration of the chloroform paraffin, and the heat will drive off the
chloroform so that after 3 to 5 days with small objects and a small
amount of chloroform paraffin, the chloroform will have evaporated
and the tissue will be infiltrated with pure paraffin. It is possible to
hasten the infiltration by pouring off the chloroform paraffin on the
second day and adding pure paraffin, but the high temperature neces-
sary for keeping the pure paraffin melted is injurious to many tissues,

§ 273. Imbedding in Pure Paraffin.—After the chloroform has
evaporated the tissue should be imbedded. One can tell whether the
chloroform has all been driven off by tasting the paraffin, It will have
the sweetish taste of chloroform if any is present. If any is present

166 PARAFFIN SECTIONING. [CH. VII.

the tissue must remain longer in the warm chamber. If the chloro-
form has all evaporated, melt some pure paraffin (that designated
‘‘ hard paraffin ’’ by the dealers is usually about the right hardness).

Make a little paper box, fill it half full of the pure melted paraffin
and then arrange the tissue in it so that sections may be made across
the short axis. Put the tissue near one end of the box, and as soon as
the paraffin has solidified on the surface place the box in cold water so
that it will cool quickly.

§ 274. Cutting the Sections.—After the imbedding mass is well
cooled, remove the paper box and trim the end in which the tissue is,
in a pyramidal form. Clamp the block of paraffin into the microtome
so that the tissue will be at about the level of the top of the microtome.
With a very sharp razor cut the sections. ‘The razor should be dry,
and the sections are made with a rapid, straight cut asin planing. Do
not try to section with a drawing cut as with collodion sectioning. If
the temperature is right for the paraffin the sections will remain flat,
and if the end is pyramidal successive sections will adhere and thus
make ribbons. If the paraffin is too cold the sections will roll. In
that case place the microtome near the steam pipes a little while.
Remember that sections must be from 5 to 15p thick to show well.
Sections of that thinness cannot be made with a dull razor nor of an
object too large.

§ 275. Fastening the Sections to the Slide.—To fasten the sec-
tions to the slide, coat the center of the slide, or the whole if a series is
to be made, with the Albumen Fixative ($ 298). Do this by adding a
little of the fixative and then with a clean finger rub the albumen over
the slide and beat or tap the slide with the finger. This will make
a very thin and even layer. Place the sections in position and press
them to the slide with the finger. Then spread over them a small
amount of a 3%4th% collodion (§ 304). Allow them to dry in the
air for 2 to 10 minutes.

§ 276. Removing the Paraffin.—Immerse the slide in a vessel of
xylene or benzin. This will dissolve the paraffin. Half an hour will
usually suffice. One can hasten the solution by moving the slide in the
benzin. In this way it may be dissolved in 3 to 5 minutes. It will do
no harm to leave the slide in the benzin or xylene over night. Two or
three days might not do any harm, but it is usually better to proceed
at once to the other operations. ;

§ 277. Removing the Benzin.—From the benzin or xylene plunge
the slide bearing the sections into a jar of 95% alcohol and leave it for
a few minutes or move it around for a half minute or so.

CH. VITJ PARAFFIN SECTIONING. 167

§ 278. 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 slide to become dry, for that would probably ruin the tissue.
Wash away the stain with 67% alcohol then dehydrate with 95% alco-
hol and clear and mount in balsam as described below.

S 279. Staining with an Aqueous Dye.—Wash away the alcohol
by plunging the slide bearing the sections into a jar of water and mov-
ing it around a moment. Then add the stain to the sections with a
pipette and allow the slide to rest horizontally on the bars of the waste
bowl (Fig. 134) or preferably immerse the slide in a jar of the stain
($ 264). The sections are usually well stained in from 1 to 10 min-
utes when they should be thoroughly washed with water.

§ 280. Staining with a General Dye.—If it is desired to give a
general stain after the nuclear dye, carmine stained preparations can be
stained half a minute or more with picric-alcohol (§ 315) and the hem-
atoxylin stained specimens with eosin (§ 305). It usually takes only
10 to 20 seconds for this. Then wash away the stain with 67% alcohol.

§$ 281. Dehydration of the Stained Sections.—Place the slide
with the stained sections in a jar of 95% alcohol for a few minutes or
wave it around for a minute or two and the preparation will be ready
for clearing and mounting.

§ 282. Clearing the Sections.—Drain off the alcohol and add a
drop or two of clearer (Carbol-turpentine (§ 303). If any whiteness
or cloudiness remains the sections were not sufficiently dehydrated.
Complete the dehydration by washing away the clearer with 95’% alco-
hol and soaking the slide a few minutes in a vessel of the same.

§ 283. Mounting in Balsam.—For this the clearer is drained away
by standing the slide nearly vertically on some blotting paper or by
putting it in the funnel of the waste bowl. ‘The balsam is then placed
on the sections and the cover-glass added or the balsam is spread on
the cover-glass.

For labeling and cataloging and storing the preparations see § 291-

295.

168 PARAFFIN SECTIONING. [CH. VII.

ORDER OF PROCEDURE IN MAKING MICROSCOPICAL PREPARATIONS BY
THE PARAFFIN METHOD.

S$ 284. It will be seen from this table and from sections 268 to 283
that it requires from 7 to 10 days to get a microscopical preparation by
the paraffin method if one starts with the fresh tissue. Depending on
the method of fixing and hardening, the time may be much greater.
Unless much time is lost in waiting one must plan ahead in histological
work.

1. Fixing and hardening the tissue or! 11. Washing away the superfluous stain,

organ (% 269), 4 days or niore. (@ 278-279).
2. Dehydrating the object to be cut in ' 12. Staining with a general dye (7 280),
95% or stronger alcohol (% 270), 5. 10 seconds to Io minutes.

to 24 hours. |

3. Saturating the tissue with chloroform |
(2 271), 4 to 24 hours.
4. Infiltrating the tissue with chloroform
paraffin (¢% 272), 3 to 10 days.
5. Imbedding in pure paraffin (2 273),
Io minutes.
6. Cutting the sections (% 274), Io min-
utes. | 16. Mounting in balsam (3 283), 2 to 5
7. Fastening the sections to a slide. minutes,
(@ 275), 5 minutes. | 17. Sealing the cover-glass (% 238), 2
8. Removing the paraffin (% 276), 10° qUteEs,
minutes to 24 hours. i
9g. Washing in 95% alcohol to remove
the benzin (% 277), 2 minutes.
10. Staining with an alcoholic or aque- | 19- Cataloging the preparation (% 294),
ous dye (¢ 278-279), 2 minutes to 24 5 to ro minutes.
hours.

13. Washing the sections with water or
alcohol (% 280), 3-5 minutes.
'14. Dehydrating the stained sections in
95% alcohol (% 281), 5 minutes to
24 hours.
15. Clearing the sections (7 282), 5 min-
utes to 2. hours. .

18. Labeling the preparation (2 291), 2
minutes.

SERIAL SECTIONS.

S$ 285. In histological studies it is frequently of the greatest advan-
tage to have the sections in serial order, then an obscure feature in one
section is frequently made clear by the following or preceding section.
While serial sections may be very desirable in histological studies, they
are absolutely necessary for the solution of morphological problems
presented in complex organs like the brain, iu embryos and in minute
animals where gross dissection is impossible.

$ 286. Arrangement of Tissues for Sections in Histology.—
They should be so arranged that the exact relations of each part to the
organ can be readily determined. For example, an organ like the in-
testine, a muscle or a nerve, should be so arranged that exact transec-

CH. VIL) SERIAL SECTIONS. 169

tions 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.

With cylindrical objects, especially botanical specimens, one may cut
tangential sections, 7. e., sections at right angles to a radius, or parallel
with the radii (radial sections), or transections, 7. ¢., sections across
the long axis.

§$ 287. Arrangement of Serial Sections.—The numerical order
may be very conveniently like the words on a printed page, from the
upper left hand corner and extending from left to right, top to bottom
CPig, 135}

The position of the various aspects of the sections should be in gen-
eral such that when they are under the compound microscope the rights
and lefts will correspond with those of the observer. This may be ac-
complished as follows for sections made in the three cardinal sectional
planes, 7vansections, Frontal Sections, Sagittal Sections :

(A) Transections, 7. e., sections across the long axis of the embryo
or animal dividing it into equal or unequal cephalic and caudal parts.

(a) In accordance with the generally approved method of numbering
serial parts in anatomy, the most cephalic section should be first (No.
1 of Fig. 135).

(b) The caudal aspect of the section should face upward toward the
cover-glass, the cephalic aspect being next the slide.

(c) The ventral aspect should face toward the upper edge of the
slide (Fig. 135).

This arrangement may be easily accomplished in transections in two
ways: (1) The embryo or animal is imbedded in such a way that the
sectioning shall begin at the cephalic end. In this case the first section
is placed in the upper left hand corner of the slide (No. 1 of Fig. 135),
but it must be turned over so that the caudal aspect shall face up.
The ventral aspect must be made to look toward the upper edge of the
slide, then under the compound microscope the dorsal side will appear
toward the upper edge of the slide and the right and left correspond
with the observer.

(2) The embryo or animal is imbedded so that the sectioning begins
at the caudal end, then the sections are not turned over, as they are al-
ready caudal face up, but they must be put on the slide in reverse order,
z. é., the first section made is put in the lower right hand corner (No.
10 of Fig. 135). In this way the most cephalic section will be number
one as before. As in the previous case the ventral side of the section
should be toward the upper edge of the slide (Fig. 135).

170 SERIAL SECTIONS. (CH. VII.

(B) Frontal sections, 7. e., sections lengthwise of the embryo or ani-
mal and from right to left (dextral and sinistral), so that it is divided
into equal or unequal dorsal and ventral parts.

The embryo is so imbedded and arranged in the microtome that the
dorsal part is cut first. The first section is then placed in the upper
left hand corner (No. 1, Fig. 135) dorsal side up, and the cephalic end
toward the lower edge of the slide. The microscopic image will then
appear with right and left as in the observer.

(C) Sagitlal sections, 7. c., sections lengthwise of the embryo or ani-
mal, and from the ventral to the dorsal side, thus dividing it into equal
or unequal right and left parts. For these the left side of the embryo is
placed up so that it is cut first. The first section is placed in the upper
left hand corner of the slide, the left aspect facing away from the slide
and the head to the right end, the ventral side toward the upper edge
of the slide. Under the microscope the dorsal side will then appear
toward the upper edge of the slide and the head to the left.

§ 288. For serial sections with collodion imbedded objects it is a
great advantage 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 im-
bedded that the cardinal aspects, dextral and sinistral, dorsal and ven-
tral, cephalic and caudal, shall be known with certainty.

UPPER EDGE OF SLIDE.

Series No. 75. Cover .15 mm.
4 u Zi Slide No. 1.
. 3 4 5 Transections of a Diemyctylus
fy Embryo.
ip Sections 1-10.
g Total thickness of Sections, 1 mim.
q 6 7 8 9 Io
May 20, 1892.
Fic. 135.—Labeled Slide of Serial Sections. .

§ 289. Thickness of Cover.Glass and of Serial Sections.—It is
a great advantage to use very thin cover-glasses (44° to 48, mm.) for
serial sections, then the cover will not prevent the use of high powers.
When the ordinary slides (25 x 76 mm., 1 X 3 inch) are used cover-
glasses 23 X 55 mm, may be advantageously employed.

The combined thickness of the sections on a slide is easily determined
by noting carefully the position of the microtome screw at the first and
last sections, and measuring the elevation. Then if the sections are

CH. VII.) LABELING AND CATALOGING. 171

uniform the thickness of each may be easily found. The average
thickness may be easily determined in any case.

§ 290. Labeling Serial SeCtions.—The label of aslide on which se-
rial sections are mounted should contain at least the following : (1) The
number of the series ; (2) The number of the slide in the series (if the
series required more than one slide) ; (3) Kind of sections (transections,
etc.) and the name of the object from which derived ; (4) The number
of the first and last section on the slide ; (5) The total thickness of all
the sections on the slide ; (6) The date of the series.

LABELING, CATALOGING AND STORING MICROSCOPICAL PREPARATIONS.

§ 291. Every person possessing a microscopical preparation is inter-
ested in its proper management ; but it is especially to the teacher and
the investigator that the labeling, cataloging and storing of microscop-
ical preparations are of importance. ‘‘ To the investigator, his speci-
mens are the most precious of his possessions, for they contain the facts

‘which he tries to interpret, and they remain the same while his knowl-
edge, 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 wri-
ter that every preparation should possess two things: viz., a label anda
catalog or history. This catalog should indicate all that is known of a
specimen at the time of its preparation, and all of the processes by
which it is treated. It is only by the possession of such a complete
knowledge of the entire history of a preparation that one is able to
judge with certainty of the comparative excellence of methods, and
thus to discard or 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.’’

§ 292. Labeling Ordinary Microscopical Preparations.—The
label should possess at least the following information (see § 290 for
serial sections) :

EXAMPLE.
(1) The number of the preparations, the No. 475. = 15.
thickness of the cover-glass and ecs. 8 uu

of the sections under it.

(2) The name and source of the prepara-
tion.

(3) The date of the specimen (2 of
catalog.) October 15, 1894.

Striated Muscle; transection of the
Sartorius of the Cat.

172 LABELING AND CATALOGING, (CH. VII.

§ 293. Cataloging Preparations.—It is believed from personal ex-
perience, and from the experience of others, that each preparation
(each slide or each series) should be accompanied by a catalog contain-
ing at least the information suggested in the following formula: This
formula is very flexible, so that the order may be changed, and num-
bers not applicable in a given case may be omitted. With many ob-
jects, especially embryos and small animals, the time of fixing and
hardening may be months or even years earlier than the time of im-
bedding. So, too, an object may be sectioned a long time after it was
imbedded, and finally the sections may not be mounted at the time they
are cut. It would be well in such cases to give the date of fixing under
2, and under 5, 6 and 8, the dates at which the operations were per-
formed 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.

@ 294. General Formula for Catalog- _ A Catalog Card Written Accord-ng to
ing Microscopical Preparations : this Formula :

1. The general name and_ source. Mosewlat Fillets: Cah

: : : ; C. 15.
Thickness of cover glass and of sections. Pihevsiso ta ao pe thick:

2. The number of the preparation and 2. No. 475. (Drr. IX) Oct. 1, 1891. S.
the date of obtaining and fixing the H. G., Preparator.

specimen ; the name of the Peeperarer | 3. Tendinous and intra-muscular ter-

minations of striated muscular fibers
tion and the common and scientific | frown the Sertoriue ol Ue cat (eels dos

name of the object from which it is de- | mestica.) :
rived. Purpose of the preparation. | 4. Cat eight months old, healthy and
-well nourished. Fasting and quiet for
4. The age and condition of the object | 12 hours.
from which the preparation is derived.) 5, Muscle pinned on cork with vas-
Condition of rest or activity ; fasting elined pins and placed in 20 per cent.
or full fed at the time of death. nitric acid immediately after death by
chloroform. Left 36 honrs in the acid ;
temperature 20°C. In alum water (%
sat. aq. sol.) 1 day.
6. Fibers separated on the slide with
6. The mechanical treatment, —im-/ needles, Oct. 3.
bedded, sectioned, dissected with nee- 7. Stained 5 minutes with Delafield’s
dles, etc. Date at which done. hematoxvlin.

7. The staining agent or agents and S Dehydrated with og 7. alcohol §
the time required for staining. minutes, cleared 5 minutes with carbol-
2 turpentine, mounted in xylene balsam ;
8. Dehydrating and clearing agent, Sealed with Bieta,

mounting medium, cement used for 9: Use 18 mm. for the general appear-
sealing. ance of the fibers, then 2 or 3 mim. ob-
jective for the details of structure. Try

_9- The objectives and other accesso- | the micro-polariscope (% 209).
ries (micro-spectroscope, polarizer, etc.) 10, The nuclei or muscle corpuscles
for studying the preparation, are very large and numerous; many of
: ; the intra-inuscular ends are branched.
io. Remarks, including references to) See S. P. Gage, Proc. Amer. Micr. Sci.,

original papers, or to good figures and_ 1890, p. 132; Ref. Hand-book Med., Sci.,
descriptions in books, Vol. V., p. 59.

3. The special name of the prepara-

5. The chemical treatment, —the
method of fixing, hardening, dissociating |
etc., and the time required.

CH. ITT.) LABELING AND CATALOGING. 173

@ 295. 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 very well. As pur-
chased, 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.

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.

Fic. 136. Writing diamond for writing numbers and labels on glass slides, cut-
ting cover-glasses, etc. (Queen & Co.). :

The author has found stiff cards, 1213x713 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
addel 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 Alling and by Ward (Jour. Roy. Micr. Soc., 1887, pp. 173, 348; Amer.
Mouthly 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 very strong belief,
practically amounting to acertainty, 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, 189), pp. 271-289 ; Hodge, on nerve cells in rest and
fatigue, Jour. Morph., vol. VII. (1892), pp. 95-168; Jour. Physiol., vol. XVIL.,
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; Sci-
ence, vol. II., Aug. 23, 1895, pp. 209-218).

CABINET FOR MICROSCOPICAL PREPARATIONS.

2 296. While it is desirable that microscopical preparations should be properly
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 couvenient 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 :

(1). The cabinet should allow the slides to lie flat, and exclude dust and light.

(2). Each slide or pair of slidesshould 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. 140). It is also desira-
ble 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

174 LABELING AND CATALOGING. (CH. VII.

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.

A B

No. 96 1850
Nerve fibers |
BAe a

Fig. 140.—A—.Part of a cabinet drawer seen
Jrom above. Incompartment No. 96 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. The sealing
cement ts 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.
(4 229, 234).

B.—This represents a section of the same
part of the drawer. (a) Slide resting asin
A. No. 96 The preparation is seen to be
above a groove in the floor of the compart-
ment. (6) One end of the slide is seen to be
uplifted by depressing 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. 141).

Fig. 141.—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.
Lt 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).

CH. VIL) PREPARATION OF REAGENTS. 175

REAGENTS FOR FIXING, MOUNTING, ETC.

@ 297. Albumen Fixative (Mayer’s).—This consists of equal parts of well-beaten
white of egg and glycerin. To each 5o0cc. of this r gram of salicylate of soda
is added to prevent putrefactive changes. Probably a small amount of formal-
dehyde, say I cc. of the 40%, to 50 or 100 ce. of the fixative would suffice ; if too
strong the albumen would be precipitated. For method of use see ¢ 275.

% 298. Alcohol (Ethylic) —Ethyl alcohol is mostly used for histological pur-
poses. (A) Absolute alcohol (i. e. alcohol of 99-100%) is recommended for many
purposes, but if plenty of 95% alcohol is used it answers every purpose in histology.

(B) 82% alcohol made by mixing 5 parts of 95% alcohol with 1 part of water.

(C) 67% alcohol made by mixing 2 parts of 95% alcohol with 1 part of water.

2299. Alum Solution.—For muscle dissociated in nitric acid (% 249) a saturated
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 vessel 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 cooling a part
will crystallize out, leaving a cold saturated solution). The saturated solution may
be used but, if a half saturated solution is employed, it will answer all the pur-
poses. Fora half saturated solution take 100 cc. of water and 100 ce. of saturated
alum water and mix the two.

