HE METRIC SYSTEM. 
TIMETERS DIVIDED INTO IOO MILLIMETERS. 
5 Centimeters. 
5 Centimeters divided into 
% Centimeters. 
UNITS. 
Centimeter (c.m.), i-iooths Meter; Millimeter (m.m.), i-ioooths Meter; Micron 
(/t), i-ioooths millimeter; the Micron is the unit in Micrometry (\ 157). 
Kilometer, 1000 Meters; used in measuring roads and other long distances. 
The most commonly used divisions and multiples. 
THE METER FOR 
LENGTH. . . . 
THE GRAM FOR 
WEIGHT. . . . 
Milligram (m.g.), i-ioooths gram. 
Kilogram, 1000 grams, used for ordinary masses, like groceries, etc. 
Cubic Centimeter (c.c.), i-ioooths Liter. This is more common than the cor- 
rect form, Milliliter. 
THE LITER FOR 
CAPACITY. . . 
Divisions of the Units are indicated by the Latin prefixes: deci, i-ioth ; centi, i-iooth; Milli, 
i-ioooth. 
Multiples are designated by Greek prefixes : deka, io times ; hecto, ioo times ; kilo} 1000 times ; 
myria, to,ooo times. 
TABLE OF METRIC AND ENGLISH MEASURES. 
METER = 100 centimeters, 1000 millimeters, 1,000,000 microns, 39 3704 inches. 
Millimeter (mm.) = 1000 microns, millimeter, meter, inch, ap- 
proximately. 
Micron (pi) (Unit of Measure in Micrometry) = TT,V5th mm. meter. 
(0.000039 inch) inch, approximately. 
Inch (in.) = 25.399772 mm. (25 4 mm., approx.) 
Liter — 1000 milliliters or icoo cubic centimeters, 1 quart (approx.) 
Cubic centimeter (cc. or ccttn.) = of a liter. 
Fluid ounce (8 Fluidrachms) = 29 578 cc. (30 cc., approx.) 
Gram =15 432 grains. 
Kilogram (kilo) = 2.204 avoirdupois pounds pounds, approx.) 
Ounce Avoirdupois = (4377 grains) = 28.349 grams. \ , annrox ) 
Ounce Troy or Apothecaries = (480grains) =31.103grams. | ® ’’ ’’ 
TEMPERATURE. 
To change Centigrade to Farenheit : (C. X f) 4- 32 = F. For example, to find 
the equivalent of io° Centigrade, C. = io° (io° X f) 4- 32 = 50° F. 
To change Farenheit to Centigrade: (F.—32°)/Xf = C. For example, to re- 
duce 50° Farenheit to Centigrade, F. =50°, and (50° — 320) X $ = io° C. ; or — 40 
Farenheit to Centigrade, F. = — 40° (— 40° — 320) = — 720, whence — 720 X 7 
= - 40° C. 
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 addresses in the table of tube-length, p. 15. Nearly all import for- 
eign goods. 
The Bausch & Lomb Optical Co., . . New York City and Rochester, N. Y. 
Eimer and Amend, 205-211 Third Avenue, New York City 
The Franklin Educational Co., , Harcourt St., Boston, Mass. 
J. Grunow; 70 West 39th St., New Y’ork 
The Gundlach Optical Co., Rochester, N. Y. 
Wm. Krafft, (representative of Leitz in America), 411 W'est 59th St., New York 
The McIntosh Battery and Optical Co., 521-531 Wabash Ave., Chicago, 111. 
Queen & Co., Incorporated, . 1010 Chestnut St., Philadelphia, Pa. 
Richards & Co., Limited, 30 E. 18th St., New York, 108 Lake St,, Chicago, 111. 
Edward Pennock, 3609 Woodland Ave., Philadelphia, Pa. 
Spencer Lens Co., 546 Main St., Buffalo, N. Y. 
Walmsley, Fuller & Co., 134-136 Wabash Ave., Chicago, 111. 
Williams, Brown & Earle, 10th & Chestnut Sts., Philadelphia, Pa. 
G. S. Woolman, (Queen & Co., in New York), 116 Fulton St., New York 
J. Zentmayer, 209 South nth St., Philadelphia, Pa, THE MICROSCOPE IN SECTION. 
1. Compensation ocular X12 ; it is a positive 
ocular. 
2. Draw-tube, by which the tube is lengthened 
or shortened. 
3. Main tube or body, to the lower end of which 
the objective or revolving nose-piece is 
attached. 
4. Society screw in the lower end of the 
draw-tube. 
5. Society screw in the lower end of the 
tube. 
6. Objective in position. 
7. Stage, under which is the substage with the 
substage condenser. 
8. Spring clip for holding the specimen. 
9. Screw for centering, and handle of the 
iris diaphragm in the achromatic con- 
denser (see Fig. 41). 
10. Iris diaphragm outside the principal focus 
of the condenser for use in centering 
(? 77)- 
11. Mirror with plane and concave faces. 
12. Horse-shoe base. 
13. Rack and pinion for the substage conden- 
ser. 
14. Flexible pillar. 
15. Part of pillar with spiral spring of fine 
adjustment. 
16. Screw of fine adjustment. 
17. Milled head of coarse adjustment. THE 
MICROSCOPE 
AND 
MICROSCOPICAL METHODS, 
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. Copyright, 1896, 
By Simon Henry Gage. 
All Rights Reserved. 
Printed by 
Andrus & Church, 
Ithaca, N. Y. PREFACE TO THE SIXTH EDITION. 
THE rapid advance in microscopical knowledge, and the great strides in the 
sciences employing the microscope as an indispensable tool, have reacted 
upon the microscope itself, and never before were microscopes so excellent, 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, 
Corneee University. 
October j/, 1896. preface tq the fifth edition, 
THIS 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, while 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 & Tomb Optical Co., Gundlach Optical Co., Queen & Co., and all the op- 
ticians mentioned in the table of tube-length, p. io.). I feel under special obliga- 
tion to my various classes for the enthusiasm and earnestness with which they have 
followed the instructions in the book, to mv 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. Dwell 
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 microscojpo 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 :• 
“ It 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 
have been made an integral part of the work. 
“In considering tfie reef greatness of the microscope, and the truly splendid VI 
PREFACE. 
service it lias 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, 
Ithaca, New York, U. S. A. 
February 12, 189$. CONTENTS. 
CHAPTER I. 
2 ... , . PAGE. 
& I_ 55—The Microscope and its Parts—Demonstration of the Function 
of each Part. Figures of Laboratory Microscopes, 1-32 
CHAPTER II. 
$ 56-119—Lighting and Focusing, Manipulation of Dry, Adjustable, and 
Immersion Objectives; Care of the Microscope and of the 
Eyes> 33-79 
CHAPTER III. 
$ 120-144—Interpretation of the Appearances under the Microscope, . . . 80- 91 
CHAPTER IV. 
$ 145-167—Magnification of the Microscope ; Micrometry, 92-108 
CHAPTER V. 
§168-178—Drawing with the Microscope, 109-119 
CHAPTER VI. 
§179-218—Micro-spectroscope and Micro-polariscope; Use and Applica- 
tion, 120-139 
CHAPTER VII. 
§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. 
§ 322-351—Photo-micrography and the Photography of Natural History 
Specimens in a Horizontal Position with a Vertical Camera, . 183-209 
APPENDIX. 
§ 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- 
ing, 213-219 
BOOKS AND PERIODICALS 220-225 
INDEX 227-237 UST OF ILLUSTRATIONS, 
The author extends grateful acknowledgments to the opticians and others who 
have loaned cuts for this edition. The sottrce 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, 
Frontispiece . . , 4 4 . 