2 300. Balsam, Canada Balsam, Balsam of Fir; Xylene Balsam.—This is one
of the oldest and most satisfactory of the resinous media used for mounting micro-
scopical preparations. Sometimes it is used in the natural state, but experience
has shown that it is better to get rid of the natural volatile constituents. A con-
siderable quantity, half a liter or more, of the natural balsam is poured into shal-
low plates in layers about 1 or 2 centimeters thick, then the plates are put in a
warm, dry place, on the back of astove or on a steam radiator, and allowed to re-
main until the balsam may be powdered when it iscold. This requires a long
time, the tine depending on the temperature and the thickness of the layer of
balsam. By heating the natural balsam ina tin or agate vessel over a Bunsen
burner or an alcobol lamp the time may be greatly abbreviated. The heat should
not be sufficient to boil the balsam, however.

When the volatile products have evaporated, the balsam is broken into small
pieces or powdered in a mortar and mixed with about an equal volume of xylene,
turpentine or chloroform. It will dissolve in these, and then should be filtered
through absorbent cotton or a filter paper, using a paper funnel.* The balsam is
toothin in this condition for mounting, but so made for the sake of filtering it.
After it is filtered it is evaporated slowly in an open dish or a wide-mouth bottle or
jar till it is of asyrupy consistency at the ordinary temperature. It is then poured
into a bottle with a glass cap like a spirit lamp. For use it is put into a small

spirit lamp (Fig. 137).

*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 very easily made by rolling a sheet of thick writing
paper in the form of acone and cementing the paper where it overlaps, or wind-
ing a string several times around the lower part. Such a funnelis best used in one
of the rings for holding funnels.

176 PREPARATION OF REAGENTS. [CH VIT.

The xylene is much the best substance to use for thinning the balsam. Such
xylene balsam, asit is then called, may be used for mounting any object suitable
for balsam mounting. The dehydration must be very perfect, however, as xylene
is wholly immiscible with water.

Natural balsam is liable to be slightly acid. This is of advantage for mounting
sections stained with carmine or injected with carmin gelatin or Berlin blue gela-
tin. For hematoxylin preparations and for fuchsin preparations the acid will cause
the color to fade. The balsam may be neutralized by mixing some carbonate of
soda with the thinned solution before it is thickened. In a few days all the soda
will settle and the clear balsam above will be neutral and may be poured off and
thickened. If one mounts carmine or Berlin blue preparations in the neutral bal-
sam the blue will fade and the carmine diffuse.

2 301. Chloroform Paraffin.—This is made by mixing the 4 parts of the paraffin
used for imbedding (% 314) with 1 part of chloroform. This gives a paraffin which
melts at a lower temperature than the pure paraffin. Ifit is kept warm the chloro-
form evaporates in 3 to 6 days, leaving pure paraffin.

2 302. Clarifler, Castor-Xylene Clarifier.—This is composed of castor oil I part
and xylene* 3 parts.

2 303. Clearing Mixture (% 266, 282).—-(A). One of the most satisfactory and
generally applicable clearers is carbol turpentine, made by mixing carbolic acid
crystals (.dcidum carbolicum, A. phenicum crystalizatum) 40 cc. with rectified
oil of turpentine (Olezm terebinthinae rectificatum) 63 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 asthe preceding.

% 304. Collodion.—This is a solution of soluble cottont+ or other form of pyroxy-
lin in equal parts of sulphuric ether and 95% alcohol. Three solutions are used :

*The hydrocarbon xylene (C,;H,,) 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 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 C,, H,, (NO,),O,, and cellulose pentanitrate, C,,
H,, (NO,); O,. 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. Itis 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, VII.) PREPARATION OF REAGENTS. 177

(A). 6% or thick collodion. It is made by mixing 50 cc. of sulphuric ether and
50 ce, of 95% alcohol and adding 6 grams of soluble cotton. « If this is shaken re-
peatedly the solution will be complete in a day or two.

(B). 14% or thin collodion. To prepare this 1% grams of soluble cotton are
added to 100 ce. of ether-alcoliol (% 306).

(C). 3% collodion or cementing collodion. To prepare it 3/ths of a gram of
soluble cotton is added to 109 cc. of ether-alcohol.

As both ether and alcohol are very volatile it is necessary to keep the bottles
containing them weil corked.

2 305. 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 (% 135). 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 1-1oth 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 tobe mounted in glycerin or glycerin jelly, the excess
of eosin should be washed away with distilled water.

7306. Ether, Ether-Alcohol.—Sulphuric ether is meant when ether is men-
tioned in this book. For the ether-alcohol mentioned in ¢ 254, 304, etc., a mixture
of equal volumes of sulphuric ether and 95% alcohol is meant.

4 307. Farrant’s Solution.— Take 25 grams of clean, dry, gum arabic; 25 cc. of
a saturated aqueous solution of arsenious acid ; 25 cc. of glycerin. The gum ara-
hic 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. Itis much easier to avoid
than to get rid of them in mounting.

2 308. Formaldehyde Dissociator.—This is composed of 5 cc. of a 40% solution
of formaldehyde in 995 cc. of water, to which 6 grams of common table salt
(sodium chlorid) have been added. That is, it is a ~;% solution of formalde-
hyde in normal salt solution (2 313). 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 3 245. (Gage
Micr. Bulletin and Sci. News, vol. XII. (1895), pp. 4-5).

7309. Glycerin.—(A). One should procure pure glycerin for a mounting me-
dium. 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, 1 cc.

By extreme care in mounting and by occasionally adding a fresh coat to the
sealing of the cover-glass, glycerin preparations lasta long time. They are liable
to be very disappointing, however. In mounting in glycerin care should be taken
to avoid air-bubbles, as they are difficult to get ridof. A specimen need not be
discarded, however, unless the air-bubbles are large and numerous.

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

I2

178 PREPARATION OF REAGENTS. (CH. VI/T.

usually takes about half an hour. When the gelatin is softened, as may be readily
determined by taking alittle 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 hath and it will iuelt in the water it has
absorbed. Add to the melted gelatin about 5 cc. of egg albumen, white of egg;
stir it in 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 clar-
ified 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 (7. é., 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 temperature
(18°-20° 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 tessened
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.

2 310. Hematoxylin.—Hematoxylin is one of the most useful stains employed in
histology. A very excellent solution for ordinary section staining may be made as
follows: Distilled water 200 cc., and potash alum 7} grams, are boiled together for
5 minutes, in an agate-ware or glass vessel, and sufficient boiled water added to
bring the volume back to 200cc. After the mixture is cool, 4 grams of chloral hy-
drate, and ths gram of hematoxylin crystals, previously dissolved in 20 cc. of 95%
alcohol, are added. The boiling seems to destroy any fungi present in the alum
or water, and the chloral prevents the development of any that may get in after-
ward, 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
deep purple. The deepening of the color is more rapid if the bottle is left uncorked
in the light and is shaken occasionally.

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 concen-
trated, 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 liv-
ing 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 applicable
to all hematoxylin solutions, viz. : after staining, the surplus stain must be very
thoroughly washed away with distilled 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 solution three to five minutes and filter
through paper or absorbent cotton. The addition of one or two per cent. of chlo-
ral after the boiling is also advantageous. This stain has not been tried for dyeing
in bulk. Other substances than chloral were tried, but not with so good success.
(S. H. Gage, Proc. Amer. Micr. Soc., Vol. XIV, 1892, pp. 125-127).

4 311. Liquid Gelatin.—Gelatin or clear glue, 75 to roo grams. Commercial

CH. VIL.) PREPARATION OF REAGENTS. 179

acetic acid (No. 8) 10)ce., water 1o0cc., 95% alcohol toocc., glycerin 15 to 30 ce.
Crush the glue aud 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 so-
lution 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 sup-
plies, so that it is unnecessary to prepare them one’s self, except for special pur-
poses.

4312. Nitric Acid Dissociator.—This is prepared by mixing 80 cc. of water with
20 ce. 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 (#299 and 249). (Gage, Proc. Amer. Micr. Soc., Vol. XI, (1889), pp.
34-45).

% 313 Normal Salt Solution or Saline Solution,—Pure water from its differing
density from the natural lymph acts injuriously on the tissues. The addition of a
little table salt, however, preveuts-this deleterious action, or greatly lesseus it,
hence the name of zormal salt solution. It isa 75% solution of table salt (sodium
chlorid) in water ; water 1000 cc., salt 6 grams, or water 100 cc., salt 78; gram.

@ 314. Paraffin.—Paraffiu is of various melting points, hence at the ordinary tem-
perature of a laboratory, that melting at the lowest temperature will be moderately
soft, hence soft paraffin, while that melting at a higher temperature will be hard.
For the best results one has to mix hard and soft paraffins. Usually a mixture of
9 parts hard and 1 part soft paraffin will give good results, and may be called zm-
bedding paraffin. For chloroform paraffin, 4 parts of imbedding paraffin are mixed
with one part of chloroform (% 301, 272).

2 315. 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 isa 4% solution of picric
acid in 50% alcohol). It acts quickly, in from one to three days. (@ 252, 269).
(Proc. Amer. Micr. Soc., Vol. XII (1899), pp. 120-122).

2 316. Shellac Cement.—Shellac cement for sealing preparations and for mak-
ing shallow cells (% 233) 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 oc-
casionally 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 funnel (2 300, note), into an open dish or a wide-mouth bottle. To every
50 cc. of this filtered shellac, 5 cc. of castor oil and 5 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. 134). 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,
7. é., in a week or two in a warm place, or in Jess time if the bottle is frequently.

180 MICRO-CHEMISTRY. (CH. VIT.

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 benzin 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).

ARRANGING AND MOUNTING MINUTE OBJECTS.

2 317. Minute objects like diatoms and 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, liquid gelatin (% 311) thinned with an
equal volume of 50% acetic acid answers well. A very thin coating of this is spread
on the cover with a needle, or in some other way, and allowed todry. The objects
are then placed on the gelatinized side of the.cover and carefully got into position
with a mechanical finger, made by fastening a cat’s whisker in a needle holder.
For most of these objects a simple microscope with stand (Figs. 19, 20, 130, 131)
will be found of great advantage. After the objects are arranged, one breathes
very gently on the cover-glass to soften the 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 (2 232, 240). See Newcomer, Amer. Micr. Soc.’s Proc.,
1885, p. 128; see also E. H. Griffith and H. Ll. 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.

MICRO-CHEMISTRY AND CRYSTALLOGRAPHY—EXPERIMENTS.

2318. The student of science, and especially chemistry, so frequently requires a
knowledge of the appearance of minute crystals to aid in the determination of an
unknown substance, or for his information in studying objects where crystals are
liable to occur, that a few experiments have been introduced to give him a start in
preparing and permanently mounting some of the commion crystals.

It is recommended that the crystals be made in several ways, that is, from alco-
holic solutions, aqueous solutions saturated and dilute, by spontaneous drying and
crystallization and by rapid crystallization by the aid of heat. The modifications
in crystallization under these different methods of treatment are frequently very
striking.

In every case the student is advised to study the appearance of the crystals in
the ‘‘mother liquor.” Asarule, their characteristics are more clearly shown in
the “mother liquor” than under any other conditions.

It is of very great advantage to examine all crystalline forms with polarized light
(@ 209).

@ 319. Determination of the Character of the Solid Sediment in Water. —Take
some of the sediment from a filter or allow a considerable volume of water to stand

CH. VIT.] MICRO-CHEMISTR Y. 181

in a tall glass vessel to deposit its sediment. Take a concentrated drop of this sed-
iment and mount it om a slide under a cover-glass. Study the preparation with the
microscope. Probably there will be an abundance of animal and vegetable life as
well as of solid sediment. Put a drop of dilute sulphuric acid (Acidum sulphuri-
cum, t. é., strong sulphuric acid 1 gram, water 9 grams) at the edge of the cover

Fic. 139. Czapski’s Ocular Iris-diaphragm with cross hairs
Sor examining and accurately determining the axial images 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, No. 30).

and at the opposite edge a small piece of the lens paper (Fig. 128). The acid will
gradually diffuse, and if the solid particles are carbonate of lime, minute bubbles
will be seen to be given off. If they are silica or clay no change will result. Sul-
phuric acid is recommended for this, as the microscope would be far less liable to
injury than as if some acid giving off fumes were used.

§ 320. Herapath’s Method of Determining Minute Quantities of Quinine.—For
a so-called test fluid 12 cc. of glacial acetic acid, 4 cc. of 95% alcohol, and 7 drops
of dilute sulphuric acid (§ 319) are mixed. A drop of the test fluid is put on a slide
and a very minute amount of quinine added. After this is dissolved, add an ex-
tremely minute drop of an alcoholic solution of iodine. ‘‘ The first effect is the
production of the yellow cinnamon-colored compound of iodine and quinine, which
forms as a small circular spot; the alcohol separates in little drops, which, by a
sort of repulsive movement, drive the fluid away ; after a time the acid liquid again
flows over the spot, and the polarizing crystals of sulphate of iodo-quinine are slow-
ly produced in beautiful rosettes. This succeeds best without the application of
heat.” Dr. Herapath used this method to determine the presence of quinine in
the urine of patients under quinine treatment. See Hogg, p. 150; Quarterly Jour.
Micr. Sc., vol. ii, pp. 13-18. For further papers on micro-chemistry by Dr. Hera-
path, see the Royal Society's Catalog of Scientific Papers.

2321. List of Substances for the Study of Crystallography with the Microscope.*
The substances are crystalized on the cover-glass in all cases, and in all cases, ex-
cept where otherwise stated, a saturated aqueous solution of the substances was
first prepared.

i. Ammonium chlorid ; 2. Ammonium copper chlorid ; 3. Barium chlorid ;
4. Cobalt chlorid (beautiful crystals obtained by mixing the saturated aqueous so-
lution with an equal volume of 95% alcohol). Crystallization in a current of dry

* Most of the chemicals here named were suggested to the writer by Prof. L. M.
Dennis, of the Chemical Department.

182 MICRO-CHEMISTRY. (CH. VII.

air some distance above an alcohol or Bunsen flame; mount in xylene balsam
(% 300), or one may fuse the salt with the balsam (% 300); 5. Copper acetate ;
Mount dry (? 231); 6. Copper sulphate. Crystals much more satisfactory when
examined in the ‘mother liquor.’’ 7. Lead nitrate; 8. Mercuric chlorid (corro-
sive sublimate), mount in xylene balsam (? 300, 240). 9. Nickel nitrate, obtain
crystals by heating; mount in xylene balsam (% 240, 300); 10. Potash alum; Ir.
Potassium chlorate; 12. Potassium dichromate. Compare specimen crystallized
by heat and spontaneously ; mount dry or in xylene balsam (% 300). 13. Po-
tassium iodide. Dilute with one or two volumes of water, and crystallize by heat.
14. Potassium nitrate; 15. Potassium oxalate; 16. Potassium sulphate; 17. Sali-
cin. Fuse the dry salicin on the cover-glass, mount dry, or preferably fuse in
balsam (2 300). 18. Salicylic acid. Make a 10% solution in 95% alcohol; let it
crystallize spontaneously in the air; mount dry (% 231). 19. Sodium chlorid
(common salt). Mix sat. aq. sol. with one or two volumes of water, and heat ;
mount dry or in balsam. 20. Sulphonal (7 218).

@ 322. For directions and hints in micro-chemical work and crystallography,
consult the various volumes of the Journal of the Roy. Micr. Soc. ; Zeitschrift fiir
physiologische Chemie, and other chemical journals; Wormly ; Klément & Re-
nard; Carpenter-Dallinger; Hogg; Behrens, Kossel und Schiefferdecker ; Frey ;
Dana, and other works on mineralogy ; Davis.

CHAPTER. VIM.

PHOTO-MICROGRAPHY AND PHOTOGRAPHY WITH A
VERTICAL CAMERA.*

APPARATUS AND MATERIAL FOR THIS CHAPTER.

Compound microscope with achromatic condenser; Achromatic and apochro-
matic objec ives; Oculars, ordinary and projection ; Lamp and bull’s-eye condenser
of some form; Photo-micrographic camera, and an ordinary copying camera; Fo-
cusing glass; dry plates, developer, fixer, trays, dark-room, and the other things
needed for photograply, like printing frames, etc., etc. ; Photographiic- objectives,
one for large objects and one for small objects to be magnified from two to fifteen
diameters. ;

2323. 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. Indeed, the very first pictures made on white paper and white
leather, sensitized by silver nitrate, were made by the aid of a solar microscope
(1$02). The pictures were made by Wedgwood 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.” ¢

* Considerable confusion exists as to the proper nomenclature of photography
with the microscope. In Germany and France the term micro-photography is very
common, while in English photo-micrography and micro-photography niean difer-
ent things. Thus: A pholo-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 Sutton’s Photographic Notes, Vol. III, 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 micro-scopic photograph of a macro-
scopic object.”’ See also Afedical News, Jan. 27, 1894, p. 108.

{In a most interesting paper by A. C. Mercer on ‘‘ The Indebtedness of Photog-
raphy to Microscopy, Photographic Times Almanac, 1887, it is shown that: ‘To

184 PHOTO-MICROGRAPHY. (CH. VIII.

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 photography when the time of exposure re-
quired 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.

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, mi-
croscopic objectives have been naturally constructed to give the clearest image to
the eye, that is the visual image as it issometimes called, is for microscopic obser-
vation, of prime importance. The actinic or photographic image, on the other
hand, is of prime importance for photography. Then for the majority of micro-
scopic objects transmitted light (% 50) must be used, not reflected light as in ordin-
ary vision. Finally, from the shortness of focus and the smallness of the lenses,
the proper illumination of the object is accomplished with some difficulty, and the
fact of the lack of sharpness over the whole field with any but the lower powers,
have all combined to make photo-micrography less successful than ordinary
u1acro-photography. So tireless, however, have been the efforts of those who be-
lieved in the ultimate success of photo-micrography, that now the ordinary achro-
matic objectives and ortho-chromatic or isxchromatic plates give very 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 athromatic and apochromatic condensers and
by the electric and other powerful lights now available. There still remains the
the difficulty of transmitted light and of so preparing the abject that structural de-
tails shall stand out with sufficient clearness to make a picture which shall
approach in definiteness the drawing of a skilled artist.

The writer would advise all who wish to undertake photo-micrography seriously,
to study samiples of the best work that has been produced. Among those who
showed the possibilities of photo-micrographs was Col. Woodward of the U. S.
Army Medical Museum. The photo-micrograpls 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 hy Woodward, and most of
them are vastly inferior. It is gratifying to state, however, that at the present
time (1895) many original papers are partly or wholly illustrated by photo-micro-
graphs, and no country bas 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.

briefly recapitulate, photography is apparently somewhat indebted to microscopy
for the first fleeting pictures of Wedgwood 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 illustra-
tion [Donné & Foucault, 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.