1-9. The principal axis and center of various lenses 2 
10-11. Principal focus with converging and diverging lense9 . . . 3 
12. Chromatic aberration 4 
13. Spherical aberration 4 
14-15. Real and virtual image with convex lenses. ...... 5 
16. Simple microscope and eye of observer 6 
17. Tripod magnifier 7 
18. Achromatic triplet The Bausch & Tomb Opt. Co.) . 7 
19. Lens-holder (The Bausch & Lomb Opt. Co.) 8 
20. Dissecting microscope (The Bausch & Tomb Opt. Co.) 9 
21. Principle of the compound microscope 10 
22. Dry objective II 
23. Immersion objective 12 
24. Tube-length 15 
25. Tube-length when nose piece and Ocular micrometer are used (Zeiss’ cat- 
alog, No. 30) 16 
26. Angular aperture 17 
27-29. Dry and immersion objectives (Ellenberger) 18 
30. Section of Huygeniau ocular for eye-point 22 
31. Compensation oculars (Zeiss’ catalog, No. 30) 24 
32. Projection oculars (Zeiss’ catalog, No. 30) 25 
33-34. Ocular micrometer -with movable scale (Bausch & Lomb Opt. Co.) . . 25 
35. Ocular screw micrometer (Zeiss* catalog, No. 30) . 26 
36. Triple nose-piece or revolver (Queen & Co.) 27 
37. Size of field with various objectives and oculars 29 
38. Principle of the simple microscope (Fig. 16 repeated) . 31 
39-40. Dry and immersion objectives (Figs. 22-23 repeated) ......... 34 
41. Achromatic condenser (Zeiss’ catalog, No. 30) 41 
42-43. Image of diaphragm in centering 42 
44-45. Centering the source of illumination on the object 43 
46-47, Aperture of condenser (from Nelson) . 43 
48-51. Abbe condenser, central, oblique and dark ground illumination .... 47 
52. Lamp and bull’s eye condenser 49 
53-55. Refraction diagrams (from Carpenter-Dallinger) 50 
56. Aberration produced by the cover-glass (Ross) 52 LIST OF ILLUSTRATIONS. 
57. Cover correction by changing tube-length 54 
58. Screen for face and microscope 56 
59. Ward’s eye shade (Cut loaned by Queen & Co.) 59 
60. Double eye-shade 59 
61-63. Marker, sectional view (Proc. Amer. Micr. Soc., 1894) 64 
64-66. Specimens showing the use of the marker 64 
67. Krauss’ method of marking objectives on a nose-piece (from Dr. Krauss, 
see Proc. Amer. Micr. Soc., 1895) 65 
68. Removable mechanical stage (Feitz catalog) . . ' ' 65 
69. Removable mechanical stage (Bausch & Fomb Opt. Co.) 65 
70. Zeiss’ Microscope la 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 
Franklin Educational Co.) 68 
73. BB Microscope of the Bausch & Fomb Optical Co. (B & F) 69 
74. Reichert’s microscope Illb (cut loaned by Richards & Co.) 70 
75. Queen & Co.’s microscope II of the continental pattern (Q. & Co.) ... 71 
76. Feitz’ microscope lb (cut loaned by Wm. Krafft) 72 
77. Ross eclipse microscope (cut from Walmsley, Fuller & Co.) 73 
78. AA. Microscope of the Bausch & Fomb Optical Co. (B. & F.) 74 
79. Beck’s star microscope (cut loaned by Williams, Brown & Earle) .... 75 
80. Zentmayer’s clinical microscope (Zentmayer) 76 
81. Zentmayer’s microscope, No. V (Zentmayer) 76 
82. Feitz’ demonstration microscope (from Wm. Krafft, N. Y.) 77 
83. Feitz’ microscope IV (from Wm. Krafft, N. Y.) 77 
84. Queen & Co.’s acme microscope, No. IV (Q. & Co.) 78 
85. McIntosh’s scientific microscope, No. 2 (McIntosh Battery Co.) 79 
86. mounted in stairs to show order of coming into focus 82 
87. Putting on a cover-glass 84 
88. Oil and air bubbles 85 
89. Glass rods in optical section 86 
90. Double contour 87 
91. Micrometer with ring to facilitate finding the lines 94 
92. Wollaston’s camera lucida 95 
93. Geometrical diagram showing size of object and image 96 
94. Image and object with differing tube-length 96 
95. 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 xoi 
98-99. Ocular micrometer (Figs 33-34 repeated) 103 
100. Ocular screw micrometer (Fig. 35 repeated) 104 
101. Fines of stage and ocular micrometer in getting the valuation of the ocu- 
lar micrometer 107 
102. Abbe camera lucida with 450 mirror no 
103. Geometrical figure going with Fig. 102 no 
104. Ocular showing eye-point (Fig. 30 repeated) no 
105. Wollaston’s camera lucida (Fig. 92 repeated) 111 
106. Abbe camera lucida with 350 mirror 114 
107. Geometrical figure going with Fig. 106 114 
io8. Upper view of the prism of the Abbe camera lucida 114 LIST OF ILLUSTRATIONS. 
109. Quadrant attached to the mirror of the Abbe camera lucida 1x4 
no. Inclined microscope with the Abbe camera lucida 115 
111. Drawing board for the Abbe camera (The Bausch & Lomb Opt. Co.) . . 116 
X12. Micrometer lines indicating the scale of a drawing 1x8 
113. Longisection of the Abbe micro-spectroscope (cut loaned by the Bausch 
& Lomb Opt. Co.) 121 
114. Slit mechanism of the micro-spectroscope (from B. & L.) 121 
115. Various spectrums 122 
116. Absorption spectrum of hemoglobin, etc. (Gamgee & McMunn) .... 124 
117. Section of the micro-spectroscope 126 
118. Prism showing apparent reversal of colors 126 
119. Section of a micro-polariscope 126 
120. Micrometer calipers (Brown & Sharp) 143 
121. Cover-glass measurer (The Bausch & Lomb Opt. Co.) 144 
122. Zeiss cover-glass measurer (from Zeiss’ catalog) 145 
123. Putting on a cover-glass (Fig. 87 repeated) 146 
X24. Needle holder (Queen & Co.) 146 
125. Turn table (Queen & Co.) 148 
126. Centering card 145 
127. Anchoring a cover-glass 150 
128. Irrigation, staining, etc., under the cover 150 
129. Moist chamber for fibrin, blood corpuscles, etc. (from Proc. Amer. Micr. 
Soc., 1891) 151 
130. Adjustable lens holder (Leitz, cut from Wm. Krafft) 155 
131. Adjustable lens-holder (The Bausch & Lomb Opt. Co.) 156 
132. Preparation vials (Proc. Amer. Micr. Soc., 1895) 159 
133. Pipette for stains, etc. (Whitall, Tatum & Co.) 161 
134. Waste bowl with rack and funnel (cut loaned by Wm. Wood & Co.) . . 162 
135. Round aquarium for waste bowl, rinsing jar, etc. (Whitall, Tatum & Co.) 162 
136. Glass box for cleaning slides and covers (Whitall, Tatum & Co.) .... 162 
137. Balsam bottle 164 
138. Serial section slide, showing order of arranging sections 170 
139. Writing diamond (Queen & Co.) 173 
140. Drawer of cabinet for slides (Proc. Amer. Micr. Soc., 1883) 174 
141. Cabinet for microscopical specimens (Proc. Amer. Micr. Soc., 1883) . . . X74 
142. Czapski’s iris diaphragm ocular (Zeiss’ catalog, No. 30) 181 
143. Walmsley’s large photo-micrographic camera (from Mr. Walmsley) . . . 186 
144. Leitz’ vertical photo micrographic camera (from Wm. Krafft) 188 
145. Projection oculars (Fig. 32 repeated) 189 
146. Walmsleys autograph camera in a vertical position (from Mr. Walmsley) 190 
147. Same in horizontal position 192 
148. Vertical photo-micrographic camera (the Bausch & Lomb Opt. Co.) . . 193 
149. Zeiss’ 70 millimeter projection objective (from Zeiss’ photo-micrographic 
catalog) 195 
150. Focusing screen 195 
151. Perigraphic photographic objective (The Gundlach Opt. Co.) 196 
152. Zeiss anastigmatic photographic objective (from the Bausch and Lomb 
Opt. Co.) 196 
153. F'ocusing glass (from the Gundlach Opt. Co.) 197 
154. The tripod as a focusing glass 198 xii 
LIST OF ILLUSTRATIONS. 
155- Engraving glass (The Bausch & Loinb Opt. Co.) t9g 
156. Bausch & Tomb’s chain lens-holder for use with a dissecting lens, hold- 
ing an engraving glass, etc. (B. & T.) 199 
157. Bull’s eye and lamp (Fig. 52 repeated) 200 
158. Zeiss’ vertical photo-micrographic camera (Zeiss’ catalog) 202 
159. Rack for drying negatives (Rochester Opt. Co.) 203 
160-161. Sections of the head and brain of Diemyctvlus (Mrs. Gage, from the 
Wilder Quarter Century Book) 203 
t62. Vertical photographic camera for picturing brains and other preparations 
in a horizontal position 207 
164. Abbe’s test plate 2II 
165. Abbe’s apertometer (Zeiss’ catalog) ■ . 212 THE MICROSCOPE 
AND 
MICROSCOPICAL METHODS. 
CHAPTER I. 
THE MICROSCOPE AND ITS PARTS. 
APPARATUS AND MATERIAL FOR THIS CHAPTER. 
A simple microscope ($ 2, 9) ; A compound microscope with nose-piece (Figs. 
6S-80), eye-shade (Figs. 59-60), achromatic ($ 18), apochromatic (| 20), dry ($ 15), 
immersion (£ 16), unadjustable and adjustable objectives (§ 21, 22), Huygeuian 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. 