CH. ITI] PHOTO-MICROGRA PHY. 185

As the difficulties of photo-nicrography are so much greater than of ordinary
photography, the advice is almost universal that no one shonld try to learn photo-
graphy and photo-micrography at the same time, but that one should learn the
processes of photograpliy by making portraits, landscapes, copying drawings, etc.,
and then when the principles are learned one can undertake the more difficult
problem 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 isin 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.

2 324. Camera.—For the best results with the least expenditure of time one of
the cameras especially designed for photo-micrograpby is desirable but is not by
any means necessary 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.

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 evel
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
fraine. 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 how
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.

[CH. VIII.

PHOTO-MICROGRAPA ¥.

‘HYOM WOA AAONVAAY

'VYANVO OIHAVYDOYUOIN-OLOHd '0 "dH ‘A GAAOYdNI S:ADTSNTIVM

8FIL ‘ON

Fic. 143.

CH. VIII.) PHOTO-MICROGRAPHY. 187

Fic. 143. Walmsley's Improved Photo-Micrographic Camera. In this figure is
shown an evcellent fori of photo-micrographic camera for use with a horizontal
microscope. It has the advantage of the possibility of a very long bellows or a
shorter one as the need of the specal case demands. It is arranged for photo-mt-
crographic work with the microscope or a wide angled objective or for copying and
slightly enlarging or diminishing, drawing, etc., with an ordinary photographic
objective. Lt is also arranged for making lantern slides. 1 very simple arrange-
ment has been adopted for focusing when the bellows is pulled out so far that one
cannot reach the fine adjustment, and tt works with great smoothness. ‘(A groove
7s turned in the periphery of the fine adjustment screw, around which a small cord
ts passed, and carried through a succession of screw-eyes on either side of the base-
board to the rear, where a couple of small leaden weights are attached to its ends,
thus keeping the cord taut. A very slight pull on either side, whilst the eye ts
fixed upon the image on the screen, suffices to adjust the focus with the utmost
exactness. Lf preferred, a rod running the entire length of the camera-bed, and
terminating at the rear end with a milled head, whilst on the other is a grooved
pulley, carrying a cord or belt which also passes around the groove in the milled
head of the fine adjustment, may be substituted for the cord and weights. This
arrangement [shown in the figure] is more costly, and probably no better in actual
service than that with the cord and weights.’ (From Mr. Walmsley. )

2325. Work Room.—It is almost self-evident that the camera must be in some
place free fromm vibration. Frequently a basement room where the camera table
miay rest directly on the ground or on a pier is an excellent situation. Such a sit-
uation is almost necessary for the best work with bigh 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.

2326. Arrangement and Position of the Camera and the Microscope.—For the
greater number of photo-micrographs, a horizontal camera and microscope are to
be preferred as one may then vary the length of the bellows at will and still pre-
serve steadiness (Fig. 143). For some specimens, however, it is necessary to keep
the microscope in a vertical position, hence to photograph, the camera must also
be vertical. Very excellent arrangements were perfected long ago, especially by
the French. (See Moitessier).

Vertical photo-micrographic cameras are now very 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.
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 bel-
lows (see below). (Fig. 144). It must not be forgotten, however, that penetration
varies inversely as the sguare of the power, and ouly inversely as the numerical
aperture (7 29), 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
shorter bellows. (See Carpenter-Dallinger, pp. 318-319).

2 327. Microscope.—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. In some microscopes

188 PHOTO-MICROGRAPH Y. (CH. VIIZ,

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.

i

I

ui nN : I aS :

FIG. Idd.

Fic. 144. Leitz’ Pholo- Micrographic Camera Sor Vertical Microscope (from
Leitz’ American House, New York). The camera may be raised or lowered to
adapt tt to various stands. The microscope, as shown, rests on the base of the cam-
era support, thus aiding steadiness, and the simultaneous vibration of the entire
apparatus, if any should occur.

With this instrument the Jocusing can be done with the hand on the fine adjust-
ment, as in ordinary microscopical study,

CH. Vil. PHOTO-MICROGRAPHY. 189

@ 328. 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, one of 35 and one of 70 millimeters equivalent focus are used without
any ocular (Fig. 149). 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 be has good achromatic ob-
jectives. Froim a somewhat extended series of experiments with the objectives of
many makers the good modern achromatic objectives 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 projection 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 ocu-
lars in photography, although excellent 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 specimeus. *

Fic. 145. Projection Oculars with section re-
moved to show the construction. Below are
shown the upper end with graduated circle to
indicate the autount of rotation found necessary
to focus the diaphragm on the screen. No. 2,
No. g. The numbers indicate the amount the
ocular magnifies the image formed by the ob-
jective as with the compensation oculars.
(Zeiss Catalog, No. 30).

7329. Difference of Visual and Actinic Foci.
Formerly there was much difficulty exper-
ienced in photo-micrographing on account of
the difference in actinic and visual foci. Mod-
ern objectives are less faulty in this respect
and the apochromiatics 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

*The comparative study both with projection oculars, and without an ocular were
made with the achromatic objectives 25 mm. (1 inch), 18 mm. (3¢ inch), 5 mm. (+
to }inch) and 2mm. ;¥, (inch) homogeneous immersion of the Bausch & Lomb
Optical Co.; Gundlach Optical Co.; Leitz; Reichert; Winkel and Zeiss. Good
results were obtained with all of these objectives both with and without projection
oculars. The objectives of Spencer and Smith, especially constructed for photo-
micrography, are highly spoken of by Piffard (Jour. Roy. Micr. Soc., 1892-1893).
The writer has not had the opportunity of comparing these with those mentioned.
Piffard spoke highly of the work of Wales also. From the known excellence of
the work of these opticians one would expect good results.

190 PHOTO MICROGRAPH Y. (CH. VIII.

arisen from differences in the foci. This is especially true when mono-chro-
matic 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 only mere chance. If sharp pictures cannot be
obtained with an objective when lamp light and orthochromatic plates are used
the fault may not rest with the objective but with the plate holder and focusing

Fic. 146. Walmsley’s Autograph Proto-Micrographic Camera in a vertical po-
sition. Compare Fig. 147 (from the Proc. Amer. Micr. Soc. Vol. XVII, 1895).

CA. VITTL.) PHOTO-MICROGRAPH Y, Ig!

screen. They should be carefully tested to see if there is coincidence in position
of the focusing screen and the sensitive filin as described in (4 324.)

¢ 330. 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 mir-
ror. Ifa bull's eye is used it should be as nearly achromatic as possible. The
engraving glass shown in Fig. 155 answers very well for large objects. For smaller
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 achr-matic condenser,
but preferably it should be an apochromatic condenser. Whatever the form of the
condenser it should possess diap!iragms so that the aperture of the condenser may
be varied depending upon the aperture of the objective. For along 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 center-
ing.

@ 331. Light Filters or Color Screens.—These are solutions or suitably stained
collodion or gelatin films placed between the source of illumination and the ob-
ject. It does not make much differeuce where the color screen is placed provided
no light reaches the object which has not passed through the filter. The purpose
of the color screen or filter is to take out the excessive number of blue and violet
rays so that the more slowly acting red, yellow and green may have time to pro-
duce the appropriate chemical changes in the seusitive plate. This action of the
longer waves (see under spectroscope ? 179-198), is greatly aided by the isochro-
matic or orthochromatic plates which are especially treated so that they will be
sensitive to the longer waves as well as to the shorter, blue and violet waves. This
is why itis so necessary in manipulating the plates to avoid exposing them to
any light whatsoever in putting them into the plate holder, developing, etc.

The color screen used by Dr. Leaming in preparing the negatives for the plates
in Wilson’s and Starr’s atlases was made by staining a lantern slide plate from
which all the silver salts had been removed, with an alcoholic solution of tropaeo-
Zin and then after drying, Canada balsam anda coverglass were applied. Others
have recommended collodion stained with aurantia. The purpose of these is to
filter out the greater number of the blue and violet rays and then increase the time
of exposure from 2 to 5 times, depending on the thickness of the color screen.
Color screens and color cells are furnished by various makers. None of them
answers for all preparations and for some preparations they are unnecessary.

2 332. Objects Suitable for Photo-micrographs —While almost any large object
may be photographed well with the ordinary camera and photographic objective,
only a small part of the objects mounted for microscopic study can be photo-mi-
crographed satisfactorily. Many objects that give beautiful and satisfactory images
when looking into the microscope and constantly focusing with the fine adjust-
ment, appear almost without detail on the screen of the photo micrographic cam-
era and in the photo-micrograph.

Lf 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-

192 - PHOTO-MICROGRAPHY. (CH. VII.

niteness may be readily obtained. Stained microbes also furnish favorable objects
when mounted as cover-glass preparations.

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 vezerable 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. é

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 an alco-
holic solution of one of the anilins, blue or green, then in studying the prepara-

tion one can see it even where covered by the ring

Fic. 147. Walmsley's Autograph Photo micrographic Camera in a horizontal
position, «1 microscope lamp and bull’s-eye condenser are in position. Compare
Fig. 148 in (Proc. Amer. Micr. Soc., Vol, X VII, 1895).

4 333. 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

CHO VITE| PHOTO-MICROGRA PHY.

many workers find the only opportunity for work. Following the sunlight, the
electric light is the most intense of the available lights. Then comes magnesi-
um, 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 (114 to 3 minutes). This light is also cheapest
and most available.

Fic. 148. Vertical Photo-Micrographic
Camera furnished by the Bausch & Lomb
Optical Company. (From the 15th edition
(1896) of their Catalog.)

EXPERIMENTS IN PHOTO-MICROGRAPHY.

S$ 334. The following experiments are
introduced to show practically just how
one would proceed to make photo-micro-
graphs with various powers, and be
reasonably certain of fair success. If
one consults prints or the published
figures made directly from photo-micro-
graphs it will be seen that, excepting
the bacteria, the magnification ranges
mostly beween 1o and 150 diameters.
The technical difficulties in making good
photo-micrographs of animal tissues at
a greater magnification are so great that,
while they may be used as the basis for
figures, they are, in most cases not suit-
able for direct reproduction.

$ 335. Photo-Micrographs at a Magnification of 5 to 20 Di-
ameters.—In the study of embryology and the morphology of small
animals or of individual organs like the brain, it is frequently desirable
to make pictures of the whole object in its natural setting. These ob-
jects and their surroundings are frequently from one to two centimeters
in diameter, that is of a size too great to be satisfactorily photographed
with microscopic objectives. In common with other observers the
writer has found the short focus, wide angled, photographic objectives
to give excellent results. It is necessary to have considerable length
(1% to 2 meters) of bellows for this, if a magnification of 10 to 15
diameters is desired.

Put the objective in position in the front of the camera and place the
object on some kind of support near the objective. A T-shaped board

13

194 PHOTO-MICROGRAPH Y. (CH. VIII.

with a hole of proper size is good. Then with a glass chimney on the
lamp, place the bull’s eye between the lamp and object (Fig. 157). Put
a piece of white paper over the object and mutually arrange lamp and
bull’s eye till a sharp image of the flame may be seen on the paper
covering the object (Fig. 157). Remove the paper from the object
and proceed to focus. ‘This is accomplished by sliding the whole
camera toward or away from the object, see also § 339. One must also
shorten or lengthen the bellows to get the picture of the proper size.

In case the whole specimen is not illuminated the lamp must be
turned so that the broad side of the flame is toward the object. The
mutual arrangement of lamp and bull’s eye must also be such that the
brightest part of the flame illuminates the object. If the bull’s eye
is moved a little toward the lamp, the flame will be sufficiently broad-
ened. It must be remembered, however, that the best and most
intense illumination is obtained when the object appears in the image
of the flame. When the object is evenly illuminated and the focus is
made as perfect as possible, a small diaphragm is used in the objective
(2. e., f 32 or 64) and the plate holder with the sensitive plate is put in
place of the focusing screen. Some kind of cover is put over the ob-
jective, the slide of the plate holder is withdrawn and then the
objective is uncovered. With instantaneous, orthochromatic or
isochromatic plates the exposure in most cases need not be over 30 to
go seconds, depending on the object and the diaphragm.

It sometimes occurs that the whole object cannot be satisfactorily
lighted. In that case one may use diffused day-light as follows:
Elevate the camera so that the object is against the clear sky fora
back ground. If any of the earth should form part of the back ground
that part of the object would not be sufficiently illuminated. It is best
not to use any mirror. ‘The light from the sky will evenly and com-
pletely illuminate even the largest object.

Instead of elevating the camera one might use a large reflector
covered with very white cloth or paper and set at an angle of 45 de-
grees. This reflector would then serve for back ground equally with
the clear sky.

§ 336. 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
focusing may be done with the unaided eye and on the ground glass,
but for the final focusing a clear screen and a focusing glass must be
used (Fig. 150).

CH, VII.) PHOTO-MICROGRAPHY. 195

Fic. 149. Zeiss’ Apochromatic, Projection Objective of 7o
min, equivalent focus, for photo-micography. (Zeisss’ Catalog.
Ne. 30), This, and another of 35 mm. focus, are designed for
making piclures of moderate magnification. Usually rather
large objects are photographed with them. The object may be tl-
luminated in the ordinary way. They are used without an ocular,
like a pholographic objective The one of 35 mm. is screwed
into the tube of the microscope like an ordinary objective, but
the one of 7o mm. here shown, is, by means of a conical
adapter, screwed into the ocular end of the tube.

For illuminating the object, any suitable light may be used,
but it is recommended that the light be concentrated by means
of a bull’s eye or some form of combination like the engraving glass, and that the
condenser be so placed that 1t focuses the light upon the objective, not upon the
object. The object ts then illuminated with a converging cone of light.

For the clear screen, Mr. Walmsley and others have recommended
that a pencil mark or cross be made in the center of the ground glass,
and then that a large circular or square cover-glass be put on the
ground glass with Canada balsam. ‘To do this, warm the ground glass
carefully, add a drop of rather thick balsam tothe center on the ground
side, then apply the cover and press it down firmly. After the balsam
has cooled it may be cleaned off around the cover with xylene or alcohol.
The balsam will fill up the inequalities in the glass and being of about
the same refractive power will make this part of the glass clear as if it
were unground (Fig. 150).

For using the focusing glass first carefully adjust it so that the pencil
cross in the center of the ground glass is in the best possible focus.
The image when in the best focus must then be in the same plane as

1
Fic. 150. Focusing screen with clear center
Sor the final adjustment with a focusing glass
like that shown in Fig. 153 or 154.
2 1. The ground surface of the focusing screen,

zt ts translucent but not transparent.

2. Central clear part of the screen made by
cementing acover-glass to the ground surface
with Canada balsam. In the center 1s shown
the pencil mark to indicate the plane to which
the focusing glass should be adjusted.

the ground side of the focusing screen. If the uncovered part of the
focusing screen is too opaque, rub some fine oil on it; only a little

196 PHOTO-MICROGRAPHY. (CH. VIII.

should be used. The focusing screen as thus prepared with a clear
center, serves both for the general focusing and the finest focusing, and
avoids the danger of using a double screen. That is, the fewer the
processes the less the liability to error.

Fic. 151. Pertgraphic Objective of about go millimeters equivalent focus for
making photo-micrographs ata magnification of 2 to 15 diameters. It was found
‘to give the best results when used right end to as in ordinary photography. (From
ithe Gundlach Optical Co. )

Fic. 152. Zeiss’ Anas-
itigmat Objective of about
85 millimeters equivalent
focus for photo-micro-
graphs atamagnification
of 2to 15 diameters. Ex-
cellent results were ob-
tained with this, and the
other short focused anas-
tigmats. On the whole
the results were more sat-
tsfactory when the lens
was used right end to as
in ordinary photography.
(from the Bausch and
Lomb Optical Co.)

§ 337. Development of the Negative.— After the exposure, comes
the development ; this in photo-micrography requires more judgment

CH. VIL.) PHOTO-MICROGRAPHY., 197

than for ordinary photography. ‘The ordinary negative is liable to
have too much contrast, but this is rarely the case with photo-micro-
graphs. Any good developer may be used. One can asa rule do no
better than to follow the directions accompanying the plates used.
The writer’s experience has been so satisfactory with Mr. Walmsley’s
developers that he desires to call attention tothem. The developers
are easily made, will develop anything that can be developed and one
can feel confident that if the negative is not good the fault does not lie
with the developer.

The best photo-micrographic negatives made by the writer were made-
with Cremer’s instantaneous isochromatic plates, and the image com-.
menced to appear with Walmsley’s developer in 2%4 to 3 minutes and!
the development was completed in 12 to 15 minutes. Excellent nega-
tives have been developed in less time and also when it required half
an hour to develop them. The temperature has much to do with the
development of correctly exposed negatives, so that no rule can be
given. Metol and rodinal developers have also given excellent results
and in a shorter time.

If one desires the best possible results it is necessary to avoid the
light in developing. Even the ruby light of the dark room should be
avoided as much as possible, for the plates used are made purposely
sensitive to the longer rays of the spectrum (§ 193). After the nega-
tive is developed and washed it should not be taken to the light till it
is fixed. Too much light after development and before fixing, injures
the clearness of the negative.

Fic. 153. Focusing Glass. ‘It ts achromatic, con-
sisting of a double convex crown lens and a negative
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
focusing screen (Fig. 150) 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 of the
focusing glass or the field will be restricted. (From
the Gundlach Optical Co. )

§ 338. Labeling and Care of Negatives.—The care, printing, etc.,
of negatives is like that of ordinary negatives. It is well to label them

198 PHOTO-MICROGRAPHY. [CH. VIIT.

carefully, however. This label should contain as far as possible, the
following information: (1) Kind of plate; (2) Time of exposure; (3)

Light used ; (4) Objective; (5) Ocular; (6) Magnification ; (7) Name
of object and number of preparation from which taken; (8) Date of
making the negative.

Fic. 154. 7ripod magnifier. This serves fairly well
asa focusing glass, butis inferior to the one shown in
m Fig. 153.

§ 339. Low Microscopic Objectives. — Mi-
croscopic objectives of 35 mm. and greater focal
length may be attached to the camera directly
after the manner of a photographic objective if
one has a plate made with the proper screw for
the objective. The only difficulty is in focusing
the object properly. If one has the special stand
made by the Bausch and Lomb Optical Co., or something equivalent so
that the object may be moved with a fine screw, the focusing can be
well done. Such an arrangement is also very convenient in using the
photographie objectives (Figs. 15-152) and the apochromatic objec-
tives of 35 and 70 mm. focus made by Zeiss.

Fic. 155. Engraving glass to serve as
a bull’s-eye condenser. (From the
Bausch and Lomb Optical Co. ).

§ 340. Photo-Micrographs of
20 to 50 Diameters.—For these,
low objectives are used (35 mm. to
2omm. focus). They are attached
to the camera as described in $ 339
or a microscope is used. If the
microscope is used the object is
placed on the stage and focused in
the usual wav. The mirror is pref-
erably removed or swung aside and the lamp and bull’s eye mutually
arranged to give an image of the flame as described in § 335. If the
object is small the achromatic condenser may be used (See § 342) or
one of the Steinheil magnifiers. When the light is satisfactory 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. It will be

CH. VIII.) PHOTO-MICROGRAPHY. 199

necessary to focus down considerably to make the image clear.
Lengthen or shorten the bellows to make the image of the desired size,
then focus with the utmost care. In case the field is too much
restricted on account of the tube of the microscope, remove the draw
tube. When all is in readiness it is well to wait for three to five
minutes and then to see if the image is still sharply focused. If it has
got out of focus simply by standing, a sharp picture could not be ob-
tained. If it does not remain in focus, something is faulty. When the
image remains sharp after focusing make the exposure. From 15 to
60 seconds will usually be sufficient time with instantaneous plates and
the light as described.

Fic. 156. Lens holder composed of several links and balls, thus giving the flext-
bility of a chain and enabling one lo turn the lens in any desired direction. Each
link has a screw to take up wear, thus insuring permanence. The lens is grasped
by two movable pieces which open widely enough to take a tripod or an engraving
glass, they also hold with equal facility a very much smaller lens. This ts one of
the most efficient and satisfactory lens holders ever made. It is epecially good for
holding a lens for concentrating the light on the mirror in artificial lighting
(From the Bausch & Lomb Optical Co. )

(B) Photographing with a Projection Ocular.—If the object is small
enough to be included in the field of a projection ocular, one may put
that in place of the ordinary ocular. The first step is then to focus the
diaphragm of the projection ocular sharply on the focusing screen.
Bring the camera up close to the microscope and then screw out the
eye-lens of the ocular a short distance. Observe the circle of light on
the focusing screen to see if its edges are perfectly sharp. If not, con-
tinue to screw out the eye-lens until it is. If it cannot be made sharp
by screwing it out reverse the operation. Unless the edges of the light

200 PHOTO-MICROGRAPITY. [CH. VIII.

circle, 7. ¢., the diaphragm of the ocular, is sharp, the resulting picture
will not be satisfactory. When the diaphragm is sharply focused on
the screen, the microscope is focused exactly as though no ocular were
present, that is, first with the unaided eye then with the focusing glass ;
the object should be in focus in the beginning.

Fic. 157. 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 30 mm. wide, so that the thickness of
the flame, if taken edgewise, will give an intense light. A wide flame also enables
one to geta larger image of the flame, and thus illuminate a larger object than as
though a small flame were used.

2. Bull’s-eye condenser on a separate stand. The engraving glass shown in Fig.
155, or the tripod magnifier (Fig. 154) answers fairly. The Steinheil lenses are
still better.

3. Screen showing image of the fame inverted.

The lamp and bull’s-eye stand are on blocks with screw-eyes as leveling screws.

The exposure is also made in the same way, although one must have
regard to the greater magnification produced by the projection ocular
and increase the time accordingly ; thus when the x 4 ocular is used,
the time should be at least doubled over that necessary when no ocular
is employed.

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 would aid in
making the field uniformly sharp.

CH. VIL. PHOTO-MICROGRAPHY. 201

§$ 341. Photo-Micrographs at a Magnification of 100 to 150 Di-
ameters.—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.

§ 342. 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. 39 to 46. Then for each picture the photographer
must take the necessary pains to light the object properly. An achro-
matic condenser is almost a necessity (§ 76). Whether a color-screen
should be used depends upon judgment and that can be attained only
by experience. In the beginning one may try without a screen, and
with different screens and conipare results.

A plan used by many skillful 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. Of course this metal diaphragm
must be removed while focusing the diaphragm of the projection
ocular, as the diaphragm opening would be smaller than the image of
the ocular diaphragm.

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.

S$ 343. Adjusting the Objective for Cover-Glass.—After the ob-
ject is properly lighted, the objective, if adjustable, must be corrected
for the thickness of cover. If one knows the exact thickness of
the cover and the objective is marked for different thickness, it
is easy to get the adjustment approximately correct mechanically,
then the fine or final correction depends on the skill and judg-
ment of the worker. It is to be noted too that if the objective is
to be used without a projection ocular the tube length is practically ex-
tended to the focusing screen and as the effect of lengthening the tube
is the same as thickening the cover-glass, the adjusting collar must

202 PHOTO-MICROGRAPH Y, [CH. VIII.

be turned to a higher number than the actual thickness of the cover
calls for (see § 96).

§$ 344. 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.

§ 345. Photographing with a Projection Ocular.—Proceed as de-
scribed in § 343, 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.

Fic. 158. 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) ts held. This rod ts now
graduated in centimeters and is a ready
means of delermining the length of the cam-
eva, MM. 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. Kk. The upper end
of the camera where the focusing screen and
plate holder ave situated. (From Zeiss’ Photo-
micrographic Catalog).

S$ 346. Determination of the Magnification of the Photo-Micro-
graph.—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
now the distance between two or more of the lines of the micrometer
is obtained with dividers and the distance measured on one of the steel
rules the magnification is obtained by dividing the size of the image by
the known size of the object ($ 146). If now the length of the bel-

T6r.

Pre,

160.

Fic.

CH. VIII) PHOTO-MICROGRAPH Y. 203

Fics, 160-161. Mine tint, half-lone reproductions of photo-micrographs of sections
made by Mrs. Gage, to show the possibilities of photo-micrography with photo-
graphic objectives and with low microscopic objectives without a projection ocular.

1. Frontal section of the head ofa large red Diemyctylus viridescens (red newt)
at the level of the portue of the brain, magnified 10 diameters. Negative made
with a Gundlach perigraphic objective of about 90 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. By the permission of Mrs. Susanna Phelps
Gage, from the Wilder Quarter Century Book.

lows from the tube of the microscrope is noted, say on a record table
like that in section 348, one can get a very close approximation to the
power at some other time by using the same optical combination and
length of bellows.

Fic. 159. Rack for drying negatives. (From
the Rochester Optical Co.)

S 347. Photo-Micrographs at a Mag-
KN VHTRNNi SALVA nification of 500 to 2000 Diameters.—
For this the homogenous immersion objec-
tives should be employed, and as it would
require a long bellows to get the higher
magnification with the objective alone, it is

° best to use the projection oculars.

For this work the foe given in § 342 must be followed with
great exactness. The edge of the lamp flame will be sufficient to fill
the field in most cases. With many objects the time required with
good lamp light is not excessive ; viz., 144 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 aper-
ture, so that the illuminating power of the homogeneous immersion is
great in spite of the great magnification (§ 31).

For work with high powers a stronger light than the petroleum lamp
is employed by those doing considerable photo-micrography. Very
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.

[CH. VIII.

PHOTO-MICROGRAPHY.

204

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CH. VIII.) PHOTO MICROGRAPHY. 205

PHOTOGRAPHING NATURAL HISTORY SPECIMENS WITH A VERTICAL
CAMERA,”**

S$ 349. For most natural history specimens it is inconvenient, and for
many impossible, to use a horizontal camera and to raise the objects
in a vertical position. In order to have the objects horizontal either a
mirror must be used or preferably the camera itself may be so arranged
that it may be put in a vertical position.

For the last twenty years such a camera has been in use in the Ana-
tomical Department of Cornell University for photographing all kinds
of specimens ; among these, fresh brains, and hardened brains have
been photographed without the slightest injury to them. 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 ver-
tical 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 contrasting 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. Incase the fish tends to float in the water a little mer-
cury injected into the abdomen or intestine will serve as ballast.

The photographs obtainable in water are alinost 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. In-
deed so good are the results that excellent fine tint, half tone plates
may be produced from the pictures thus made, also excellent photo-
gravures. In those cases, as in anatomical preparations, where the
photograph rarely answers the requirements of a scientific figure, still
a photograph serves as a most admirable basis for a scientific 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 di-

*Papers on this subject were given by the writer at the meeting of the American
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.
III, pp. 443. 444.

206 PHOTO-MICROGRAPH Y. (CA. VIL.

minishes the labor of the artist 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 imagi-
nation 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 :
1°. 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; 5°. Obtaining outlines for tracing upon the
drawing-paper.’’

‘© 7°. While the camera delineates rapidly, the image is liable to dis-
tortion. 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 diaphragm with
a small opening.’’

‘©2°. By placing the camiera 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. These positions are noted in
some way (on the brass guide, 3, in the apparatus here figured).

Whenever it is desired to photograph an object, natural size, for ex-
ample, the ground glass is fixed in the proper position indicated on the
brass guide (Fig. 162, 3). Then as the relative position of the objec-
tive and the ground glass must not be varied, it is necessary, in focus-
ing, to move the camera toward or away from the object, or the re-
verse. ‘To do this, the camera is fastened to a board which moves in a
frame by means of a screw Fig. 162, 7. Whenever the camera is to be
moved considerably,—as to a position for twice natural size from one
giving an image of half natural size,—the position of the camera on
the board is changed by loosening the two thumb-screws clamping it to
the movable board (Fig. 162,5, 6). The approximate position for the
various sizes being once determined and noted, it is but a moment’s
“work to set the camera for any enlargement or reduction within its
range.’’

3°. The object is placed on a horizontal support, and so arranged
that the lighting will give prominence to the parts to be especially
emphasized, For a contrasting background, black velveteen for light,
and white paper for dark, objects, have been found excellent.

CH. VIII] PHOTO-MICROGRAPH ¥. 207

5

FIG. 162.

Arrangement of a vertical or horizontal camera for making photographs of nat-
ural history objects—sectional view.

I. The photographic objective. It should be of the best quality and rectilinear
so that there may be no distortion.

2. Cone to increase the length of the camera and avoid shadows.

3. Graduated rod to support the front of the camera and hold it rigid, and also
to serve as guide to the various magnifications and reductions most commonly
destred.

4. Ground glass. It 1s an advantage 1o havea clear space in the middle for
accurate focusing, as for the photo-micrographic camera (fig. 153).

5. Camera bed fastened to the sliding focusing board, 6.

6. Focusing board to which the camera is clamped.

7. Focusing screw.

8. Solid piece connecting the focusing board and focusing screw.

9g. Hinge on which the camera swings.

ro. Drawer in which are kept the objective and other accessories.
1. Box of sand or other heavy material to serve as ballast.
The large screw eyes in the legs of the table serve as leveling screws.

fe}

4°. If the photographic prints are to be used solely for outlines, the
well-known blue prints so much used in engineering and architecture

208 PHOTO-MICROGRA PAY. (CH. VIII.

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. In whatever way the print is made, it
is blacked on the back with soft lead-pencil, put over the drawing-
paper, and the outlines traced.

OUTLINES OBTAINED DIRECTLY BY MEANS OF THE CAMERA.

§ 350. While it is desirable to make photographs of objects in many
cases, this may frequently be avoided and a tracing made directly from
the camera. The object is arranged as for a ph tograph and well
lighted. Then the ground glass is changed for a plain glass and the
tracing paper is put over the glass. If the head and screen are covered
in some way the image may be seen and traced very easily. This will
give a reversed image, but that is easily remedied by turning the paper
over in making the tracing on the drawing paper.

Frequently also one wishes to enlarge or reduce a drawing or outline
already made. If the figure or outline is placed in a good light the
image may be made of any desired size and either photographed or °
traced as just described. Tracings of any desired size may also be ob-
tained of negatives, but for this one must employ the method of light-
ing described in § 335, where the clear sky is taken for a background
and illuminant. Of course artificial light may also be used, but it is
less satisfactory unless one has abundant facilities.

An excellent method of making large diagrams is to use a photo-
graphic objective and project the image into a dark room, something
after the manner of a stereopticon, then one can trace the image directly
on the material on which the diagram is to be drawn.

§$ 35t. Prints of Photo-Micrographs and Mechanical Printing.
After one’s negatives are made, prints from them may be obtained
from a photographer, but from the author's experience, unless the pho-
tographer is familiar with the kind of printing necessary to bring out
the features most desired, the results will not be very satisfactory. It
is better to make one’s own prints, and this can be very easily done
with the excellent aristotype and platinotype paper on the market. It
may also be well done with the bromide paper. The last has the advant-
age of being capable of furnishing prints by lamp as well as daylight.

For mechanical prints, half tones and photogravures, one should get
first as good a negative as possible. And for photogravures the so-
called stripping plate should be used so that the picture will not be re-
versed, For half tones the print should have a good deal of contrast

CA. VII.) PHOTO-MICROGRAPHY. 209

and some pure white. With good printing the fine half tone work on
the larger natural history specimens is capable of giving very satisfac-
tory results as may be seen by observing Fig. 160 and 161.

For the methods of Photo-Micrography and Photography in general, consult the
works mentioned in the bibliography. Especial attention is also called to the
papers of Woodward ‘‘On an improved method of photographing histological
preparations by sunlight, 1871, and on the Application of Photography to Mi-
crometry.”

To the papers and discussions of A. C. Mercer in the various volumes of the
Proceedings of the American Microscopial Society.

To the papers and discussions on photo-micrography by Piffard ; Journal of the
Royal Micr. Soc. 1892, and 1893, also in the Amer. Jour. Med. Sciences 1893. The
paper by Dr. G. A. Piersol,—‘‘Some experiences in Photo-Micrography”’ in the
American Annual of Photography for 1890 will also be found very instructive and
helpful. In every volume, and freqeuntly in every number, of the microscopical
journals of this and foreign countries one may find articles, notes or references to
work in photo-microgaphy. Excellent papers are also frequently found in the
photographic journals and annuals.

Thomas J. Wray.—Photo-Micrography by use of ordinary objectives, practically
considered with specimens of work. Proc. Amer. Micr. Soc. Vol. XVIII, (1896).
The specimens in connection with this paper impressed one that simple apparatus
was no bar to success.

Sunlight and Heliostat.—In case one wishes to utilize sunlight for Photo-
Micrography it would be of great advantage to consult the papers of Woodward
mentioned above and also the paper of Mercer in the Jour. Roy. Micr. Soc., 1892,
pp. 305 to 318. For work with sunlight, a heliostat is almost a necessity. An ex-
cellent and inexpensive form was devised by Dr. L. Deck and described and
figured in the Proc. Amer. Micr. Soc., 1891, pp. 49-50. A good form of heliostat
is also figured and described in Van Heurck, pp. 265-266.

Sunlight is not so easily managed as some form of artificial (light, even when
one possesses a heliostat.

For samples of photo-micrographic work, see the magnificent photo prints ex-
hibited by Dr. Woodward at the Centennial Celebration at Philadelphia in 1876,
the works of Mason, Sternberg, Piersol, Wilson and Starr; Carpenter-Dallinger,
Pringle, Bousfield Nehauss, etc., besides the great embryological and morpho-
logical journals where photo-micrographic plates are frequently published.
Catalogs of photo-micrographic apparatus, in some cases, have admirable speci-
mens of photo-micrographs.

14

APPENDIX.

Abbe’s Test-Plate; The Apertometer; Experimental Determination of the
Equivalent Focus of the Objective and of the Ocular; Testing Homogeneous
Fluid ; Preparation of Diagrams ; Drawings for Photo-Engraving.

2352. For the sake of those who may have opportunity to use Abbe’s Test-
Plate the following directions from Zeiss are appended :

‘“CARL ZEISS, JENA. ON THE METHOD OF USING ABBE’S TEST-PLATE.”’

“This test-plate is intended for the examination of objectives with reference to
their corrections for spherical and chromatic aberration and for estimating the
thickness of the cover-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 o 24 mm., silvered on the under surface and cemented side by side on
aslide. The thickness of each is written ou 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 illumina-
tion is used no alteration of the adjustment should be necessary to show the con-
tours with equal sharpuess.”’

“Tf an objective fulfills these conditions with any oue 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 undercor-
rection of this zone; an alteration of the adjustment for oblique and central
illumination, that 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 overcorrectiou
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
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-

APLENDIN.) ABLES TEST-PLATE. 2rr

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 ob-
jective and a rapid interchange from oblique to central light Abbe’s Illuminating
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 illuminator by means of a drop of water, gly-
cerin 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 examination of objectives of smaller aperture (less than 40°-50°) we
may obtain all the necessary data for the estimation of the spherical and chro-
matic 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 ob-
jectives.”’

“It is of fundamental importance iu 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 trustworthy
results. Differences in the thickness of cover-glasses of &OI or 0.02 mmi. 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.”’

‘‘ 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
portion (with good color correction in the middle) are caused by unequal magnifica-
tion 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.’’

Fic. 164. Set of lines under one cover-glass in the Abbe Test-Plate.

212 APERTOMETER. [APPENDIX.

DETERMINATION OF THE APERTURE OF OBJECTIVES.

§ 353. Determination of the Aperture of Objectives with an Apertometer.—Ex-
cellent directions for using the Abbe apertometer may be found in the Jour. Roy.
Micr. Soc., 1878, p. 19, and 1880, p, 20; in Dippel, Zimmerman and Czapski. The
following directions are but slightly modified from Carpenter-Dallinger, pp 337-
338. 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. 165. Asseen by this figure

ili
Ti Hh

Fic. 165. Abbe Apertometer.

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 ina 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, aud with it the back lens of the objective, whose aperture 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 surface is re-
flected by the beveled part of the chord in a vertical direction so that in reality a
fan of 180° in air is formed. There are two sliding screens seen on either side of
the apertometer ; they slide on the vertical circular portion of the instrument.
The images of these screens can be seen in the image of the bright band. These
screens should now be moved so that their edges just touch the periphery of the
back lens. ‘They act, as it were, as a diaphragm to cut the fan and reduce it, so
that its angle just equals the aperture of the objective and no more.”’ ‘‘ This
angle is now determined by the arc of glass between the screens ; thus we get an
angle in g/ass the exact equivalent of the aperture of the objective. As the uu-
merical apertures of these arcs are engraved on the apertometer they can be read
off by inspection. Nevertheless a difficulty is experienced, from the fact that it is
not easy to 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, che limit of aperture,
for curious as the expression may appear we have found at times that the back
lens of an objective is /arger 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.

APPENDIX.) TESTER FOR IMMERSION LIQUID. 213

TESTING HOMOGENEOUS IMMERSION LIQUID.

@ 354. In order that one shall realize the full benefit of the homogeneous im-
mersion principle it is necessary that the homogeneous immersion liquid should
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 per-
fectly plain slip to act as cover. (See Proc. Amer. Micr. Soc., 18385, p. 83). It
will be readily seen that this concavity, if filled with air or any liquid of less re-
fractive 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 adapter 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 (4 46-47).

2355 The following table indicates the points with a tester prepared by the
Gundlach Optical Co., and used with a 16 mm. apochromatic objective of Zeiss, x 4
compensation ocular, achromatic condenser, 1.00 N. A. (Fig. 41):

EVATION OF THE TUBE NEC-
TESTER AND LIQUID IN THE | size or THR | MMT no RESTORE THE
: ‘ FOCUS.
No tester used... 1.825 mm. . | Standard position.
Whole thickness of (lie beater at
one end, not over the cavity. 1.85 mm.. .| No change of focus.
Tester with airin the cavity... .| .6mm. . .| Tube raised 6 mm.
Tester with water... .....{/ 075mm. .)......3%mm.
Tester with 95% alcohol .. . .| .ig5mm...|......3mm.,
. kerosene... LA Wt, . «|e «a ~ x 4 Banh,
. Gundlach oe Co! s
“hom, liquid 2 ips -| 1.825mm. .)... . . . yf mm.
Bausch & Lomb a God s thats:
liquid... 1825mm. .;.... . . pfs mim.
Leitz’ hom. Hiqnid eee ee ef 825mm. 21... 2 ay mm.
Zeiss’ hom. liquid... ..../|1.825mm. .|..... . 4% mm.