§ I. A Microscope is an optical apparatus with which one may obtain a clear 
image of a near object, the image being always larger than the object; that is, it 
enables the eye to see an object under a greatly increased visual angle, as if the 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). 
\ 2. A Simple Microscope.—With this an enlarged, erect image of an object 
may be seen. It always consists of one or more converging lenses or lens-systems 
(Figs. 16-20), and the object must be placed within the principal focus (g 9). 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. 
Figs. 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 tzuo surfaces 
of the lens. c. 1. Optical center of the lens, r-r'. Radii of curvature of the two 
lens surfaces, t-t'. Tangents in Fig. 4. 
\ 3. Principal Optic Axis.—111 spherical lenses, i. <?., 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 c-c' 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. 
§ 4. Optical Center.—The optical center of a lens is the point through which rays 
pass without angular deviation, that is, the emergent ray is parallel to the incident 
ray. It is determined geometrically by drawing parallel radii of the curved sur- 
faces, r-r' in Figs. 4-9, and joining the peripheral ends of the radii. The optical 
center is the point on the axis cut by the line joining the radii. I11 Figs. 4-5 it is CH. /.] 
MICROSCOPE AND ACCESSORIES. 
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, (r') is the radius of the curved sur- 
face and (r) of the plane surface ; and the point where a line joining the ends of 
these radii crosses the axis is at the curved surface in each case. 
By a study of Fig. 4 it will be seen that if tangents be drawn at the peripheral 
ends of the parallel radii, the tangents will also be parallel and a ray incident at one 
tangential point and traversing the lens and emerging at the other tangential point 
acts as if traversing, and is practically traversing a piece of glass which has 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. 
Figs, io, ii.—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 is 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 zvhich 
the light comes. The principal focal dis- 
tance is the distance along the axis, from 
the optical center to the principal focus 
io 
ri 
| 6. 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. io 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. n 
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. 
Fig. 12.—Double Convex Lens, Showing Chromatic Aberration. 
The ray of white light (w) is represented as dividing into the short waved, blue 
(b) and the long waved, red (r) light. The blue (b) ray comes to a focus nearer 
the lens and the red ray (r) farther from the lefts than the principal focus (/). 
Principal focus (f) for rays very near the axis, f' andf", 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 andf". 
§ 7. 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). 
Fig. 13. The ray (0) near the 
edge of the lens is brought to a 
focus nearer the lens than the 
ray (i). Both are brought to 
a focus sooner than rays very 
near the axis, (f) Princi- 
pal focus for rays very near 
the axis ; (fy) Focus for the 
ray (i), and (fu) Focus for 
the ray (0). Intermediate 
rays would cross the axis all 
the way from (f" to f). 
Fig. 13. Double Convex Lens, showing 
Spherical Aberration. 
§ 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 rays (0 i) 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. /.] 
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 tinder-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 15. 14. Convex lens 
showing the position of the object (A-B) 
outside the principal focus {F), and 
the course of the rays in the formation 
of real images. To avoid confusion 
the rays are drawn from only one point. 
A B. Object outside the principal fo- 
cus. B' A'. Real, enlarged image on 
the opposite side of the lens. 
Axis. Principal optic axis. 1, 2, 3. 
Rays after traversing the lens. They 
are converging, and consequently form 
a real image. The dotted line and the 
line (2) give the direction of the rays 
as if unaffected by the lens. (F). The 
principal focus. 
Fig. 15. Convex lens showing the po- 
sition of the object (A B) within the 
principal focus and the course of rays 
in the formation of a virtual image. 
A B. The object placed between the lens and its focus; A' B' virtual image 
formed by tracing the rays backward. It appears on the same side of the lens as 
the object, and is erect ($ 9). 
Axis. The principal optic axis of the lens. F. The principal focus. 
/, 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 is shown by the 
dotted lines. Ray (/) traverses the center of the lens, and is therefore not deviated. 
It is a secondary axis (§5). 
14. 
15- 
$ 8a. Geometrical Construction of Images.—As shown in Figs. 14-15, f°r the 
determination of any point of an image, or the image being known, to determine 
the corresponding part of the object, it is necessary to know the position of the- 
principal focus (and there is one on each side of the lens, § 6), and the optical! 
center (Figs. 1-9) of the lens. Then a secondary axis, (2) in Fig. 14, (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.. [CH. I. 
6 
MICROSCOPE AND ACCESSORIES. 
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. 
SIMPIyE 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). 
Fig. i 6. Diagram of the simple microscope show- 
ing the course of the rays and all the images, and 
that the eye forms an integral part of it. 
A1 Bx. The object within the pruicipal focus. A3 
B3. The virtual image on the same side of the lens 
as the object. It is indicated with dotted lines, as it 
has no actual existence. 
B1 A2. Retinal image of the object (A1 B). The 
vir tual image is simply a projection of the retinal 
image in the field of vision. 
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 
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 CH. I.] 
MICROSCOPE AND ACCESSORIES. 
point represents approximately the principal focus (Figs, io, 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 
object must be within the principal focus. Com- 
pare (§ 49.). 
Fig. 17. 
Tripod, Magnifier. 
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 
Fig. 18. Achromatic Triplet for the pocket. As shown in the left hand figure it 
is composed of three lenses, one of crown and two of flint glass. The whole is 
protected by a metal covering when not in 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. 
% io. A Compound Microscope.—This enables one to see an enlarged, inverted 
image. It always consists of two optical parts—an objective, 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. 
\_CH. I. 
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). 
Fig. 19. Lens Holder with adjustments for focusing 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.). Fig. 20. Dissecting Microscope with hand rests and nose-piece for several lenses of 
different power. (Bausch & Lomb Optical Co.). 10 
MICROSCOPE AND ACCESSORIES. 
[CH. I. 
MECHANICAL PARTS. 
§11. 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. 
| 12. Microscopic Objective.—This 
consists of a converging lens or of one 
or more converging lens-systems, which 
give an enlarged, inverted, real image of 
the object (Figs. 14, 21). And as fortlie 
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 \\ 9, 
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 general, 
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 lenses are of crown 
and the concave lenses of flint glass 
(Figs. 22, 23). 
Fig. 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 
(A2 B'1), and the projection of the retinal 
image into the field of vision as the 
virtual image {BA A3). 
A B. The object. A1 B'1. The retinal 
image of the inverted real image, 
formed by the objective. BiA?’. The in- 
verted virtual image, a projection of 
the retinal image. CH. /.] 
MICROSCOPE AND ACCESSORIES. 
Axis. The principal optic axis of the microscope and of the eye. 
Cr. Cornea of the eye. 1*. Crystalline lens of the eye. R. Single, ideal, re- 
fracting surface at which all the refractions of the eye may be assumed to take 
place. 
F.F. The principal focus of the positive ocular and of the objective. 
Mirror. The mirror reflecting parallel rays to the object. The light is central. 
See Ch. II. 
Pos. Ocular. An ocular in which the real image is formed outside the ocular. 
Compare the positive ocular with the simple microscope (Fig. 16). 
§ 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 fz in. or 2 mm., indicates that the objective produces a 
real image of the same size as is produced by a simple converging lens whose prin- 
cipal focal distance is y 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 that the relative size of object and image vary directly as their distance 
from the center of the lens (Figs 14, 15, see Ch. IV,) it follows that the less the 
focal distance of the simple lens or of the equivalent focal distance of the 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. 
| 14 Numbering or Lettering Objectives.—Instead of designating objectives by 
their equivalent focus, many Continental opticians use letters or figures for this 
purpose. With this method the smaller the number, or the earlier in the alpha- 
bet the letter, the lower is the power of the objective. (See further in Ch. IV, for 
the power or magnification of objectives). This method is entirely arbitrary and 
does not, like the one above, give direct information concerning the objective. 
\ 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 (i. <?., )4th in. or 3 mm. and lower powers) are 
dry. 
Fig. 22. Section of a dry objective showing 
working distance and lighting by reflected 
light. 
Axis. The principal optic axis of the ob- 
jective. 
B C. Back Combination, composed of a 
plano-concave lens of flint glass [F), and a 
double convex lens of crown glass (t). 
F C. Front Combination. 
C, 0, si. The cover-glass, object and slide. 
Mirror. The mirror is represented as above 
the stage, and as reflecting parallel rays from 
its plane face upon the object. 
Stage. Section of the stage of the microscope. 
W. The Working Distance, that is the distance from the front of the objective to 
the object when the objective is in focus. 
NOMENCLATURE OR TERMINOLOGY OF OBJECTIVES. 12 
MICROSCOPE AND ACCESSORIES. 
[CH. I. 