214. DETERMINATION OF EQUIVALENT FOCUS. (APPENDIX.

It will be seen by glancing at the above table that whenever the Jiquil 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 (% 152), and the field is smaller
than when uo tester is used. It will also be seen that as the liquid in the con-
cavity 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 were used in the tester the field
would be larger, 7. é., the magnification less, and one would have to turn the tube
down instead of up to restore the focus. The tester used in these experiments
was made by the Gundlach Optical Company of Rochester.

EQUIVALENT FOCUS OF OBJECTIVES AND OCULARS.

@ 356. 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.

2 357. 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 someway on the 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. Remove the field lens from a micrometer ocular, thus making
a positive ocular of it (Fig. 21). Pull out the draw-tube until the distance between
the ocular micrometer and the back lens is 250 millimeters. Use a stage microm-
eter as object aud focus carefully. Make the lines of the two micrometers
parallel (Fig. ror), 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 7 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. Theimage, A'B! 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 (4; mm.), that of the image directly
measured (1° 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 : 250. Supplying the
known values, O= 75, 1=4+2 then 2,m:1mm::F:250 whence F=somm. That
is, the equivalent focus is approximately 50 millimeters.

§ 358. Determination of Initial or Independent Magnification of the Objective.
The initial magnification means simply the magnification of the real image (A! B’,
Fig. 21) unaffected by the ocular. It may be determined experimentally exactly

1PPENDIN.] PREPARATION OF DIAGRAMS. 215

as described in 4 357. For example, the image of the object (,2;mm.) measured
by the ocular micrometer, at a distance of 250 mm. is 1% mm., 2. é., it is five times
magnified, hence the initial magnification of the 50 mm. objective is approxi-
mately five.

Knowing the equivalent focus of an objective, one can determine its initial mag-
nification by dividing 250 mm. by the equivalent focus in millimeters. Thus the
initial magnification of a5 mm. objective is 232—50; of a3 mm., 252—83.3; of
a2mm.,, 2329125, etc.

2359 Determining the Equivalent Focus of an Ocular.—If one knows the ini-
tial magnification of the objective (§ 358) the approximate equivalent focus of the
ocular can be determined as follows:

The field lens must not be removed in thiscase. The distance between the posi-
tion 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 2 357, 358, then
by the aid of Wollaston’s camera lucida the magnification of the whole microscope
is obtained, as described in 3 149. 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 a50 mm. objective, its initial power will be approximately 5.
If with this objective and an ocular of unknown equivalent focus the magnifica-
tion of the whole microscope is 50, then the real image or initial power of the ob-
jective must have been multiplied 10 fold. Nowif the ocular multiplies the real
image ro fold it kas the same multiplying power asa simple lens of 25 mm. focus
for, using the same formula as before; 0 = 5 :i=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 Microscopic Ob-
jectives, Franklin Inst. 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. I. 1893, p. 142. See pp. 1037to 1049 of Carpenter-Dallinger for
mathematital formula; also Daniell, Physics for medical students; Czapski,
Theorie der optischen Instrumente; Dippell, Nageli und Schwendener, Zimmer-
mann.

PREPARATION OF DIAGRAMS.

2 360. For class room work many diagrams are needed. Those which are pur-
chased soon get out of date, but from their cost one does not feel like throwing
them away. It is afact, however, that so much of one’s education comes through
the eye that it is not safe to have an incorrect diagram for students to study. The
visual impression is liable to outlive the verbal correction. To avoid incorrect di-
agrams or those that no longer represent the present state of knowledge one may
use blackboard diagrams. By means of the flexible, or roll blackboards, one may
make the diagrams anywhere and hang them in the lecture-room. This also is of
advantage where several must use the same lecture-room.

For permanent diagrams which shall be as easy to make as the ordinary black-

216 DRAWINGS FOR PHOTO-ENGRAVING. (APPENDIX.

board drawings and so cheap that there is no temptation to preserve them after their
usefulness is past, one may adopt the suggestion of Dr. Dunnington of the Uni-
versity of Virginia and make the diagrams on manilla paper with ordinary black-
board crayons, and then fix them so that they will not rub by hanging them where
the face cannot touch and putting the fixative on the back with a brush or asponge.
The fixative may be readily prepared by mixing one liter (1000 cc.) of ordinary
painter’s turpentine with 100 cc. of dammar varnish. If these are well shaken to-
gether the dammar will be dissolved in the turpentine, and then if the mixture is
put on the dack of the diagram it will soak through the paper and upon drying,
will fix the crayon lines so that they will not rub. At the present day black black-
board crayons are manufactured and they are somewhat easier to shade with, and
very much cheaper for all black work than the Condé crayons. The Condé cray-
ons are better for lettering, however.

It requires only about 12 hours for the fixative to dry. Diagrams may be used
in less time, but they should not be rolled much sooner. If one wishes to have
rollers, and they are very convenient, they are easily made by using what the car-
penters call ‘‘ half-round.’”’ If two of these are used, the paper being put between,
and then the sticks nailed together, very neat looking diagrams are produced. Of
course if itis desired, water colors may be used upon these diagrams either before
or after the crayon has been fixed.

In making diagrams from figures in books, if one desires to enlarge a definite
number of times the drawing paper should be laid out in squares. If these are
made lightly in pencil they will not show in the finished diagram unless one scans
it closely.

DRAWINGS FOR PHOTO-ENGRAVING.

(WRITTEN BY MRS. GAGE).

4 361. The inexpensive processes of reproducing drawings bring within the
reach of every writer upon scientific subjects the possibility of presenting to the
eye by diagrams and drawings the facts discussed in the text. Though artistic
ability is necessary for perfect representation of an object, neatness and care will
enable any one to make a simple illustrative drawing, from which an exact copy is
obtained and a plate prepared for printing.

4 362. A shaded drawing prepared by washes of India ink can be reproduced by
the ‘‘ half-tone process,’’ which is the same as that used in the case of photographs.
(See 2 351, Figs. 160-161). The process usually called photo engraving is that by
which all the line drawings in this book were reproduced, and is much less expen-
sive than the ‘‘half-tone’’ process. For photo-engraving, only pure black and
white can be used in the drawing, as shades of gray are not successfully repro-
duced.

2 363. Outfit for Drawings.—A perfectly squared drawing board; a T-square ;
thumb tacks; a right-line pen; a circle pen; an assortment of smooth pointed
pens, including for fine work a lithographic pen; very soft, hard, and medium
pencils; fine scissors; fine forceps; a sharp-pointed knife or scalpel; smooth,
white bristol board; perfectly black ink. To test the ink draw extremely fine
lines and look at them with a magnifying glass. Most of the water-proof and
liquid India inks on the market answer well.

4 364. Size of Drawing.—It is first necessary to decide upon the scale at which
the drawings are to be made. It is always recommended that they be made large,

APPENDIN.] DRAWINGS FOR PHOTO-ENGRAVING. 217

in order that in the photo engraving they may be reduced in size, and thus lessen
the apparent imperfections of the drawings. The amount of reduction must be de-
termined by each individual, depending upon whether a fine and close or coarse
and broad style is natural. In the former case a reduction of \% is sufficient ; in
the latter, 4% or even % is desirable. The most generally useful reduction is found
to be 4%*.

If one knows the size of the page upon which the figure or plate is to be printed,
it is easy to plan exactly as to the size of the drawings. For example, a finished
plate is to be 10x 16 cm., and the reduction is 4, then the drawing should be made
's larger than the plate; that is, 15 x 24 cm. and \% reduction will produce the ex-
act size needed. Then the eularged page so determined can be outlined by the
T-square and the drawings artistically arranged in the space. If one does not
know at the outset the size of the page, the drawings may be made upon separate
papers, closely trimmed, arranged on card board, care being taken that they do
not overrun the limit of the page, as above determined, and pasted in position.
This is an exceedingly practical method of procedure, even when the size of the
page is known. Care should be taken to keep all straight lines representing per-
pendicular and horizontal directions upon the individual drawings, in correct rela-
tions to the corresponding outlines of the completed plate. For instance, the scale
of magnification (¢ 177) should be parallel with the bottom of the page, and this is
easily determined by the T-square.

2 365. For mechanical drawings, the exact plan is carefully plotted, dots are
placed for intersections and endings of lines; the lines are lightly put in with a
sharp-pointed, medium pencil, and then with a right-line pen and a circle pen.

¢ 366. Fora large natural history object, a blue print (% 349) or a tracing made
from a cainera (4% 350) is obtained ; or for a microscopic object, a camera lucida or
embryograph drawing is made (2176, 178). The outlines are carefully corrected
by comparison with the object, and sharply defined. The back of this photograph
or drawing is blackened with a soft lead pencil, and rubbed gently with a bit of
paper to make an even coating. To transfer the drawing it is placed over the
drawing paper and secured in the desired position with thumb tacks. All the out-
lines are then traced with an ivory point, or a very hard, round pointed lead pen-
cil, and the outlines will appear on the drawing paper. These are again lightly
retouched with a pencil to complete defective lines. If it is desired to reverse a
drawing, as in the case of a tracing made with the camera (3 350), it is placed
against a well lighted window pane, its outlines followed upon the back with a pen-
cil, and then its original outlines retraced with a soft pencil. It is placed face down
upon the drawing paper and the lines upon the back traced with a hard point, and
the outlines will be transferred to the drawing paper.

A. Ifa diagram or outline drawing is desired, the outlines thus made are fol-
lowed by the pen, heavy, light, and interrupted lines being used to indicate differ-
ent classes of facts, according to the special need.

B. If ashaded drawing is to be made, only those outlines are traced in ink which
indicate cut edges of tissue, or for some reason need to be emphasized in an espe-
cial manner. Then the shading is put in, the deep shadows and high lights being
strongly marked and the gradations carefully determined. This is more easily

*In any case, the amount of reduction should be clearly indicated to the photo-

engraver.

218 DRAWINGS FOR PHOTO-ENGRAVING. [APPENDIX.

done if a pencil or wash drawing is first made. The shading may either be dove
by dotting the surface with a pen (stipple) or by lines. It is mucli easier to pro-
duce acceptable results by stippling, because a slight erasure may he made or a
deeper shadow obtained by adding a few carefully placed dots. This method is
well adapted for showing the structure of histological speciniens.

C. If lines are to be used for shading, the beginner will find it more satisfactory
to use the fine lithographic pen, the line extending from the shadow toward the
high light and ending in a series of dots. The deeper shadow is reinforced by a
second or even third series of lines, and these should rarely cross each other at
right angles. They should rather follow the contour of the surface represented
and in crossing should form diamond shaped spaces. <A study of good copper or
steel engravings will aid one in securing a proper method and instances of excel-
lent pen work abound in the first-class illustrated magazines from which valuable
suggestions can be obtained

D. It is sometimes desirable to be able to put white lines over a dark shadow.
In this case a white ink may be used.

E. Occasionally a very dark picture is needed. In this case a specially prepared
paper may be used and covered with an even wash of black ink. Lines are cut
out with a sharp instrument, thus leaving white lines on a black background.

F. Paper with a raised stipple is sometimes used. Upon this the shading is done
by wax crayons, the crayon adhering to the elevations and leaving the depressions
white. Lines and deep shadows are put in with ink. In this way more excellent
drawings can be made than by the more laborious method with pen and ink, but
as a rule the results of the photo-engraving are not so satisfactory.

G. In case it is necessary to show different colors, the drawing is made in dif-
ferent colored inks exactly as desired, pale colors being avoided. The photo-en-
graver makes a plate for each color, and the accuracy necessary in their production
and printing makes the process many times more expensive than the simple repro-
duction of a black and white drawing.

2 367. Lettering.—A halftone engraving from a photograph may have letters
placed upon it by a second printing.

The most carefully prepared drawing may be artistically ruined by placing upon
it ill-formed and irregular lettering, and as even, artistic lettering is almost a trade
by itself, it is recommended that either the drawing he sent to the photo-engraver
with directions to have the letters properly put in, or what is more satisfactory, to
have the letters, abbreviations and words needed, printed and separately placed on
the drawings.

To do this, type of the proper size for the reduciion determined upon is chosen.
Numbers of the following size have been found convenient for numbering the
separate figures of a plate for '4 reduction ;

’

60, Gi, 62,
of the following size for serial parts of the individual drawings,
1, 2 3, 4, 5,

while italic letters of the following size show what has proved most satisfactory for
% reduction,

APPENDIX.) DRAWINGS FOR PHOTO-ENGRAVING. 219

R. bc Lig. t. Filament of Necturus C. Leucocytes,

For ' reduction the following are large enough,
Meten, mic. mtp. mitpr. PF 2 3g Be

It is convenient to have the complete alphabet both in capitals and small letters
and series of numbers in different sizes. The printing should be done on firm, but
light weight, white paper (20 lb, Demy has been found satisfactory ).

The slips thus printed are pinned upon a board and coated upon the back with
liquid gelatin (¢ 311) and dried. With fine scissors the words are cut out neatly,
moistened and with fine forceps placed upon the drawing and pressed down with
blotting paper. Upon a large and complicated plate, the letters and words should
be placed so as not to offend the eye. Those which are intended to be parallel to
each other and to an edge of the plate should be strictly so, as determined in ad-
justing them hy means of the T-square. The numbers of the different figures of
a plate can be arranged so as to lend to the general harmony and still be kept
close to the designated figure. If a great deal of lettering and numbering is ap-
plied a careful plan of the exact place of each word must be determined before
any are fastened, otherwise confusion results. A word may be made to follow the
arcof acircle, by snipping the slip ou which it is printed with the scissors and
curving it after it is moistened.

2368. After the letters and words are thus fastened, if any of them are not
exactly upon the parts to which they refer, a line from the part to the word should
be drawn with the right line pen and T-square. Dotted or full lines may be used,
according to which will show most clearly—and in passing over a deep shadow,
it may be scratched out with a sharp scalpel, leaving a white line.

4 369. After and only after every part of the drawing is as complete as possible
should the eraser be lightly used to remove pencil marks and the sharply pointed
knife or scalpel to remove slight defects, as the overrunning of an inked line or to
pick out shadows which are too deep. This is to avoid roughening the surface of
the drawing paper aud thus making it impossible to add clear cut lines with the
pen.

2370. Considerable modification of a photo-engraving can be made by a skillful
engraver with his tools. Most of the illustrations of the current magazines are
half tone or photo-engravings retouched by the engraver. Slight defects of a plate,
a line a trifle too long, a complete line which should be interrupted, a superfluous
line can be easily remedied, but a line cannot easily be added. A deep shadow
can be lightened or removed, but it is cheaper to renew the drawing than to under-
take extensive changes in the plate. In the case of a plate made up of drawings
on separate papers, the edge of each paper casts a shadow which would appear in
the plate except that it is cut out by the photo-engraver, and in the same way the
edges of the slips used for lettering cast shadows which the engraver removes. If
auy such lines should appear in the proof they can be indicated and removed be-
fore used in printing.

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 Index Medicus, the Zoologischer Anzeiger, and the Zeitschrift fiir wissenschaftliche
Mikroskopie, Anatomischer Anzeiger, Biologisches Centralblatt and Physiologisches Central-
blatt, and the smaller microscopical journals taken together 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 Socrety’s Catalog of
Sctentific 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, London, 1771. Also Essays, 1787.

Angstr6m.—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. Pp. 95, illustrated. Rochester, 1891.

Beale, L. $S.—How to work with the microscope. sthed. Pp. 518, illustrated. London, 1880.
Structure and methods.

Beauregard, H., et Galippe, V.—Guide de 1’éléve et du praticien pour les travaux pratiques de
micographie, comprenant la technique et les applications du microscope 4a l’histologie végétale,
Ala physiologie, a la clinique, 4 la hygiéne et a la médicine légale. Pp. 904, 570 Fig. Paris, 1880.

Behrens, J. W,—The microscope in botany. A guide for the microscopical investigation of
vegetable substances. Translated and edited by Hervey and Ward. Pp. 466, illustrated. Bos-
ton, 1885.

Behrens, W., Kossel, A., und Schiefferdecker, P.—Das Mikroskop und die Methoden der mikro-
skopischen Untersuchung. Pp. 315, 193 Fig. Braunschweig, 1889 +.

Brewster, Sir David.—A treatise on the microscope. From the 7th ed. of the Encye. Brit., with
additions. Illustrated, 1837.

Brewster, Sir David.—A treatise on optics. Illustrated. New edition. London, 1853.
Browning, J.—How to work with the micro-spectroscope. London, 1894.
Bousfield, KE. C.—Guide to photo-micrography. 2ded. Illustrated. London, 1892.

Carnoy, J. B., Le Chanoine.—La Biologie Cellulaire ; Etude comparée de la cellule dans les deux
régnes. Illustrated (incomplete). Paris, 1884. Structure and methods.

Carpenter, W. B.—The microscope and its revelations, 6th ed. Pp. $2, illustrated. London,
and Philadelphia, 1881, Methods and structure.

Carpenter-Dallinger.—The microscope and its revelations, by the late William B. Carpenter.
7th edition, in which the first seven chapters have been entirely re-written, and the text through-
out reconstructed, enlarged and revised by the Rev. W. H. Dallinger. Iondon and Philadelphia,
1891.

BIBLIOGRAPHY. 221

This work deals very satisfactorily with the higher problems relating to the microscope, and
is invaluable as a work of reference.

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. PartI. The microscope and instructions
foritsuse. Part II. Microscopic objects, how prepared and mounted. Illustrated. 2d edition.
London, 189s.

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, r892.

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. E.—Practical microscopy. 3d ed. Illustrated. London, 1895.

Dippel, L.—Handbuch der allgemeinen Mikroskopie. Illustrated. 2d ed. Braunschweig, 1882.

Dippel, L —Grundziige der allgemeinen Mikroskopie. Pp. 524, 245 Fig. Braunschweig, 1885.
Excellent discussion of the microscope and accessories.

Dodge, Charles Wright.—Introduction to elementary practical biology ; a laboratory guide for
high school and college students. New York, 1894.

Ebner, V. v.—Untersuchungen iiber die Ursachen der Anisotropie organischer Substanzen.
Leipzig, 1882. Large number of references.

Ellenberger, W.—Handbuch der vergleichenden Histologie und Physiologie der Hausséuge-
thiere. Berlin, 1884 +.

Fol, H.—Lehrbuch der vergleichenden mikroskopischen Anatomie, mit Einschluss der ver-
gleichenden Histologie und Histogenie. Illustrated (incomplete). Leipzig, 1884. Methods and
structure,

Foster, Frank P.—An illustrated encyclopedic 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 Bacterieu-Kunde. Berlin, 1889 +.

Francotte, P.—Manuel de technique microscopique. Pp. 433, 110 Fig. Brussels, 1886.

Francotte, P.—Microphotographie appliquée a Il’histologie, l'anatomie comparée et l’embryolo-
gie. Brussels, 1886.

Frey, H.—The microscope and microscopical technology. Translated and edited by G. R. Cut-
ter. Pp. 624, illustrated. New York, 1880. Methods and structure.

Gage, A. P.—The principles of physics. Illustrated. Boston and London, 1895.

Gamgee, A.—A text-book of the physiological chemistry of the animal body. Part I, pp. 487,
63 Fig. London and New York, 1880. Part II, 1893.

Gibbs, H.—Practical histology and pathology. Pp. 107. London, 1880, Methods.

Giltay, Dr. E.—Sieben Objecte unter dem Mikroskop. Einfiihrung in die Grundlehren der
Mikroskopie. Leiden, 1893. This is also published in the Holland (Dutch) and French lan-
guage.

Goodale, G. L. Physiological botany. Pp 499+36, illustrated. New York, 1885, Structure and
methods.

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. London, 1883.

Gould, G. M.—The illustrated dictionary of medicine, biology and allied sciences, Illustrated,
3d ed., Philadelphia, 1896. This is recognized as the best single volume medical dictionary. It
is especially satisfactory for the worker with the microscope.

Halliburton, W. D.—A text-book of chemical physiology and pathology. Pp. 874, 104 illus.
London and New York, 1891.

Harker, A.—Petrology for students ; an introduction to the study of rocks under the micro-
scope. Cambridge, 1895.

222 BIBLIOGRAPHY.

Harting, P.—Theorie und algemeine Beschreibung des Mikroskopes. 2nd ed, 3 vols. Braun-
schweig, 1866.

Hogg, J.—The microscope, its history, construction and application. New edition, illustrated.
Pp. 764. London and New York, 1883. Much attention paid to the polariscope.

Huber, G. Carl—Directions for work in the histological laboratory. More especially arranged
for the use of classes in the University of Michigan. 2d ed. Illustrated. Aun Arbor, 1895.

James, F. L.—Elementary microscopical technology. Part I, the technical history of a slide
from the crude material to the finished mount. Pp. 107, illustrated. St. Louis, 1887.

Jeserich, P.—Die Mikrophotographie auf Bromsilbergelatine bei natiirlichem und kiinstlichem
Lichte. Figs. and Plates. Pp. 245. Berlin, 1888.

King, J.—The microscopist’s companion. A popular manual of practical microscopy. Illus”
trated. Cincinnati, 1859. ,

Klément and Renard.—Réactions microchemiques 4 cristaux et leur application en analyse
qualitative. Pp. 126, 8 plates. Bruxelles, 1886.

Kraus, G.—Zur Kentniss der Chlorophyllfarbstoffe. Stuttgart, 1872.

Latteux, P.—Manuel de technique microscopique. 3d ed, Paris, 1887.

Le Conte, Joseph.—Sight—an exposition of the principles of monocular and binocular vision.
Pp. 275, illustrated. New York, 1881.

Lee, A. B.—The microtomist’s vade-mecum, A hand-book of the methods of microscopic
anatomy. 4thed. Philadelphia, 1896,

Lehmann, C. G.—Physiological chemistry. 2 vols. Pp. 6484547, illustrated. Philadelphia,
1855.

Lehmann, O.—Molekularphysik mit besonderer Beriicksichtiguug mikroskopischer Unter-
suchungen und Anleitung zu solchen, sowie einem Anhang iiber mikroskopische Analyse. 2
vols. Illustrated. Leipzig, 1888-1889.

Lockyer, J. N.—The spectroscope and its application, Pp. 117, illustrated. London and New
York, 1873.

M’ Kendrick, J. G.—A text-book of physiology. Vol. I, general physiology. Pp. 516, 318 illus.
New York, 1888.

Macdonald, J. D.—A guide to the microscopical examination of drinking water. Illustrated.
London, 1875. Methods and descriptions.

MacMunzn, C. A.—The spectroscope in medicine. Pp. 325, illustrated. London, 1885.

Martin, John H.—A manual of microscopic mounting with notes on the collection and examin-
ation of objects. 2d ed. Illustrated. London, 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 Lott, F. FE. The microscope in the brewery and the malthouse. Illus-
trated. London, 1889.

Mayall, Jr., John.—Cantor lectures on the microscope, delivered before the society for the
encouragement of arts, manufactures and commerce. Nov.-Dec., 1885. History of the micro-
scope, and figures of many of the forms used at various times.

Moitessier, A.—La photographie appliquée aux recherches micrographiques. Paris, 1866.

Nageli und Schwendener.—Das Mikroskop, Theorie und Anwendung desselben 2d ed. Pp.
647, illustrated. Leipzig, 1877.

Neuhaus, R.—Lehrbuch der Mikro-photographie. Illustrated. Braunschweig, 1890.

Pelletan, J.—Le microscope, son emploi et ses applications. 278 figures, 4 plates. Paris, 1878.

Phin, J.—Practical hints on the selection and use of the microscope for beginners. 6th ed.
Illustrated. New York, 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.

Queckett, 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. : -

BIBLIOGRAPHY. 223

Ranvier, ..—Traité technique @’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.

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.

Richardson, J. G.—A hand-book of medical microscopy. Pp. 333, illustrated. Philadelphia,
1871. Methods and descrip ions.

Robin, Ch.—Traité du microscope et des injections. 2d ed. Pp. 1101, illustrated. Paris, 1877.
Methods and structure.

Roscoe, Sir Henry.—Lectures on spectrum analysis, 4th ed. London, 1885.
Rosenbusch, K. H. F., translated by Iddings, P.—Microscopical physiography of the rock mak-
ing minerals. Illustrated. New York, 1889. .

Ross, Andrew.—The microscope. being the article contributed to the ‘‘ Penny Cyclopaedia.”’
Republished in New York in 1877. Illustrated.

Rutherford, W.—Outlines of practical histology. 2d ed. Illustrated. Pp. 194. London and
Philadelphia, 1876. Methods and structure.

Satterthwaite, F. E. (editor).—A manual of histology. Pp. 478, illustrated. New York, 1881.
Structure and methods.

Schafer, E. A.—A course of practical histology, being an introduction to the useof the micro-
scope. Pp. 304, 40 Fig. Philadelphia, 1877. Methods.

Schacht, H., translated by F. Currey.—The microscope in its special application to vegetable
anatomy and physiology. Illustrated. London, 1853.

Schellen, H.—Spectrum analysis, translated by Jane and Caroline Lassell. Edited with notes
by W. Huggins. 13 plates, including Angstrém's and Kirchhotf’s maps. London, 1585.

Science Lectures at South Kensington. 2 vols. Pp. 290 and 344, illustrated. One lecture on
microscopes and one on polarized light. Ijondon, 1878-1879.

Sedgwick and Wilson.—General biology. 2ded. 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.

Starr, Allen M., with the codperation of Oliver S. Strong and Edward Leaming.—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.

Sternberg, G. M.—Photo-micrographs, and howto make them. Pp. 204, 20 plates. Boston, 1883.

Sternberg, G. M.—A manual of bacteriology. Pp. 886, 268 Fig. viii plates. New York, 1892.
Bibliography on photographing bacteria.

Stokes, A.—Microscopical praxis. Illustrated. Portland, Conn., 1894.

Stokes, A.—Aquatic microscopy for beginners, or common objects from the ponds and ditches.
Illustrated. Portland, Conn., 1896.

Stowell, Chas. H.—The students’ manual of histology, for the use of students, practitioners and
microscopists. 3d ed. Pp. 368. Illustrated. Ann Arbor, 1884. Structure and methods.

Strasburger, E.—Das botanische Practicum. Anleitung zum Selbststudium der mikroskopis-
chen Botanik, fiir Anfanger und Fortgeschrittnere. Pp. 664, illustrated. Structure and meth-
ods. Also English translation.

Suffolk, W. T.—On microscopical manipulation. 2d ed. Pp. 227, illustrated. London, 1870.
suffolk, W. T.—Spectrum analysis applied to the microscope. Referred to in Beale.

‘Thomas, Mason B., and Wim. R. Dudley.—A laboratory manual of plant histology. Illustrated,
Crawfordsville, Ind., 1894.

Trelease, Wm.—Poulsen’s botanical micro-chemistry ; an introduction to the study of vegetable
histology. Pp. 118. Boston, 1884. Methods.

Trutat, M.—La photographie appliquée a l’histoire naturelle. Pp. 225, illustrated. Paris, 1884.
valentin, G.—Die Untersuchung der Pflanzen und der Thiergewebe in polarisirtem Licht.
Leipzig, 1861.

224 BIBLIOGRAPHY.

Vierordt.—Die quantitative Spectralanalyse in ihrer Anwendung auf Physiologie, 1876.

Vogel, Conrad.—Practical pocket-book of photography. Pp. 202, Figs. TI,ondon, 1893.

Vogel, H. W.—Practische Spectralanalyse irdischer stoffe ; Anleitung zur Benutzung der Spec-
tralapparate in der qualitativen und quantitativen chemische Analyse organischer und unorgan-
ischer Kérper. 2d ed. Figs. Berlin, 1889. P

Wethered, M.—Medical microscopy. Pp. 406, Figs. Wondon and Philadelphia, 1892.

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. 2zded. New York and Chicago,
1886.

Wilson, Edmund B., with the codperation of Edward Leaming.—An atlas of fertilization and
Karyokinesis. Columbia University Press, New York, 1895. This atlas marks an era in embry-
ological study. It has admirable text 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, nodate. Upwards of 400
figures of pretty objects for the microscope, also brief descriptions and directions for preparation.

Wormly, IT. G.—The micro-chemistry of poisons. 2d ed. Pp. 742, illus. Philadelphia, 1885.

Wythe, J. H.—The microscopist ; a manual of microscopy and a compendium of microscopical
science. qth*ed. Pp. 434, 252 Fig. Philadelphia, 1880.

Zeiss. R.—Special-catalog iiber Apparate fiir Mikro-photographie. Jena. Instructions for
using the apochromatic objectives and projection oculars, etc.

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 microscopy and popular science. Illustrated. New York, 1876-1881.
The American monthly microscopical journal. Illustrated, Washlngton, D. C., 1880+.
American naturalist. Illustrated. Salem and Philadelphia, 1867 +-.

American quarterly microscopial journal, containing the transactions of the New York micro-
scopial society. Illustrated. New York, 1878.

American microsqppical society, Proceedings. 1878+.

Anatomischer Anzeiger. Centralblatt fiir die gesammte wissenschaftliche Anatomie. 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 société belge de microscopie. Bruxelles, 1874+.

Archiv fiir microscopische Anatomie. Illustrated. Bonn, 1865+.

Centralblatt fiir Physiologie. Unter Mitwirkung der physiologischen Gesellschaft zu Berlin
Herausgegeben von S. Exnerund J. Gad. Leipzig und Wien. 1887+. Brief extracts of papers
having a physiological bearing. Full bibliography of current literature.

English mechanic. London, 1866}. Contains many of the papers of Mr. Nelson on lighting,
photo-micrography, etc.

Index Medicus. New York, 1879}. Bibliography, including histology and microscopy.
International journal of microscopy and popular science. London, 1890+.

Journal of anatomy and physiology. Illustrated. London and Cambridge, 1867+.
Journal de micrographie. Illustrated. Paris, 1877-1892.

Journalof microscopy and natural science. London, 1885+.

*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 fora full list
of periodicals.

BIBLIOGRAPHY. 225

Journal of the New York microscopical society. Illustrated. New York, 1885+.
Journal of physiology. Illustrated. London and Cambridge, 1878+.

Journal of the American chemical society. New York, 1879+.

Jonrnal of the chemical society. London, 1848-+.

Journal of the royal microscopical society. Illustrated. London, 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 Queckett microscopical club. London, 1868+.

The Lens, a quarterly journal of microscopy, and the allied natural sciences, with the tran-
sactions of the state microscopical society of Illinois. Illustrated. Chicago, 1872-1873.

The Microscope. Illustrated. Washington, D. C., 1881+.

Microscopical bulletin and scienc? 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. London, 1869-1877.

Nature. Illustrated. London, 1869-+-.

The Observer. Portland, Conn., 18go+.

Philosophical Transactions of the Royal Society of London. Illustrated. London, 1665+.
Proceedings of the American microscopical society, 1878+.

Proceedings of the Royal Society. London, 1854+,

Quarterly journal of microscopical science. Illustrated. London, 1853+.

Science Record. Boston, 1883-4.

Zeitschrift fiir Instrumentenkunde. Berlin, 1881+-.

Zeitschrift fiir physiologische Chemie. Strassburg, 1877+.

Zeitschrift fiir wissenschaftliche Mikroskpie und fiir 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.

IND

EOL:

A i

Abbe apertometer, 212.

Abbe camera lucida, 111; arrangement
of, 112; drawing with, 115-118; fig-
ures, 95, I10, 114; hinge for prism,
117; inclined microscope with, 115.

Abbe condenser or illuminator, 44, 47;
amount of light for, 43 ; dark-ground
illumination with, 48; experiments, |
46; laboratory microscope with, 61 ;
light, axial and oblique, 46; mirror
with, 46.

Abbe’s test-plate, method of using, 210.
Aberration, chromatic, 4; by cover-glass,
51; negative, 52; spherical, 4, 210.
Absorption spectra. 122, 135; amount of
material necessary and its proper ma-
nipulation, 130; Angstrom & Stokes’
law of, 123; banded, not given by all
colored objects, 133 ; of blood, 131 ;
of colored bodies, 123, 133 ; of color-
less bodies, 134; of minerals, 134;

of permanganate of potash, 131.

Acetylene light, 36, 48.

Achromatic condenser, 40, 41; object-
ives, 12, 61; oculars, 23 ; triplet, 7.

Achromatism, 13.

Actinic focus, 189 ; image, 184.

Adjustable objectives, 12, 13, 52; experi-
ments, 51; and micrometry, 106;
and photo-micrography, 201.

Adjusting collar, graduation of, 52.

Adjustment, of analyzer, 136; coarse or
rapid, 60; fine, 60; focusing, 60;
of objective, 12, 13.51; of objective
for cover-glass, specific directions,
53; testing, 60; with graduated col-|
lar, 53. i

Aerial image, 30, 31.

Air bubbles, 84, 85; with central and
oblique illumination, 84, 85 ; air and
oil, distinguished optically, 85; by
reflected light, 85.

Albumen fixative, Mayer’s, 175.

Albuminous material, removal of, 58.

Alcohol, ethylic, 175.

Alcoholic dye, staining sections with, 167.

Alum solution, 175.

Amici prism, 120, 126.

Amplifier, 97, 214. |

Amplification of microscope, 92.

Analyzer, 126, 136; adjustment and put-
ting in position, 136.

Anastigmatic objective, 196.

Angle of aperture, 16, 17.

Angstrom and Stokes’ law of absorption
spectra, 123.

Angular aperture, 16, 17.

Anisotropic, 137.

Apertometer, Abbe’s, 212.

Aperture of objective, 17-22, 212; angu-
lar, 16, 17; formula for, 17, 18; of
illuminating cone, 44; numerical,
17, 20, 212; numerical of condenser,
43; and optical section, 87; signifi-
cance of, 20.

Aplanatic cone, 44; objectives, 12; ocu-
lar, 23.

Apochromatic condenser, 40 ; objectives,
12, 61, 195. :

Apparatus and material, 1, 33, 80, 92, 109,
120, 140, 183 ; for drawing, 216; for
photo-micrography, 185, ror.

Appearances, interpretation, 80, 91.

Aristotype paper, 208.

Arrangement of condenser, 45 ; of lamp,
bull’s eye and microscope, 49; mi-
nute objects, 180; serial sections,
169 ; tissue for sections, 168.

Artifacts, 81. J

Artificial illumination, 36, 46, 48; for
photo-micrography, 200.

Autograph, photo-micrographic camera,
190-192.

Avoidance of diffusion currents, 162 ; of
distortion, III.

Axial light, 35, 84; experiments, 39; with
Abbe illuminator, 46,

Axial point, 16; ray, 35. :

Axis, optic, 2, 3, 11; of illuminator, 47 ;
secondary, 5, 47.

B

Back combination or system of objective,
10-13.

Bacillus tuberculosis, 56.

Balsam, 175 ; bottle, 164; balsam, mount-
ing in, 163, 167; preparation of, 175;
removal from lenses, 58; natural,
176; neutral, 176; removal from
slides, 141 ; xylene, 175, 176.

Banded absorption spectra not given by
all colored objects, 133.

Bands, absorption, 123.

Base, circular, of microscope,, 73..

' Bed, camera, 207.

228

Benzin, removing of from sections, 165. |
Biaxial crystals, 138.
Bibliography, 32, 91, 108, 119, 135, 139,
173, 182, 209, 215. :
Binocular vision, for getting magnifica-
tion, 93.

Blood, absorption spectrum of, 131; or
other albuminous material, removal,
58; velocity of current, 83.

Body of microscope, Frontispiece.

Bottle for balsam, glycerin or shellac, 164.

Bowl, waste, 162.

Bread crumbs, examination of, go.

Bromide paper, 208.

Brownian movement, 88.

Brunswick black, removal from lenses.55.

Bubble, air, 84-85.

Bull’s-eye, 49, 191, 209; engraving, glass
for, 198.

Burning point, 6; finding of, 30.

Butterfly scales, go.

iC

Cabinet for microscopical preparations, |
173, 174.

Calipers, micrometer, 143, 144; pocket,
143.

Camera bed, 207 ; photographic, for draw-
ing, 208; long and short bellows,
187, 200; photo-micrographic, 186,
188, 190-193, 202. 205, 207; testing,
185; vertical, 188, 293, 202, 205, 207.

Camera lucida, Abbe, 111 ; arrangement
of, 112, 113; drawing with, 114, 115,
118; hinge for prism, 112; with in-
clined microscope, 115; laboratory
microscope with, 61; magnification
of microscope with, 92, 118.

Camera lucida, definition, 109; figures
of, 98, I10 111, 114; Wollaston’s, 95,
IIl.

Canada balsam, 175; mounting in, 163,
167; preparation of, 175; removal
from lenses, 58; removal] from slides,

141.

Carbol-xyleue, 176.

Carbon-monoxide hemaglobin, spectrum
of, 133.

Carbonate of lime, pedesis, 89.

Card catalog, 173 ; centering, 149.

Care of, eyes, 58; microscope, mechan-
ical parts, 57 ; optical parts, 57 ; neg-
atives, 197 ; water immersion object-
ives, 55.

Carmine to show currents and pedesis,
89; spectrum of, 133.

Castor-xylene clarifier, 176.

Catalog, cards, labels, ink for, 173.

Cataloging, formula, 172; preparations,
171-172.

Celloidin for coating glass rod, 87.

INDEN.

Cells, deep, thin, 148; isolated, prepara-
tion of, 155; mounting, 148; stain-
ing, 155.

Cement, shellac, 179.

Cementing collodion, 177.

Center, optical, 2, 3.

Centering and arrangement of illumina-
tor, 41, 45; card, 149; condenser for
photo-micrography, 201; dia-
phragm, 42; image of source of illu-
mination, 42

Central light, 35, 84; with a mirror, 39.

Chain lens holder, 199

Chamber, moist, 151.

Chimney, metal for lamp, 48.
Chloroform, parafin, 176; infiltrating
with, 165 ; saturating with, 165.
Chromatic aberration, 4; correction, 12

13; correction, test for, 210.