\ 16. Immersion Objectives.—An immersion objective is one with which there 
is some liquid placed between the front of the objective and the object or cover- 
glass. The most common immersion objectives are those (A) in which water is 
used as the immersion fluid, and (B) where some liquid is used having the same 
refractive and dispersive power as the front lens of the objective. Such a liquid 
is called homogeneous, as it is optically homogeneous with the front glass of the 
objective. It may consist of thickened cedar wood oil or of glycerin 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. 
Fig. 23. Sectional view of an Immersion, Ad- 
justable Objective, and the object lighted with 
axial or central and with oblique light. 
Axis. The principal optic axis of the objective. 
B C, M C, F C. The back, middle and front 
combination of the objective, hi this case the 
front is not a combination, but a single piano 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. 
I 17. Non-Achromatic Objectives.—These are objectives in which the chro- 
matic aberration is not corrected, and the image produced is bordered by colored 
fringes. They show also spherical aberration and are used only on very cheap 
microscopes. (§§ 7, 8, Figs. 12, 13). 
$ 18. 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 (£ 19). 
That is the various spectral colors come to the same focus. 
$ 19. 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. (§ 7, 8). 
§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, Apocliromatic, 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 apocliromatic ob- 
jectives without using the fluorite Some of the apochromatics 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 two different colors could be brought to the same focus. 
(2) In these objectives the correction of the spherical aberration is obtained for 
two different colors in the brightest part of the spectrum, and the objective shows 
the same degree of chromatic correction for the marginal as for the central part of 
the aperture. I11 the old objectives, correction of the spherical aberration was 
confined to rays of one color, the correction being made for the central part of the 
spectrum, the objective remaining under corrected spherically for the red rays and 
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 apocliromatic objectives 
give as clear images as the same power water immersion objectives of the old form. 
§2i. Non-Adjustable or Unadjustable Objectives.—Objectives in which the 
lenses or lens systems are permanently fixed in their mounting so that their rela- 
tive position always remains the same. Tow power objectives and those with 
homogeneous immersion are mostly noil-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). 
§ 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). 
§ 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. 
[CH. 1. 
screw collar the combinations may be separated, diminishing the power or ap- 
proximated and thereby increasing it. 
§ 25. Projection Objectives.—These are designed especially for projecting an 
image on a screen and for photo-micrography. 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). 
§ 26. 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 
microscopist must know and use this distance or ‘ microscopical tube-length ’ to 
obtain the best results in using any objective in practical work.” Unfortunately 
different opticians have selected different tube-lengths and also different points 
between which the distance is measured, so that one must know what is meant by 
the tube-length of each optician whose objectives are used. See table. 
The thickness of cover-glass used on an object (see Ch. VII, on mounting), 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. /.] 
MLCROSCOPE AND ACCESSORIES. 
Length in Millimeters and Parts included in “ Tube-Length ” by 
Various Opticians * 
Pts. included 
in “ Tube- 
length.” 
See Diagram. 
“ Tube-length ” in 
Millimeters. 
Grunow, New York 203 mm. 
E. Leitz, Wetzlar 170 mm. 
Nachet et P'ils, Paris . . * * 146 or 200 mm. 
Powell and Lealand, London ..... 254 mm. 
C. Reichert, Vienna 160 to 180 mm. 
Spencer Lens Co., Buffalo 235 or 160 mm. 
W. Wales, New York 254 mm. 
a-d 
Bausch & Lomb Opt. Co., Rochester. . 216 or 160 mm. 
Bezu, Hausser et Cie, Paris 220 mm. 
Klonne und Muller, Berlin i6o-i8oor254mm. 
W. & H. Seibert, Wetzlar 190 mm. 
Swift & Son, London 165 to mm. 
C. Zeiss, Jena 160 or 250 mm. 
b-d 
Gundlach Optical Co., Rochester . . . 254 mm. 
R. Winkel, Gottingen 220 mm. 
Ross & Co., London 254 mm. 
R. & J. Beck, London 254 mm. 
J. Green, Brooklyn 254 mm. 
Hartnack, Potsdam 160-180 mm. 
Verick (Stiassnie) Paris 160-200 mm. 
Watson & Sons, London 160-250 mm. 
a-g 
a-g 
c-d 
c-e 
c-f 
Fig. 24. 
Thickness of Cover-Glass for Which Non-Adjustable Objectives are Corrected by 
Various Opticians. 
J. Green, Brooklyn. 
J. Grunow, Brooklyn. 
Powell and Lealand, London. 
Spencer Lens Co., Buffalo. 
W. Wales, New York. 
Watson & Sons, London. 
Klonne und Muller, Berlin. 
E. Leitz, Wetzlar. 
R. Winkel, Gottingen, Germany. 
Ross & Co., London. 
Bausch & Lomb Optical Co., Rochester. 
C. Zeiss, Jena. 
C. Reichert, Vienna. 
Gundlach Optical Co., Rochester. 
W. & H. Seibert, Wetzlar. 
R. &J. Beck, London. 
J. Zentmayer, Philadelphia. 
Nachet et Fils, Paris. 
Bdzu, Hausser et Cie, Paris. 
Swift and Son, London. 
TffVnm. 
iV8onim- 
xV^mm. 
-^fff-mm. 
x^mtn. 
Wmm. 
ll(5XSmm. 
if^Hum. 
V^-nnn. 
mmm. 
*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. i6 
MICROSCOPE AND ACCESSORIES. 
[CH. I. 
§ 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 
[i. 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). If the table in this edition is compared with the original table or with that in 
the previous edition of this book some differences will be noted, the changes being 
in the direction of uniformity and in general in the direction recommended by the 
writer and Mr. Bauscli and the committee of the American Microscopical Society. 
The recommendations of the committee, published in the Proceedings, Vol. XII, 
p. 250, are as follows : 
“Believing in the desirability of a uniform tube-length for microscopes, wTe 
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 i—Be, for the short 
or continental tube, 160 mm., or 6.3 inch- 
es, and 216 mm., or 8% 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. inch) and shorter focus 
should be such as to bring the optical 
center of the objective I'/i. inches below 
the shoulder, and that all objectives be 
marked with the tube-length for which 
they are corrected. 
5. That non-adjustable objectives be 
corrected for cover-glass from to 
mm. (x}x to x|x inch) in thickness. 
These recommendations give a distance 
of 10 inches (254 mm.) between the par- 
focal plane of the ocular and the optical 
center of the cbjective for the long tube, 
and are essentially in accord with the 
actual practice of opticians. 
At the request of the committee, a joint 
conference was held with the 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.” 
Fig. 25. The tube of a microscope with 
ocular micrometer and nose piece in 
position to show that in measuring 
tube-length one must measure from 
the eye lens to the place where the ob- 
jective is attached. {Zeiss' Catalog, 
No. jo). 
(Signed) Simon H. Gage, 
A. Clifford Mercer, 
Charles E. Barr. 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- 
Fig. 26. Diagram illustrating the angular aperture of a 
microscopic objective. Only the front lens of the objective is 
shown. 
Axis, the principal optic axis of the objective. 
BA, B C, the most divergent rays that can enter the objective, 
they mark the angular aperture. A B D or C B D half the 
angular aperture. This is designated by u in making Nu- 
merical Aperture computations. See the table, \ 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). I11 order 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 “ Numerical Aperhtre,” (N. 
A.) 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. e., 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 18 
MICROSCOPE AND ACCESSORIES. 
[C/I. I. 
is N. A. = 71 sin u ; N. A. representing numerical aperture, 7i the in- 
dex of refraction of the substance in front of the objective, and 7i the 
semi-angle of aperture. 
For example, take three objectives each of 3 mm. 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 
1060, the water immersion of 940 and the homogeneous immersion of 
90°. Simply compared 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 
Ellenberger, and are introduced to illustrate 
the relative amount of utilised light, with dry, 
water immersion and homogeneous immer- 
27 sion objectives of the same equivalent focus. 
The point from which the rays emenate is 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). 
Fig. 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. 
Fig. 28. Rays from the axial point of the 
object traversing a cover of the same thick- 
ness as in Fig. 26, and entering the front lens 
of a water immersion objective. 
Fig. 29. Rays from an axial point of the 
29 object traversing a cover-glass and entering 
the font of a homogeneous immersion objec- 
tive. 
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 1060, N. A. = n sin u. In the case of dry ob- 
jectives the medium in front of the objective being air, the index of 
refraction is unity, whence n = 1. Half the angular aperture is -f-° = 
530. By consulting a table of natural sines it will be found that the 
sine of 530 is 0.799, whence N. A. = n or 1 X sin u or 0.799 = 0.799. 
With the water immersion objective in the same way N. A. = n sin u. 
I11 this case the medium in front of the objective is water, and its index 
of refraction is 1.33, whence 11 = 1.33. Half the angular aperture is 
V'° — 47°j and by consulting a table of natural sines, the sine of 470 is CH. /.] 
MICROSCOPE AND ACCESSORIES. 
19 
found to be 0.731, i. <?., sin u = 0.731, whence N. A. —n or 1.33 X sin 77 
or 0.731 = 0.972. 
With the oil immersion in the same way N. A. = n sin u ; 71 or the 
index of refraction of the homogeneous fluid in front of the objective is 
1.52, and the semi-angle of aperture is \°-° = 450. The sine of 450 is 
0.707, whence N. A. = 71 or 1.52 X sin u 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 (i. 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. = 71 sin u one has N. A. = 0.80 for 
all objectives. 
For the dry objective 71 — 1 (Refractive index of air). 
“ water immersion objective 71 = 1.33 (Refractive index of water). 
“ homogeneous immersion objective ti = 1.52 (Refractive index 
of homogeneous liquid). And 2 u is to be found in each case. 
For the dry objective, substituting the known values the formula 
becomes 0.80 = 1 sin zi, or sin u = 0.80. By inspecting the table of 
natural sines (3d page of cover) it will be found that 0.80 is the sine of 
53 degrees and 8 minutes. As this is half the angle the entire angular 
aperture of the dry objective must be 530 8' X 2 = 106° 16'. 
For the water immersion objective, substituting the known values 
in the formula as before : 0.80 = 1.33 sin u, or sin u = Q = 0.6015. 
i-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' 
X 2 = 730 58'. 
For the homogeneous immersion objective, substituting the known 
values, the formula becomes : 0.80 = 1.52 sin u whence sin u — 
= 0.5263. And by consulting the table of sines it will be found 
that this is the sine of 310 whence 2 u or the entire angle (balsam 
or oil angle) is 63° 31'. 
That is, three objectives of equal resolving powers, each with a nu- 
merical aperture of 0.80 would have an angular aperture of 1060 16' in 
air, 730 58' in water, and 63° 31' in homogeneous immersion liquid. 
For the apparatus and method of determining aperture, see appendix. 20 
MICROSCOPE AND ACCESSORIES. 
[CH. I. 
§ 30. Table of a Group of Objectives with the Numerical Aper- 
ture (N. A.) a?id 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 and n = 1, for water 
immersions n — 1.33, and for homogeneous immersions n = 1.52. (For 
a table of natural shies, see third page of cover). 
Objective. 
Angular 
Aperture 
(2 u) 
Naturae Sine 
of half the angular 
aperture. 
(sin u). 
Index of 
Refraction 
of the medi- 
um in front 
of the objec- 
tive. (n). 
Numerical Aperture 
(N.A.) = 11 sin u. 
25 mm. 
(Dry.) 
20° 
Sin — =0.1736 
2 
n — 1 
N.A. = 1X0.1736 = 0.173 
25 mm. 
(Dry.) 
40° 
Sin = 0.3420 
2 
n = 1 
N.A. = 1 Xo 3420 = 0.342 
12y2 mm. 
(Dry.) 
420 
Sin =0.3583 
2 
n — 1 
N.A. = 1 X 0.3583 = 0.358 
\2]/2 mm. 
(Dry.) 
IOO° 
Sin I<3C) = o 7660 
2 
« = 1 
N. A. = 1 X 0.7660 = 0.766 
6 mm. 
(Dry.) 
75° 
Sin = 0 6087 
2 
n = i 
N. A. = 1X0.6087 = 0.608 
6 mm. 
(Dry.) 
136° 
Sin = 0.9272 
2 
n = 1 
N.A. = 1X0.9272=0.927 
3 mm. 
(Dry.) 
1150 
Sin1 = 0.8434 
2 
« = 1 
N. A. = 1 X 0.8434 — 0.843 
3 mm. 
(Dry.) 
163° 
Sin = 0.9890 
2 
n = 1 
N.A. = 1 X0.9890 = 0.989 
2 mm. 
Water. 
Immersion. 
96°I2/ 
Sin 96° I2'_ 07443 
2 
« = i-33 
N.A. = 1.33 X 0.7443 = o-99 
2 mm. 
Homogeneous 
Immersion. 
iio°38' 
Sin iio038'_o.8223 
2 
« = 1.52 
N.A. = 1.52 X0.8223 = 1.25 
2 mm. 
Homogeneous 
Immersion. 
1340 IOr 
SinI34°io'_0 2IO 
2 
n =1.52 
N.A. = 1.52 Xo.92io= 1.40 
§ 31- Significance of Aperture.—As to the real significance of 
aperture in microscopic objectives, it is now an accepted doctrine that— 
the corrections in spherical and chromatic aberration being the same— 
(i) Objectives vary directly as their numerical aperture in their ability 
to define or make clearly visible minute details (resolving power). For 
example an objective of 4 mm. equivalent focus and a numerical aper- 
ture of 0.50 N. A. would define or resolve only half as many lines to CH. /.] 
MICROSCOPE AND ACCESSORIES. 
21 
the millimeter or inch as a similar objective of i.oo N. A. So also an 
objective of 2 mm. focus and 1.40 N. A. would resolve only twice as 
many lines to the millimeter as a 4 mm. objective of 0.70 N. A. Thus 
it is seen that defining power is not a result of magnification but of 
aperture, otherwise the 2 mm. objective would resolve far more than 
twice as many lines as the 4 mm. objective. 
(2) The illuminating power of an objective of a given focus is found 
to vary directly as the square of the numerical aperture (N. A. )2. Thus 
if two 4 111m. 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 0.202 : o.402= 0.0400 : o. 1600 or 1 : 4. That is the 
objective of 0.20 N. A. would have but 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 3 mm. and a 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 62 : 32 or 36 to 9 or 4 : i, 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 111m. is 42 
and of the 3 mm. 84, whence the illuminating power of the two objec- 
tives are as 42s : 842 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 111m. 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 —- 
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 42 : 22 or 16 : 4 or 4 : 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 OCUEAR. 
\ 32 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. 
Fig. 30. Sectional view of a Hnygenian ocular (Hg. ocu- 
lar), to show the formation of the Eye-Point. 
Axis. Optic axis of the ocular. D. Diaphragm of the 
ocular. E. L. Eye-Lens. F. L. Field-Lens. 
E.P. Eye-point. As seen in section, it appears something 
like an hour-glass. When seen as in looking into the ocu- 
lar, i. e., 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. 
\ 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 
absent (Fig. 21). As the field-lens of the ocular aids in the 
formation of the real image it is considered by some to form 
a part of the objective rather than of the ocular. The upper 
or eye lens of the ocular magnifies the real image. 
§ 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 CH. /.] 
MICROSCOPE AND ACCESSORIES. 
23 
eye-lens toward the object and looking into the field-lens an image may be seen, 
however. 
Special names 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 § 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 Tolies and 
is constructed on true stereotomic principles, both fields being equally illuminated. 
His ocular is also erecting. Campani's Ocular (see Huygenian Ocular). Com- 
pound Ocular ; Fr. Oculaire compose ; Ger. zusammengesetztes Okular. An ocu- 
lar of two or more lenses, e. g., the Huygenian (see Fig. 30). Continental Ocular. 
An ocular mounted in a tube of uniform diameter as in Fig. 31. Deep Ocular, 
see high ocular. Erecting Ocular ; Fr. Oculaire redresseur ; Ger. bildumkeh- 
rendes Okular. An ocular with which an erecting prism is connected so that the 
image is erect as with the simple microscope. Such oculars are most common on 
dissecting microscopes. Filar micrometer Ocular ; Screw m. o. Cobweb m. o. 
Ger. Okular-Schraubenmikrometer. A modification of Ramsden’s Telescopic Cob- 
web micrometer ocular. Goniometer Ocular ; Fr. Oculaire a goniometre ; Ger. 
Goniometer-Okular. An ocular with goniometer for measuring the angles of mi- 
nute crystals. High Ocular, sometimes called a deep ocular. One that magnifies 
the real image considerably, i. e., 10 to 20 fold. Huygenian Ocular, Huygens’ O., 
Campani’s O., Airy’s O. ; Fr. Oculaire d’Huygens, o. de Campani ; Ger. Huy- 
gens’sches Okular, Campaniches Okular, see \ 35. Index Ocular ; Ger. Spitzen- 
O. An ocular with a minute pointer or two pointers at the level of the real image. 
The points are movable and serve for indicators and also, although not satisfacto- 
rily for micrometry. Kellner's Ocular, see orthoscopic ocular. Low Ocular, also 
called shallow ocular. An ocular which magnifies the real image only moderate- 
ly, i. e., 2 to 8 fold. Micrometer or micrometric Ocular ; Fr Oculaire microme- 
trique ou a micrometre; Ger. Mikrometer-Okular, Mess Okular, Bdneches O., 
Jackson m. o., see § 38. Microscopic Ocular ; Fr. Oculaire microscopic ; Ger. mi- 
kroskopisches Okular. An ocular for the microscope instead of one for a telescope. 