Chemical focus, 13 ; rays, 13.

Clarifier, 176 ; castor-xylene, 176.

Cleaning back lens of objective, 58 ; ho-
niogeneous objectives, 56; mixtures
for glass, 145; slides and cover-
ylasses, 140-142; water immersion
objectives, 55.

Clearer, clearing, 153, 164, 167, 176.

Clearing mixture, preparation of, 176.

Clinical microscope, 76.

Clothes moth, examination of scales, go.

Cloudiness of objective and ocular, how
to determine, 82; removal, 58.

Coarse adjustment of microscope, Front-
ispiece ; testing. 60.

Cob-web micrometer, 104.

Collective, 24.

Collodion, 176 ; for coating glass rod, 87 ;
cementing, 177; clarifying, 158; cot-
ton, 176; hardening, 158; method,
157, 164; thick, 158, 177; thin, 157,
177.

Color correction, 13 ; images, 51, 56; law
of, 124; production of, 138; screens,
IQI, 50.

Colored minerals, absorption spectra of,
134 ; substances, spectra of, 133.

Colorless bodies, spectra of, 134.

Coma, 53.

Combination of lenses, back and front,
10-12, 52; optical, 105.

Comparisou prism, 127, 128; spectrum,
128.

Compensating ocular, 24-26.

Complementary spectra, 124.

Compound microscope, see under micro-
scope.

Concave lenses, 3 ; mirror, use of, 36.

Condenser, 39-49; Abbe, 44-48; achro-
matic, 40, 191, 201 ; apochromiatic, 4o,
191; bull’s-eye, 49, 198, 200; center-
ing, 41, 45; illuminating cone with,
44; mirror for, 46; non-achromatic,

,

INDEN.

2209

44; numerical aperture of, 43-41;' Designation of oculars, 26; of wave

optic axis of, 41, 45; for photo mi-
crography, 40, 191, 201 ; substage, 39.
40. See also illuminator.

Condensing lens, 35.

Cone, aplanatic, 44; illuminating, 43.

Conjugate foci, 3.

Construction of images, geometrical, 5.

Continuous spectrum, 123.

Contoured, doubly, 87.

Converging lens, 3, 5; lens-system, 10.

Convex lenses, 3, 5.

Corn starch, examination of, go.

Correction, chromatic, or color, 13, 210;
cover-glass, 15, 52-53; cover, tube-
length for, 53-54; over and under,
13.

Cost of microscope, 63.

Cotton, collodion, 176; examination of,
g0; gun, 176; soluble, 176.

Cover-glass, or covering glass, 141; ab-
erration by, 41; adjustment, spe-
cific direciions, 53; adjustinent for,

in pho o micrography, 201 ; adjust-.

ment and tube-length, 13, I4, 53;
anchoring, 150; cleaning, 141, 142;
correction, 52, 53; effect on rays
from object, 52; gauges, 143-145:
larger than object, 146; measurer,

143-145 ; measuring thickness of,

143; non adjustable objectives. table
of thickness 15; No 1, variation of.
thickness, 143; putting on, 84, 146;!
sealing, 148, 150; size of, 146; thick- |

ness of, 14, 15, 143, 170; tube-length
with, 14, 53, 54; wiping, 142; with
serial sections, 170.

Crayons, 216.

Crystals, biaxial, depolarizing, 138 ; from |

frog for pedesis, 89.

Crystallization under microscope, 180.

Crystallography, 48, 180; list of substan-
ces for, 181.

Currents, diffusion, avoidance of, 162;
in liquids, 88.

Cutting sections, 160, 166.

D

Dammiar, for fixing crayon drawings,
216; removal from lenses, 58.

Dark-ground illumination, 36, 47, 48;
with Abbe illuminator, 48; with
mirror, 48

Daylight, lighting with, 34.

Defining power, 21.

Dehydration, 157, 162, 167.

Demonstration microscope, 77.

Depolarizing crystals, 138. |

length, 129.

Determination of field of microscope, 29 ;
magnification, 92, 94, 214; of sedi-
ment in water, 180; of working dis-
talice, 38.

Developers for photo-micrography, 197.

Development of negative, 196.

Diagrams with photographic objectives,
208 ; preparation of, 215.

Diamond, writing, 173.

Diaphragms and their employment, 35,
44; central stop, 36, 47; eccentric,
42, 48; iris, 40 ; ocular, 28; pin-hole,
41, 45, ; size and position of opening,
35. 443 with condenser, 44.

Diffraction, grating, 122; illusory ap-
pearances due to, 91.

Diffusion currents, avoidance of, 162.

Direct, light, 34; vision spectroscope,
120.

Dispersing prism, 122.

Displacements, in mounting objects in
resinous media, 153.

Dissecting microscope, 9.

Dissociator, formaldehyde, 177; nitric
acid, 179.

Distance, principal focal, 3, 30; standard
at which the virtual image is meas-
ured, 97; working d. of simple mi-
croscope or objective, 38; working
d, of compound microscope, 11, 33,
38,

Distinctness of outline, 85.

Distortion in drawing, avoidance of, III.

Diverging lens, 3. :

Dividers, measuring spread of, 93.

Double spectrum, 128; vision, 92, 93.

Doubly contoured, 87 ; refracting, 137.

Draw-tube, Frontispiece ; pushing in, 37.

Drawing, with Abbe camera lucida, 114-

115; board for Abbe camera lucida,
115-116; distortion avoidance of, 111;
embryograph for, 119; with micro-
scope, 109; photographic camera
for, 119; for photo engraving, 216 ;
scale and enlargement of, 118 ; tran-
ferring, 217.

, objectives, 11, 17-19; for laboratory
microscope, 61; light utilized, 18 ;
dry mounting, 147; numerical aper-
ture, 18; dry plates, discovery by
Maddox, 184.

Drying negatives, rack for, 203.

Dust, of living rooms, examination of,
go; on objectives and oculars, how
to determine, 82 ; removal, 58.

Dye, general, staining with, 167; aque-
ous, 161, 167; alcoholic, 161, 167.

230

E

Eccentric diaphragm, 42, 48.

Embryograph, rg.

Engraving ylass for bull’s eye condenser,
198.

Erect image, I.

Equivalent focal length, 11, 214; focus of
objectives, 11, 26, 214; focus of ocu-
lar, 26, 214.

Elements, histological, isolation of, 154.

Kosin, 177.

Ether, sulphuric, 177.

Ether-alcohol, 177; saturated with, 157.

Ethylic alcohol, 175.

Examination of dust of living rooms,
bread crumbs, corn starch, fibres of
cotton, linen, silk, human and ani-

mial hairs, potato, rice, scales of but- |

terflies and moths, wheat, 9c.

Experiments, Abbe illuminator, 46; with
adjustable and immersion objectives,
51 ; compound microscope, 26 ; crys-
tallography and micro-chemistry,
180; homogeneous immersion ob-
jective, 55; lighting and focusing,
36; in micro-chemistry, 180; with
micro-spectroscope, 131 ; with micro-
polariscope, 137; i1 mounting, 146-
161 ; pboto-micrography, 193; simple
microscope, 6.

Exposure of photographic plates, 194,
199, 200, 203.

Extraordinary ray of polarized light, 136.

Eye and microscope, I, 6. 10, 31.

Eyes, careof, 58; muscz volitantes of, 90

Eye-lens of the ocular, 22.

Eye-piece, 22 ; micrometer; 102.

Eye-point, 7, 22, 110; of ocular, demon-
stration, 32.

Eye-shade, Ward’s, 59 ; double, 59.

F

Farrant’s solution, in mounting objects,
151; preparation of, 177.

Feather, examination of, go.

Fibers, examination of, 90

Field, 27; with camera lucida, 95; illu
mination of, 44, 49; with orthoscopic
ocular, 23; with periscopic ocular,
24; of view with microscope, 27, 29,
93. 109; size of, with different object
ives and oculars, 27, 29

Field-lens, of ocular, 22; action of, 31;
dust on, 82.

Filar micrometer ocular, 23 ; ocular mi-
crometer, 103.

Filter paper, Japanese, 57.

Filters, light, r9r.

Filtering balsam, etc., paper funnel for,
175.

INDEX.

| Fine adjustment, Frontispiece ; testing,

0.

Fir, balsam of, 175.

Fixative, albumen, Mayer’s, 175.

Fixing, reagents for, 175 ; tissue, 157.

Flame, image of, 43.

Fluid, immersion, 55, 56; testing, 213.

Focal distance, or point, principal, 30;
length, equivalent, IT.

Focus, 6; always up, 35; actinic, 189;
chemical, 13; conjugate, 3; of ob-
jectives, equivalent, 11, 26, 214; of
oculars, equivalent, 26,214 ; optical,
13; principal, 3,5; visual, 189

Focusing, 6, 33 ; adjustments, testing, 60;
with compound microscope, 33; ex-
periments, 36; glass, 197-198 ; with
high objectives, 37 ; with low object-
ives, 36; objective for micro-spectro-
scope, 130; for photo micrography,
194, 199, 201; screen for photo-mi-
crography, 194-195 ; with simple mi-
croscope, 31; slit of micro spectro.
scope, 131.

Form of objects, determination of, 83.

| Formal, 177.

Formaldehyde dissociator, 177 ; for iso-
Jation, 154.

Formula for cataloging, 172.

Fraunhofer lines, 123.

Front combination or lens of objective,
10, II.

Frontal sections, 170.

Function of objective, 29, 39; of ocular,

30.
Funnel, paper, 175.

G

Gauge, cover-glass, 143, 145.

| Gelatin, liquid, preparation of, 178.

| Geometrical construction of images, 5.

| Glass, cleaning mixture for, 145; focus-

: ing, 197-198; ground, 29; for object-

| ives, 12, 63; rod appearance under

; microscope, 86, 87; slides or slips,

t 140.

Glue, liquid, preparation of, 178.

Glycerin, bottle for, 164; mounting ob-
jects in, order of procedure, 149. 177;
removal, 58.

Glycerin jelly, mounting objects in, or-
der of procedure, 150; preparation
of, 177

Gold size, removal froin lenses, 58.

Goniometer ocular, 23.

Graduation of adjusting collar, 53.

Grating, diffraction, 122.

_Ground glass, preparation of, 29.

/Gun cotton, 176.

INDEX.

H

Hairs, examination of, go.

— from photo-micrographs, 208,
216.

Hardening collodion, 158; tissue, 157,
164.

Heliostat, 209.

Hematoxylin, 178.

Hemoglobin specirum, 132, 133.
Herapath’s method of determining mi-
nute quantities of quinine, 181.
Histological elements, isolation of, 154.

Histology, physiological, 173.

History of photo-micrography, 184.

Holder, lens, 8; needle, 146.

Homogeneous immersion objective, 17-
19; cleaning, 56; experiments, 55;

231

Imbedding, 158, 165.

Immersion fluid, 55, 213; illuminator,
45 ; liquid, 55; objective, 12, 55, 56,
61.

Incandescence or line spectra, 123.
Incident light, 34.
| Index of refraction, 50; of medium in
front of objective, 17-18.
‘Infiltration with chloroform, 165; col-
lodion, 157, 158; ether, 157; paraffin,
165.
Initial magnification, 214.
Ink for labels and catalogs, 173;
| drawing, 216.
Interpretation of appearances under the
| microscope, 80-91.
| Inverted image, 7, 29.
| Iris diaphragm, 40, 181.

for

for laboratory microscope, 61 ; light Irrigating with reagents, I50.
utilized, 18, 21; numerical aperture, | Isochromatic plates, 194, 197.
21. Isolation, 154; with formaldehyde, 154 ;
Homogeneous liquid, 12, 213 ; tester for, nitric acid, 155.
55. 213. ‘Isotropic, 137.
Huygenian ocular, 22, 24, 31. Japanese filter or tissue paper, 57, 160.
Jelly, glycerin, 177.
I-J Jena glass, 63.
Jurisprudence, micrometry in, 108.
Illuminating, cone, aperture of, 43, 44;
for condenser, 44; objective, 14; L
power, 21, 22.
INumination, for Abbe camera lucida, | Labels and catalogs, ink for 173; prep-
116; artificial, 26, 48; artificial for aration of, 179.
photo-micrography, 194; centering) Labeling microscopical preparations,
image of source of, 41-45; central 171; photographic negative, 197 ;
with air andoil, 84, 85 ; dark-ground, serial sections, 171.
26, 47, 48; daylight, 34; of entire) Laboratory conipound microscope, 61.
field, 49 ; lamp for, 48; methods of, | Lamp, microscope, 48 ; spirit, 164.
34, 47; for micro-polariscope, 137;} Lamp-light, 36.
for micro-spectroscope, 129 ; oblique, | Law.of color, 124.
with air and oil, 84, 85; for photo- Lens, concave, 3; converging, 3; con-
micrography, 192; for Wollaston’s vex, 3; eye, 22; field, 22, 26; holder,
camera lucida, IIT. 8, 155, 156, 199; paper, 57; system,
Mluminator, 39-40; Abbe, 44, 47; Abbe, 10.
axial and oblique light, 36; Abbe,’ Lens-systems, 10.
experiments, 46; Abbe, mirror and! Letters in stairs, 82; for photo-engrav-
light for, 46; achromatic, 40; cen-| ing, 218.
tering and arrangement, 45 ; immer-! Lettering oculars, 26.
sion, 45. See also condenser. Light, with Abbe illuminator, 46; ace-
Image, actinic, 184; aerial, 30, 31; cen-. tylene, 36, 48; artificial, 46; axial,
tering i. of source of illumination, | 35, 39; axial with Abbe illuminator,
42; color, 49, 51. 56; with dry apo- | 46; direct, 34; central, 35, 39; filters,
chromatic and water immersion ob- 191; incident, 34; with mirror, 39;
jectives, 13; erect, 1; inverted, 7; oblique, 35, 39; oblique with Abbe
inverted, real of objective, 29; of. illuminator, 46; lamp, 36; for pho-
flame, 43 ; formation of, 3; geomet- | to-micrography, 192; polarized, 136 ;
rical construction of, 5 ; and object, reflected incident or direct, 34 ; trans-
size and position, 5, II, 96; real, 5, mitted, 35 ; utilized with different ob-
10, 22, 29-31, 92; refraction, 49, 55; jectives, 13; wave length of, 129.
retinal, 6, 10, 31; swaying of, 46; Lighting, 34; for Abbe camera lucida,
virtual i. and standard distance at! 115; artificial, 48 ; experiments, 36;
which measured, 6, 31, 92. and focusing, 33, 36; for micro-
Image-power of objectives, 19. polariscope, 137; for micro-spectro-

230

scope, 129; with a mirror, 36; with |
daylight, 34; for photo- -micrography, | |
192.
Line spectrum, 123.
Linen, examination of, go.
Liquid, currents in, 55; homogeneous, ,

‘555, 203: |
M |

Macro-photography, 184. |

Magnification of compensating oculars, |
26 ; effect of adjusting objective, 105 ; |
determination of, 92, 94; expressed
in diameters, 92; initial or indepen-
dent, 214; method of binocular or!
double vision in obtaining, 93;)
of microscope, 92; of microscope’
with Abbe camera lucida, 115; of |
microscope, compound, 94; of micro- |
scope, simple, 93; of photo-micro- ,
graphs, determination of, 202; real
images, 92 ; table of, with ocular mi- |
crometer, 99; varying with com-,
pound microscope, 97 ; and velocity,
383.

Magnifier, tripod, 7, 198.

Marker for preparations, 63, 64.
Marking objects, 63, 64, 94, 101 ;
tives, 197; objectives, 65.
Material and apparatus, I, 33, 80, 92, 120,

140, 175-181, 183, 210, 216.

Measure, unit of, in micrometry, 100 ; of
wave length, 128.

Measurer, cover-glass, 143-145.

Measuring the spread of dividers, 93 ;
thickness of cover- glass, 143.

Mechanical parts of compound micro |
scope, 61; of microscope, care of, |
67% testing, 60

Mechanical stage, 61, 65-66.

Medium, mounting, 147.

Met-hemaglobin, spectrum of, 122, 133.

Methods, collodion, 157 ; paraffin, 164.

Micro-chemistry, 180.

Micrometer, 92; calipers, 143, 144; cob-.
web, 104; filarm. ocular, 103; filling
lines of, 94; net, 113; lines, arrange-
ment of ocularand stage, 107 ; object
or objective, 94; ocular or eye-piece, |
102, 103; ocular, micrometry with, ,
305; ocular, ratio, 106 ; ocular, valu-
ation of, 103; ocular, varying valua-
tion of, 105 ; screw ocular, 103 ; stage,
94; table of magnification, 99.

Micrometry, definition, 100-102; with
adjustable objectives, 106 ; compari-
son of methods, 107 ; with compound
microscope, 100; and jurisprudence,
108 ; limit of accuracy in, 107; with
ocular micrometer, 105 ; with simple

INDEX.

microscope, 100; 106 ;
unit of measure in,

'Micro-millimeter, 101.

Micron, 100; for measuring wave-length
of light, 129.

Micro-photograph, 183.

Micro-photography, distinguished from
photo-micrography, 183.

Micro-polariscope, 89, 136; ‘experiments,
137; for laboratory microscope,
lighting for, 137; objectives to use
with, 136; purpose of, 137; selenite
plate with, 138 ; sulphonal with,
139.

Micro-polarizer, 136.

Microscope, definition, 1; amplification
of, 92; clinical, 76; demonstration,
77; dissecting, 9; care of, 57; eye
and, I, 6, 10; field of, 27, 29; focus-
ing, 31 ; magnification, 92 ; for photo-
micrography, 187; polarizing, 136;
price of, 61, 63; putting an object
under, 27; screen, 56.

Microscope compound, definition, 7;
drawing with, 110; figures, Io, 65;
focusing, 36-37; for laboratory, 61 ;
lamp, 48 ; magnification or magnify -
ing power, 94; magnification and
size of drawing with Abbe camera
lucida, 118; mechanical parts of, 61 ;
miicrometry with, 100; optic axis of,
10; optical parts of, 10, 61 ; polariz-
ing, pedesis with, 89; varying magui-
fication, 97 ; working distance of, 38 ;
testing, 60.

"Microscope, simple, definition, 1; exper-
iments with, 6; figures, 6-8, 31, 155-
156, 197-199; focusing with, 33;
maguihcation of, 93; micrometry
with, 100; working distance of, 33.

Microscopic objective, 10 ; objective low.
attached to camera, 195; objects,
drawing, 109; ocular, 22; slides or
slips, 140.

Microscopical preparations, cabinet for,
174; cataloging, 172; labeling, 171;
mounting, 146-170 ; tube- -length, 14,
15.

Microscopy and photography, 183.

remarks on,
100.

Micro-spectroscope, 120-121 ; adjusting,
125; experiments, 131 : focusing,
130; focusing the slit, 125 ; for labo-

ratory microscope, 61 ; lighting for,
‘129 ; objectives to use with, 130; re-
versal, apparent, of colors in, 120;
slit, mechanism of, 121, 125.

Micrum, roo.

Mikron, Ico.