Negative Ocular, see § 33. Nelson’s screw-micrometer ocular. A modification of 
the Ramsden’s screw or cob-web micrometer in which positive compensating ocu- 
lars may be used. Orthoscopic Oculars ; also called Kellner’s Ocular ; Fr. Ocu- 
laire orthoscopique ; Ger. Kellner’sches oder orthoskopisches Okular. An ocular 
with an eye-lens like one of the combinations of an objective (Figs. 22, 23) and a 
double convex field lens. The field-lens is in the focus of the eye-lens and there 24 
MICROSCOPE AND ACCESSORIES. 
[CH. I. 
| 35. 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 
Fig. 31. Compensating Oculars of Zeiss, with section removed to show the con- 
struction. The line A-A is at the level of the upper end of the tube of the micro- 
scope while B-B represents the lower focal points. It will be seen that the mount- 
ing is so arranged that the lower focal points in all are in the same plane and 
therefore the microscope remains in focus upon changing oculars. (The oculars are 
par focal). The lower oculars, 2, 4 and 6 are negative, and the higher ones, 8, J2 
18, are 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. Par-focal Oculars, a series 
of oculars so arranged that the microscope remains in focus when the oculars are 
interchanged (Peunock, Micr. Bulletin, vol. iii, p. 9, 31). Periscopic Ocular ; Fr. 
Oculaire periscopique ; Ger. periskopisclies 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 $ 37. 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 § 36. 
Shallow Ocular, see low ocular. Solid Ocular, holosteric O. ; Fr. Oculaire liolo- 
stere ; Ger. holosterisches Okular, Vollglass-Okular. A negative eye-piece de- 
vised by Tolies. 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 wThere a high ocular is desired. Spectral or 
spectroscopic Ocular ; Fr. Oculaire spectroscopique ; Ger. Spectral-Okular, see Mi- 
crospectroscope, Cli. 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. Arbeits Okular, 
see \ 36. CM. /.] 
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, magnify ing 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). 
£ 37. 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. 
Fig. 32. Projection Oculars with section re- 
moved to show the construction. Below are 
shown the zipper ends with graduated circle to in- 
dicate the amount of rotation found necessary to 
focus the diaphragm on the screen. No. 2, No. 
f. The numbers indicate the amount the ocular 
magnifies the image formed by the objective as 
with the compensation oculars. (Zeiss' Catalog, 
No. jo], 
\ 3S. Micrometer Ocular.—This 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. 
Figs. 33-34 Ocular Micrometer with movable scale. Fig. 33 is a side view of 
the ocular while Fig. 34 gives a sectional end view, and shows the ocular microme- 
ter in position. In both the screw which moves the micrometer is shown at the 
left. (From Bausch & Lomb Opt. Co.) 26 
MICROSCOPE AND ACCESSORIES. 
{CH. I. 
Fig. 35- Ocular Screw-Micrometer with 
compensation ocular 6. The upper figure 
shows a sectional view of the ocular and the 
screw for moving the micrometer at the right. 
At the left is shown a clamping screw to 
fasten the ocular to the upper part of the mi- 
croscope tube. Below is a face view, showing 
the graduation on the wheel. An ocular 
micrometer like this is in general like the 
cob web micrometer and may be used for 
measuring objects of varying sizes very accu- 
rately. With the ordinary ocular microme- 
ter very small objects frequently fill but a part 
of an interval of the micrometer, but with this 
the movable cross lines traverse the object (or 
rather its 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. 
§ 40. Equivalent Focus.—As with objectives, some opticians designate the ocu- 
lars by their equivalent focus (§ 13). With this method the power of the ocular, 
as with 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. 
Atner. Micr. Soc., 1883, p. 175, 1884, p. 228). 
$ 4r. 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. 
\ 42. 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 the}' multiply the 
real image of the objective. Thus for the short or 160 mm. tube-length they are, 
X 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. VIII. 
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 CH. /.] 
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. 
§ 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. Gi-66). 
§ 46. Field or Field of View of a Microscope.—This is the area 
visible through a microscope when it is in focus. When properly lighted, 
and there is no object under the microscope, the field appears as a circle 
of light. When examining an object it appears within the light circle, 
and by moving the object, if it is of sufficient size, different parts are 
brought successively into the field of view. 
In general, the greater the magnification of the entire microscope, 
whether the magnification is produced mainly by the objective, the 
ocular, or by increasing the tube-length, or by a combination of all 
three (see Ch. IV, under magnification), the smaller is the field. 
Fig. 36. Triple nose-piece 
or revolver for quickly chang- 
ing objectives (Queen & Co.). 28 
MICROSCOPE AND ACCESSORIES. 
[CH. I. 
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 in Fig. j 7. See also § 4.6. 
Equivalent 
Diameter 
Focus and 
N. A. of 
of Field 
Ocular. 
Objective. 
in mm. 
154 
37 /4 m m. 
85 mm.. . . 
10 6 
25 
Huygenian. 
8-3 
12 “ 
7.0 
37)4 mm. 
45 mm. . . . 
5-0 
25 
Huygenian. 
4.0 
I2}4 “ 
3-0 
37/4 mm. 
17 mm. . . . 
2.0 
25 
Huygenian. 
1.6 
12 >4 “ 
5-7 
180 mm. 
N.A. =0.25 
2 8 
1.4 
45 
15 
Compensation. 
0.97 
10 “ 
0541 
37)4 mm. 
5 mm. . . . 
0 371 
25 
Huygenian. 
0.290 
12)4 “ 
0.850 
180 mm. 
N.A. =0 92 
0.501 
0 250 
45 
15 
Compensation. 
0.173 
10 “ 
0.270 
37)4 “ 
2 mm. . . . 
0.186 
25 
Huygenian. 
0.147 
12)4 “ 
0.450 
180 mm. 
N.A. = 1.25 
0.251 
0.125 
45 
15 
Compensation. 
0.088 
10 “ CH. /.] 
MICROSCOPE AND ACCESSORIES. 
29 
45 m m 
17 mm 
Fig. 37. Figures showing approximately the actual size of the field with ob- 
jectives of 85 mm., 45 mm., 77 mm., 5 mm., and 2 mm., equivalent focus, and 
ocular of 37'fi. mm., equivalent focus in each case. This figure shows graphically 
what is also very clearly indicated in the table (§ 47). 
8 j m m 
§ 48. The size of the field of the microscope as projected into the 
field of vision of the normal human eye (z. 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. Pat a 2-in. (50 mm.) objective on the microscope or screw off 
the front combination of a 16 mm., 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 011 the screen. 
The image can be more clearly seen if the object is in a strong light 
and the screen in a moderate light, i. <?., 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 
for a few minutes. If the glass becomes too opaque, it may be rendered more 
translucent by rubbing some oil upon it. MICROSCOPE AND ACCESSORIES. 
[CII. I. 
30 
To demonstrate that the object must be outside the principal focus 
with the compound microscope, remove the screen and turn the tube of 
the microscope directly toward the sun. Move the tube of the micro- 
scope with the coarse adjustment until the burning or focal point is found 
(§ 6). Measure the distance from the paper object on the stage to the 
objective, and it will represent approximately the principal focal dis- 
tance (Figs, io, n). Replace the screen over the top of the tube, no 
image can be seen. Slowly raise the tube of the microscope and the 
image will finally appear. If the distance between the object and the 
objective is now taken, it will be found considerably greater than the 
principal focal distance (compare § 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. Uook at the image 
with a simple microscope (Fig. 17 or 18) as if the image were an object. CH. /.] 
MICROSCOPE AND ACCESSORIES. 
Observe that the image seen through the simple microscope is merely 
an enlargement of the one on the screen, and that the letters remain 
inverted, that is they appear as with the naked eye (§ 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 10 to 20 milli- 
meters from the eye-lens and look into the microscope. The letters 
will appear as when the simple microscope was used (see above), the 
image will become more distinct by slightly raising the tube of the 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 
Fig. 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. 
A1 Bl. The object within the principal focus. A3 
B3. The virtual image on the same side of the lens 
as the object. It is indicated with dotted lines, as 
it has no actual existence. 
BL A2. Retinal image of the object (A1 Bl). The 
virtual image is simply a projection of the retinal 
image in the field of vision. 
Axis. The principal optic axis of the microscope 
and of the eye. Cr. Cor nea of 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. 
certain definite lines and not in all directions ; 
hence, in order to see this aerial image with 
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. 
[67/. /. 
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). Tight the object brightly, focus the micro- 
scope, shade the ocular, then hold some ground-glass or a piece of the 
lens paper above the ocular and slowly raise and lower it until the 
smallest circle of light is found. By using different oculars it will be 
seen that the eye-point is nearer the eye-lens in high than in low ocu- 
lars, that is the eye-point is nearer the eye-lens for an ocular of small 
equivalent focus than for one of greater focal length. 
REFERENCES FOR CHAPTER I. 
In the appendix will be given a bibliography, with full titles, of the works and 
periodicals referred to. 
For the subjects considered in this chapter general works on the microscope 
may be consulted with great advantage for different or more exhaustive treatment. 
The most satisfactory work in English is Carpenter-Dallinger. For the history of 
the microscope, MayalPs Cantor Lectures on the microscope are very satisfactory. 
See also Beale, E Bausch, Beherens, 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, mo; same, 1890, p.480; Zeit. f. In- 
strumentenk., 1890, pp. 1-6 ; Micr. Built., 1891, pp. 6-7. 
Tube-length, etc. Gage, Proc. Amer. Soc. Micrs., 1887, pp. 168-172 ; also in the 
Microscope, the Jour. Roy. Micr. Soc., and in Zeit. wiss. Mikr., 1887-8, Bausch, 
Proc. Amer. Soc. Micrs., 1890, pp. 43-49 ; also in the Microscope, 1890, pp. 289-296. 
Aperture. J. D. Cox, Presidential Address, Proc. Amer. Soc. Micrs., 1884, pp. 
5-39. Jour. R°y- Micr- Soc., l88r> PP- 303, 348, 365, 388 i l882- PP- 300, 46o ; 1883, 
p. 790 ; 1884, p. 20. CHAPTER II. 
LIGHTING AND FOCUSING; MANIPULATION OF DRY, 
ADJUSTABLE AND IMMERSION OBJECTIVES; CARE 
OF THE MICROSCOPE AND OF THE EYES; 
LABORATORY MICROSCOPES. 
APPARATUS AND MATFRIAB 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 ($ 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 
($ 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. 
I 56. Focusing is mutually arranging an object and the microscope so that a 
clear image may be seen. 
With a simple microscope (§ 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 the tube or 
body of the microscope is raised or lowered (frontispiece). 
In general, the higher the power of the whole microscope whether simple or 
compound, the nearer together must the object and objective be brought. With 
the compound microscope, the higher the objective, and the longer the tube of 
the microscope, the nearer together must the object and the objective be brought. 
If the oculars are not par-focal, the higher the magnification of the ocular, the 
nearer must 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 X^1 in., or 6 mm. objective would not be X^1 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 34 
LIGHTING AND FOCUSING. 
\CH. II. 
high objectives, it would be necessary to use very thin cover-glasses over the ob- 
ject. (See \ 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. 
l 58. Unmodified sunlight should not be employed except in special cases. 
North light is best and most uniform. When the sky is covered with white clouds 
the light is most favorable. To avoid the shadows produced by the hands in 
manipulating the mirror, etc., it is better to face the light ; but to protect the eyes 
and to shade the stage of the microscope some kind of screen should be used. 
The one figured in (Fig. 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. 
Figs. 39-40. For full explanation see Figs. 22 and 23 
\ 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. //.] 
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 and for special purposes, special illuminating appa- 
ratus has been devised (£ 26). (See also Carpenter-Dallinger, p. 278). 
\ 60. 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 ($ 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. 
\ 62. Oblique Light.—This is light in which parallel rays from a plane mirror 
form an angle with the optic axis of the microscope (Fig. 40). Or if a concave 
mirror or a condenser is used, the light is oblique when the axial ray of the cone 
of light forms an angle with the optic axis (Fig. 49). 
DIAPHRAGMS. 
§ 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 some cases an iris diaphragm is used, and then the same 
one is capable of giving a large range of openings. The object of a diaphragm, in 
general, is to cut off all adventitious light and thus enable one to light the object 
in such a way that the light finally reaching the microscope shall all come from 
the object or its immediate vicinity. The diaphragms of a condenser serve to vary 
its aperture to the needs of each object and each objective. 
$ 64. 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 diaphragm should be as close as possible to the 
object in order, (a) that it may exclude all adventitious light from the object; (b) 
that it may not interfere with the most efficient illumination from the mirror by 
cutting off a part of the illuminating pencil. If the diaphragm is a 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 unless it swings with the mirror ; (3) it cuts off a part 
of the illuminating cone from a concave mirror. On the other hand, even with a 
small diaphragm, the whole field will be lighted. 
With an illuminator or condenser (Figs. 41, 48), the diaphragm serves to narrow 36 
LIGHTING AND FOCUSING. 
[CH. II. 
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 
(I 88; Fig. 51). 
ARTIFICIAL ILLUMINATION. 
$65. 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 : EXPHRIMENTS. 
§ 66. Lighting with a Mirror.—Place a mounted fly’s wing under 
the microscope, put the 16 mm. 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 i-nto position and light the field with it. As the con- 
cave mirror condenses the light, the field will look brighter wTith 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 If16 greatest illumination is to be obtained from the concave 
mirror, its position must be such that its focus will be at the level of the 
object. This distance can be very easily determined by finding the 
focal point of the mirror in full sunlight. 
§ 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 111m. (fi 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. //.] 
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 a cm. 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. Put a high ocu- 
lar in place of the low one (§ 40). 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. 
(yi in.) objective as indicated. Put the 3 mm. (j4 in.) or a higher ob- 
jective in place, and use a low ocular. 
Eight well, and employ the proper opening in the diaphragm, etc. 
(§ 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 may 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 changingoculars, the instrument nearly always needs slight focusing.. 
With low powers this may not be necessary. 38 
LIGHTING AND FOCUSING. 
[CH. II. 
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 011 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 10 cm. long which shall be exceedingly thin 
at one end and about 20 mm. thick at the other. Place a slide on the 
stage and some dust on the slide. Do not use a cover-glass. Focus the 
dust carefully first with the low then with the high objective. When 
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 iths mm. and 
the tripod to see the divisions. Or one may use a 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, //.] 
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 is in focus. The distance the objective was 
raised plus the thickness of the cover-glass represents the working dis- 
tance. For example, a 3 mm. objective after being brought in contact 
with 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 .01 mm., consequently the objective was 
raised .16 mm. As the cover-glass on the specimen used was .15 mm. 
the total working distance is .16 + .15 = .31 mm. 
CENTRAE AND OBRIQUE EIGHT 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 a 3 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 Pleurasigma 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. Ill, under Air-bubbles). 
CONDENSERS OR IEEUMINATORS.* 
§ 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. LIGHTING AND FOCUSING. 
40 
[CH. II. 
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.—It 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-micrograpliy 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 Uealand (Car- 
He says of illumination in general : “ The art of illuminating microscopic objects 
is not of less importance than that of preparing them for observation.” ‘‘The eye 
should be protected from all extraneous light, and should not receive any of the 
light which proceeds from the illuminating center, excepting that portion of it 
which is transmitted through or reflected from the object.” So likewise the value 
and character of the substage condenser was thoroughly understood and pointed 
out by him as follows : ‘‘I have uo 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, and the Edin- 
burgh Journal of Science, new series, No. 11 (1831), p. 83. CH. //.] 
LIGHTING AND FOCUSING. 
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, i. e., 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. 
Fig. 4r. Zeiss' Achromatic Conden- 
ser. c.s c.s. Centering screws for 
changing the position of the condenser 
and making its axis continuous with 
that of the microscope. A segment of 
the condenser is cut away to show the 
combinations of lenses. For very low 
powers the upper lens is sometimes 
screwed off. There is an iris dia- 
phragm between the middle and lower 
combinations. (Zeiss' Catalog, No. 
30) 
§ 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, 
i. e., at the apex of the cone of light given by the condenser. 
The centering is most conveniently accomplished as follows although 
daylight may be used with almost equal facility : 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. 
[CM. II. 
the small circle uncovered by the metal diaphragm. For the hole a 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. 
Fig. 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 of the field of the microscope. 
Fig. 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. 
Fig. 43. 
§ 78. Centering the Image of the Source of Illumination.—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 (Figs. 34, 52). CH. //.] 
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. 
Fig. 44. Shows the image of the 
flame (FI.) in the center (C) of the 
field of the microscope and illuminat- 
ing the object. 
Fig. 45. Shows the image of the 
flame (FI.) at one side of the center 
(.Exc.) and not properly illuminating 
the object. 
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. 
Fig. 44. 