Minerals, colored, absorption spectra of,

_ 134
Minute objects, arrangement of, 180,

INDEN.

Mirror, 10, 11 ; for Abbe illuminator, 46;
of camera lucida, arrangement for
drawing, 112; concave, use of, 36;
dark-ground illumination, 47; light
with, central and oblique, 39; light- |

_ ing with, 36; plane, use of, 36. |

Mixture, clearing, 176.

Moist chamber, 151.

Molecular movement, 88.

Monazite sand, spectrum of, 134.

Mono-refringent, 137.

Mounting, cells, preparation of, 148 ; me-
dia and preparation of, 175-'79 ; ob-
jects for polariscope, 137; perma-
nent, 147; temporary. 146.

Mounting objects, dry in air, order of
procedure, 147; in glycerin, order of
procedure, 149 ; in glycerin jelly, or-
der of procedure, 150; in media mis-
cible with water, 149; minute ob-
jects, 180; permanent, 147; in resin-
ous media, 152; in resiuous media,
by drying or desiccation, order of
procedure, 152; in resinous media,
by successive displacements, order
of procedure, 153 ; temporary, 146.

Movement, Brownian, or molecular, 88.

Muscae volitantes, go

Muscular fibers, isolation of, 155.

N

Natural balsam, 176.

Needle-holder, 146. :

Negative, aberration, 52; development |
of, 196; labeling and care of, 197;
marking, 197; oculars, 22; rack for
drying, 203.

Net-micrometer, 113.

Neutral balsam, 176.

Nicol prism, 136.

Nitrate of uranium, spectrum of, 135.

Nitric acid, dissociator, 179.

Nomenclature of objectives, 11

Non-achromatic condenser, 44; object-
ives, 12.

Non-adjustable objectives, 13 ; thickness
of cover glass for, table, 15. i

Normal salt solution, 179. :

Nose-piece, 9, 27; marking objectives
on, 65. :

Numerical aperture of condenser, 43 ; ob-
jectives, 17, 212; table of, 20; reso- |
lution aid, 21.

O

233

crometer, 94; mounting, 146; put-
ting under microscope, 27 ; shading,
56; suitable for photo-micrography,
191; transparent with curved out-
lines, relative position in microscopic
preparations, 83.

Objective, 7, 10; achromatic, 10, 12, 195 ;

adjustable, 12, 13,52; adjustable, ex-
periments, 51; adjustable, microm-
etry with, 106; adjustable, photo mi-
crography with, 201 ; adjustment for,
51; aerial image of, 30; anastigmat,
196; aperture of, 21, 22, 212; apla-
natic, 12; apochromatic, 12, 189 ;
back combination of, 12; cleaning
back lens of, 58; collar, graduated
for adjustment, 53; cloudiness or
dust, how to determine, 82; desig-
nation of, I1; dry, If, 17-19 ; equiv-
alent focus of, 11, 26, 214; field of,
28, 29; focusing for micro-spectro-
scope, 130; front combination of, 10,
11; function of, 29, 30; glass for,
11-13, 63; high, focusing with, 37;
homogeneous immersion, 17-19 ; ho-
mogeneous immersion, cleaning, 56;
homogeneous immersion, ex peri-
ments, 51, 55; illuminating, 14; im-
age, power of, 19; immersion, 12 ;
index of refraction of medium in
front of, 17, 18; inverted, real image
of, 29; for laboratory microscope, 61 ;
lettering, 11; light utilized with, 18;
low, attached directly to camera, 198;
low, focusing with, 36; magnifica-
tion of, 214; marking, by Krauss’
method, 65 ; touse with micro-polar-
iscope, 136 ; microscopic, 10; to use
with micro-spectroscope, 130; for
micro-spectroscope, focusing, 130;
nomenclature of, 11 ; non-achromat-
ic, 12; non-adjustable, 13 ; non-ad-
justable, thickuess of cover-glass for,
table, 15; numbering, II; numeri-
cal aperture, 21, 22, 212; oil immer-
sion, 12; panto-chromatic, 13; para-
chromatic, 13; perigraphic, 196; for
photo-micrography, 189; projection,
14, 195; putting in position and re-
moving, 26; semi-apochromatic. 13 ;
table of field, 28; terminology of, 11 ;
unadjustable, 13 ; variable, 13; vis-
ual and actinic foci of, in photo-mi-
crography, 189; water immersion,
17-19. 55; experiments, 51; work-
ing distance of, 11, 33, 38.

Oblique light, 35, 39; with Abbe illum1-

Object, determination of form, 83 ; hav-
ing plane or irregular outlines, rela- |

nator, 46; experiments, 39, 46; with
a mirror, 39.

tive position in a microscopical prep- | Ocular, 22; achromatic, 23; Airy’s, 23;

aration, 82; and image, size of, 5,11, ,
96; marking parts of, 63, 64; mi-'

aplanatic, 12, 23; binocular, 23 ;
cloudiness, how to determine and re-

Oil, and air, appearances and distinguish-

Oil-immersion objectives, 12.
Optic axis, 2,5; of condenser, or illumi- |

Order of procedure in mounting objects, |

INDEX.

move, 82; Campani’s, and cob-web
micrometer, 23; compensating, 24,
26 ; compound, 23 ; continental, 23;
deep, 23; designation by magnifica-
tion or combined magnification and |

equivalent focus, 26; diaphragm, 28;

dust, how to determine, 82; equiva-: Pa

lent focus of, 26; erecting, 23; eye-
point of, demonstration, 32; field--
lens, 31; filar micrometer, 23, 26,.
104; focus, equivalent of, 26; func-
tion of, 30, 31 ; goniometer, 23 ; high,

Over-correction, 5.
Oxy-hemoglobin, spectrum of, 122, 132.

P

Pautachromatic objective, 13.
per, aristotype, 208; bibulous, filter,

leis, or Japanese, 57, 160; bromide,
208 ; for cleaning oculars and object-
ives, 57, 160; funnel, 175; Usago,
160

Parachromatic objective, 13.

23; holosteric, 23; Huygenian, 22-! Paraffin, 179; chloroform, 176; chloro-

24, 31; iris diaphragm, 181 ; index, |
23; Jackson micrometer, 23; Kell-
ner’s, 23; lettering of, 26; low, 23; :
micrometer, 23, 25, 102, 105; mi-—
crometer, micrometry with, 105 ; mi-
crometer ratio, 106; table of magni- |

F ‘ |
fication of, 99; micrometer, valua--

form, infiltrating with, 165; hard,
166; imbedding in, 165; method,
164; removal from lenses, 58; re-
moving from sections, 166.

Parfocal oculars, 16, 24, 37.
Parts, optical and mechanical of micro-

scope, 10, 61 ; testing, 60.

tion of, 102; micrometer, varying Pedesis, 88; compared with currents,

valuation, 105 ; micrometer, ways of
using, 105 ; micrometric, 23 ; micro- |

88 ; with polarizing microscope, 89 ;
proof of reality of, 89.

scopic, 22, 23; negative, 22,23; num-) Penetrating power, 21, 22.

bering, 26 ; orthoscopic, 23, 28; par- | Penetration of objective, 21.

focal, 16, 24, 37 ; periscopic, 24, 28; Perigraphic objective, 196.

for photo-micrography, 25, 189; pho- Periscopic ocular, field with, 28.
to-micrography with and withouto.,' Permanent mounting, 147 ; preparations

198, 199; pointer, 63; positive, 22 ;!

of isolated cells, 155.

projection, 25, 189; projection, des- | Permanganate of potash, absorption spec-

ignation of, 189; projection, use of

trum of, 122, 131.

in photo-micrography, 199, 202 ; put-: Picric-alcohol, 179.

ting in position and removing, 27;

Pin hole diaphragm, 45.

Ramsden's, 24; screw micrometer, . Pipette, 16r.
26, 103; searching, 24, 25 ; shallow, | Photo engraving, 216, 219; drawing for,

24; solid, 24 ; spectral, 24, 106; spec-

216-219 ; lettering for, 218.

troscopic, 24, 120; staurose“pic, 24;|! Photographic negatives, marking, 197;

stereoscopic, 24; table of field of, 28;
working, 25.

ing optically, 85; removal, 58; re-
moval from sections, 161.

Oil-globules, with central illumination,

84; with oblique illumination, 85. |

nator, 35 ; of microscope, 6

‘Pho

objectives, 196 ; for photo microgra-
phy, 196.

tography, basis for figures, 206 ; com-
pared with photo-micrography, 183 ;
indebtedness to microscopy, 183,
184; lighting large objects for, 194 ;
objectives for, 196 ; of objects in alco-
hol or water, 205; with a vertical
camera, 205. 207.

i ' Photogravures from photo-micrographs,
Optical center, 2, 3; combination, 104, |

208,

105 ; focus, 13; parts of compound Photo-micrograph, 183; developers for,

microscope, 10, 61; parts of micro- |
scope, care of, and testing, 57, 60;:
section, 87 |
dry or in air, 147; in glycerin, 149; /
in glycerin jelly, 150; in resinous |
media by desiccation, 152; in resin- |
ous media by successive displace-
ments, 153

197; determination of magnification
for, 202 ; at 5-20 diameters, 193; 20-
50 diameters, 198; 100-150 diame-
ters, 201 ; 50-2000 diameters, 203 ;
objects suitable for, 191; prints of,
208 ; plates for, 194; reproductions
of, 208 ; with and without an ocular,
198

_ Photo-micrographic camera, 186, 188, 190,

Ordinary ray with polarizer, 136.
Orthochromatic plates, gr.
Orthoscopic ocular, field with, 28. |
Outline, distinctness of, S85.

192, 193, 202. :

Photo-micrography, 183-204 ; cover-glass
correction, 201 ; apparatus for, 185 ;
compared with ordinary photogra-

INDEX. 235
phy, 184; condenser for, 40, 191 ; Q—R
distinguished from micro-photogra-
Py ; SXPERMUEIS; 193 , €xpos- | Quadrant for camera lucida, 113.
ure: oe 193, 194, 199, 200, 203 ; fo- Quinine, Herapath’s method of determin-
cuss Ob. 195i) focusing screen for, ing minute quantities of, 181,
195 5 nehting, wie 193, 195, 203 ; ob- | Rack for drying negatives, 203.
eee and oculars tor, 14, 189, 195 ; | Ratio, ocular micrometer, 106.
vertical camera with, 205 ; visual and | Reagents for fixing, 175 ; irrigation witb,

actinic foci in, 189; with long and ; i
, . § 150 ; for n t 175.
short bellows, 187; with and without Real pine re oe ; magunifica-
ocular, 189-199; record table for, 204. | tion,o2, ,
Physiological histology, 173. Record table for photo-micrography, 204.
Plane mirror, use of, 36. ' Reflected light, 34 a
1 A Sel a
Plates, pe pee ite ae 193, 194, (199) 209, Refraction, 50; images, 49, 55; index of,
203 ; gelatino-bromude, 184 ; isochro- | 50; of medium in front of objective,
matic, or orthochromiatic, 189, 191, 20
197. tie p pings ;
Pleochroism, 138. mae oe tg7y higtlys. 875
yee pe neato 39 Relative position of objects, 82.
Salone axial, 16; buruing, 6. Resinous media, mounting objects in,
olariscope, 127, 136. é fi order of procedure, by drying or
Polarized light, extraordinary and ordi- desiccation, 152; by a series of dis-
1 , , =

dinary ray of, 136. sd eh wif placements, 153.
Polarizer and analyzer, putting in posi- | Resolution and numerical aperture, 21.
tion, 136. os ‘Resolving power, 20.
Polarizing microscope, pedesis with, 89. | Retinal image, 6, 10
Position of objects or parts of same ob- Revolver, 27,0 |)
ject, $2. -Revolving nose-piece, marking objec-
Positive oculars, II, 22. tives on, 65.
Power of microscope, 92; illuminating, Rice examination of. 90
penetrating, resolving, 21; of object- p Pan
lve, 19, 215; of ocular, 26, 215. :

Preparation of Canada balsam, Farrant’s Ss
solution, glycerin, glycerin jelly, etc. |
175-179. . Sagittal sections, 170.

Preparation of clearing mixture, liquid | Salicylic acid, crvstallization, 48.
gelatin and shellac cement, 175-179. | Salt solution, normal, 179.
Preparations. cataloging, 171, 172; cali | Scale of drawing, 118; of wave lengths,

net for, 173-174; labeling, 171 ; per- | 128,

manent, of isolated cells, 155; stor- Scales of butterflies and moths, examin-

ing, 171. ation of, go.
Preparation of diagrams, 215 ; of ground | Screen, color, 191 ; focusing s. for photo-

glass, 29; vials, 159. micrography, 194, 195; of ground
Price of American and foreign micro- glass, 29 ; for microscope, 56.

scope, 61, 63. Screw, society, 62; micrometer, 26, I04.
Principal focus, 3, 5; focal distance, 3, ‘Sealing cover-glass, 148. .

30; optic axis, 2, 5. Searching ocular, 25.

Prism of Abbe camera lucida, 111 ; Am-) Secondary axis, 3.
ici, 126; comparison, 127, 128; dis-| Section, optical, 87.
persing, 126; Nicol, 136; and slit of Sections, arrangement of tissue for, 168 ;
micro-spectroscope, mutual arrange- clearing, 167; cutting, 160, 166;

ment, 125; of Wollaston’s camera | fastening to slide, 160, 166; frontal,

lucida, Ti. 170; removing benzin, oil and
Prints and mechanical printing of photo | paraffin from, 166, 161 ; sagittal, 170;

micrographs, 208. serial, 168-171; staining, 161, 167 ;
Projection objective, 14, 195; ocular, 25, | transferring, 160.

26, 189; designation of, 189; in pho- Sediment in water determination of
to micrography, 199, 202. character, 180.
Putting on cover-glass, 146; an object Selenite plate for polariscope, 138.
under microscope, 27; an objective Semi-apochromatic objective, 13.
and ocular in position, 26, 27. ' Serial sections, 168 ; arranging and label-
Pyroxylin, 176. ing, 169, 171; determining thickness

236 INDEN.

of, 170; stage for, 65; thickness of! IL
cover-glass for, 170.
Shellac cement, preparation of, 179; re- Table, for immersion fluid, 213 ; of mag-

moval from lenses, 53. . ‘ nification and valuation of ocular
Sight, injury or improvement in miicro- | micrometer, 99; of tube-length and

scopic work, 59. | thickness of cover-ylasses, 15 ;
Significance of aperture, 20. natural sines, third page of cover;
Silk, examination of, go. , | of weights aud measures, second
Simple microscope, see under micro- page of cover; of numerical aper-

scope, I. ; | ture, 20; record, for ploto-microg-
Slides, 140; cleaning, 14o. | raphy, 204; size of fields, 28 ; of val-
Slips, rq4o. uations of ocular micrometer, gg.
Slit mechanism of micro-spectroscope, | Temporary mounting, 146.

121. Terminology of objectives, 11.
Society screw, 62. Test of chromatic and spherical aberra-
Sodium, lines and spectrum, 122, 123. | tion, 210.
Solar spectrum ors. of sunlight, 122. | Tester, cover-glass, 144, 145 ; for homo-
Soluble cotton, 176. geneous liquids, 55.
Solution, alum, 175; Farrant’s, 177. ‘Testing a camera, 185 ; ink, 216; a mi-
Spectral, colors, 123; ocular, 120, 126. croscope and its parts, 60.
Spectroscope, direct vision, 120. Test-plate, Abbe’s, method of using, 210.
Spectroscopic ocular, 24, 120. | Textile fibers, examinaticn of, co.
Spectrum, 122; absorption, 123; amount ‘Thickness of cover glass for non-adjust-

of material necessary and its proper | able objectives, table, 15; of serial

manipulation, 130; analysis, 135;. sections, 170.

Angstrom and Stokes’ law of, 1133 Tissues, arranging for sections, 168; fix-

banded, not given by all colored’ ing or hardening, 157, 164.

objects, 133; of blood, 131; of car-, Tolles-Mayall mechanical stage, 65.
bon monoxide bemaylobin, 133; of Transections, 169.

carmine solution, 133; of colored | Transfering drawings, 217 ; sections, 160.
minerals, 134; of colorless bodies, Tyansmitted light, 35.

134; coniparison, 128; complemen- ‘Transparent objects having curved out-
tary, 124; continuous, 123; double, lines, relative position in microscop-
128; incandescence, 123; line, 123; ic preparations, 83.
met-hemaglobin, 133 ; monazite | Triplet. achromatic, 7.

sand, 134; nitrate of uranium, 135 ; | Tripod, 7; as focusing glass, 19S.
oxy-hemaglobin, 132; permanganate Tuhe of mnicroscope, Frontispiece.

of potash, 131 ; single-bhanded of he- ‘Tube-length, 14-16, 54; for cover-glass

maglobin, 132; sodium, 122, 123 ; so- | adjustinent, 53, 54; importance of,
lar, 122, 123 ; two-handed of oxy-he- 53, 54; microscopical, 14, 15; of
maglobin, 132. | various opticians, table, 15 ; and op-
Spherical aberration, 4; test for, 210. | tical combinations, 104.
|

Stage, 61; mechanical, 61, 65, 66; mi-

. Turn-table, 148.
crometer, 94; for serial sections, 65.

F 2 i
Stain, alcoholic, 161; aqueous, 161. |

Staining cells, 155; sections, 161, 167. U—V—W—X

Stand, of microscope, 61 ; for laboratory |
microscope, 61. Unadjustable objectives, 13.
Standard distance (250 mm.) at which | Under-correction, 5.
the virtual image is measured, 97. | Unit of measures, in micrometry, 100 ;
Starch, examination of, go. of wave length, 129.
Stokes and Angstroms law of absorption | Uranium nitrate spectrum of, 135.
spectra, 123. | Usago paper, 160.
Storing preparations, 171. Valuation of ocular micrometer, 102,
Substage, 67. | 103; table, 99
Substances for crystallography, 18r. | Variable objective, 13.
Sulphonal with polarizer, 139. Varying magnification of compound mi-
Sulphuric ether, 177. croscope, 97.
Swaying of image, 46. Varying ocular micrometer valuation,

System, back, front, intermediate, of 105.
lenses, 10. ' Velocity under microscope, 88.

INDEX. 237

Vertical camera, 188, 190, 192, 193, 202, | Wave lenzth, designation of, 129; scale
207 of, 128.
Vials for preparation 159 _ Weights and measures, see 2d p. of cover.
Virtual image, 5, 6, 10, 31 ; standard dis-. Wollaston’s camera lucida, 95, 111.
tance at which measured, 97. Work-room for photo-micrography, 187.
Vision, double or binocular, 92, 93. -Work-table, position, etc., 59.
Ward's eye shade, 59. , Working distance of microscope or ob-
Waste bowl, 162. jective, 11, 33; determination of, 38 ;
Water immersion objective, 17, 19, 553. oculars, 25.
light utilized, 18; numerical aper- Writing diamond, 173.
ture, 20 Nylene, 159, 176; balsam, 175, 176.
Water, for immersion objectives, 55; re- Nylol, German form of xylene, 159, 176.
moval, 58; solid sediment in, 180.

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