Fig. 45. 
Fig. 46. 
Fig. 47. 
Figs. 46-47. Figures showing the dependence of the objective upon the illumi- 
nating cone of the condenser. [Nelson.) 
Fig. 46 if). The illuminating cone from the condenser [Ilium). This is 
seen to be just sufficient to fill the objective [Obj.). 
(B). The back lens of the objective entirely filled with light, showing that the 
numerical aperture of the illuminator is equal to that of the objective. 
Fig. 47. (A). In this figure the illuminating cone from the condenser [Ilium.) 
is seen to be insufficient to fill the objective [Obj.). 
[B). The back lens of the objective only partly filled with light, due to the re- 
stricted aperture of the illuminator. 44 
LIGHTING AND FOCUSING. 
{CH. II. 
§ 8o. 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 16 mm. objective and opening the diaphragm of the condenser till the 
back lens is just filled with light. Then if one uses a 3 or 4 mm. ob- 
jective it will be seen that the back lens of the higher objective is only 
partly filled with light, and to fill it the diaphragm must be much more 
widely opened. 
With a condenser, then, the diaphragm has simply to regulate the 
aperture of the illuminating cone, and has nothing to do with lighting 
a large or a small field. 
With the condenser, there are two conditions that must be fulfilled,— 
the proper aperture must be used, and that is determined by the dia- 
phragm, and secondly the whole field must be lighted. The latter is 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. //.] 
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 : Put a 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 small drop of ink, as from a pen- 
point, 011 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 may 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 arrangements of the 
illuminator are 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 Uealand 
were selected as models the condensers need not be so bulky, and still 
retain all their efficiency. 46 
LIGHTING AND FOCUSING. 
[CH. II. 
§ 84. Mirror and Light for the Abbe Condenser.—It is best to 
use light with parallel rays. The rays of daylight are practically par- 
allel ; it is best, therefore, to employ the plane mirror for all but the 
lowest powers. If low powers are used the whole field might not be 
illuminated with the plane mirror when the condenser is close to the 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 nun. (}i 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 § 104). CH. //.] 
LIGHTING AND FOCUSING. 
47 
§ 88. Dark-Ground Illumination.—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- 
5°- 
51- 
Figs. 48-51. Sectional views of the Abbe Illuminator of 1.20 N. A. showing 
various methods of illumination (§ 8f). 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 is centered, that is its optic axis is a prolongation of the optic axis of the 
microscope. 
S. Axis. Secondary axis. In oblique light the central ray passes along a sec- 
ondary axis of the illuminator, and is therefore oblique to the principal axis. 
D D. Diaphragms. These are placed in sectional and in face views. The dia- 
phragm is placed between the mirror and the illuminator. In Fig. 49 the opening 
is eccentric for oblique light, and in Fig. 5/ the opening is a narrow ring, the 
central part being stopped out, and thus giving rise to dark-ground illumination 
(2**). 
Obj. 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. II. 
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. (fi 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) With 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 gives a 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). CH. //.] 
LIGHTING AND FOCUSING. 
49 
Fig. 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 COEOR IMAGES. 
$ 92- Refrattion 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 \ 79). LIGHTING AND FOCUSING. 
[CH. II. 
50 
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 is a 
great advantage. This combination is generally taken advantage of in histology. 
Fig. 89 is an example of a purely refractive image. 
Figs. 53-55.—Diagrams illustrating refraction in different media and at plane 
and curved surfaces. In each case the denser medium is represented by line shad- 
ing and the perpendicular or normal to the refracting stirface is represented by the 
dotted line N-N', the refracted ray by the bent line A C. 
\ 93. 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 toward 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 cy. 
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 
C//. By comparing Figs. 54, 55, in 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, aud 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 index of refraction. In 
the figures, f^J^— — index of refraction. Worked out completely iu Fig. 39, 
b Sin CBN' F 3 * 
A B N= 40°, CBN' = 28° 54'and -Sln 4°°- = = I>33 tf>> the index of 
Sin 28° 54' 0.48327 
refraction from air to water is 1.33. (See \ 30). 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 the sine in CH. II] 
LIGHTING AND FOCUSING. 
51 
$ 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 § 101). 
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 
also by increasing the tube-length for covers thinner 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 1 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 1 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 represent the desired angle of the sine. Or if 
the sine whose angle is to be found is nearer in size to the sine just greater proceed 
exactly as before, getting the difference in the sines, but subtract the number of 
minutes of difference and the result will give the angle sought. For example take 
the case in 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° 45A,. 
and o 48481, the sine of 290. Evidently then the angle sought must lie between 
28° 45' and 290. If the difference between 0.48481 and 0.48099 be obtained, 0.48481 
— o 48099 = 0.00382, and this increase for 15' be divided by 15 it will give the in- 
crease for 1 minute; 0.00382-7-15 = 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 x minute 
it will give the total minutes above 28° 45/ ; 0.00228-7-0.000254 = 9. That is the 
angle sought is 9 minutes greater than 28° 45' = 28° 54L 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 0.00154-7-0.000254 = 6. That is the 
sine of 290 is too great for the sine whose angle is to be found by 6 minutes, hence 
290 less 6/ = 2S0 54/, the angle sought. 52 
LIGHTING AND FOCUSING. 
[CH. II. 
and by shortening the tube-length for covers thicker than the standard 
(Fig- 57)- 
Fig. $6.—Effect of the cover-glass on 
the rays from the object to the objective 
{Ross). 
Axis. The projection of the optic 
axis of the microscope 
F. 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). 
It is for the correction of this disturbance, or so called “ negative aberration f 
produced by the cover-glass that objectives are fixed in their mounting for a given 
thickness of cover, or that the combinations miking up the objective are made ad- 
justable ; that is, so that the back combinations miy be brought nearer the front 
lens or combination as the cover thickens, and separated with a thinner cover or 
for uncovered objects. ? he thicker the cover, the nearer the front and back combi- 
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. 40). 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, i. e., the adjusting collar is CH. //.] 
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 briskl}r backwards and forwards in both directions from the first 
position. Observe the expansion of the dark outline of the object, both 
when within and when without the focus. If the greater expansion or 
coma is when the object is without the focus, or farthest from the ob- 
jective [i. e., in focusing up] , the lenses must be placed further asunder, 
or toward the mark uncovered [z. e., the adjusting collar is turned 
toward the zero mark as the cover-glass is too thin for the present ad- 
justment]. If the greater expansion is when the object is within the 
focus, or nearest the objective, [i. e., in focusing down] , the lenses must 
be brought closer together, or toward the mark covered, [z. e., the ad- 
justing collar should be turned away from the zero mark, the cover- 
glass being too thick for the present adjustment].” In most objectives 
the collar is graduated arbitrarily, the zero ( O) mark representing the 
position for tincovered objects. Other objectives have the collar graduated 
to correspond to the various thickness of cover-glasses for which the object- 
ive ?nay be adjusted. This seems to be an admirable plan ; then if one 
knows the thickness of the cover-glass on the preparation ( Ch. VIII) the 
adjusting collar may be set at a corresponding mark, and one will feel 
confident that the adjustment will be approximately correct. It is then 
only necessary for the observer to make the slight adjustment to compensate 
for the mounting medium or any variation from the standard length of 
the tube of the microscope. In adjusting for variations of the length of 
the tube from the standard it should be remembered that : (A) If the 
tube of the microscope is longer than the standard for which the ob- 
jective was corrected, the effect is approximately the same as thicken- 
ing the cover-glass, and therefore the systems of the objective must be 
brought closer together, i. e., the adjusting collar must be turned away 
from the zero mark. (B) If the tube is shorter than the standard for 
which the objective is corrected, the effect is approximately the same as 
diminishing the thickness of the cover-glass, and the systems must 
therefore be separated (Fig. 40). 
In using the tube-length for cover correction Shorten the tube for 
too thick covers and Lengthen the tube for too thin covers. 54 
LIGHTING AND FOCUSING 
[CH. II. 
Fig. 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) it will be seen that the effect of the 
cover glass is to bring the object nearer the objective, and the thicker the cover the 
nearer is the object brought to the objective. As shortening the tube serves to re- 
move the object, it neutralizes the effect of the thick cover, and if the cover is so 
thin that it does not elevate the object enough for the corrections of the objective, 
then an 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. //.] 
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 hiriiself 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 011 the cover-glass. If 
the objective is adjustable, follow the directions given in § 95. 
When one is through using a wrater 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. 
§ 9$- 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. (yyth 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 111m. 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. II. 
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 Bacillus 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. 
Fig. 58.—Screen for shading the microscope and the 
face of the observer. This is very readily constructed 
as shown in the figure by supporting a wire in a disc 
of lead, iron, or heavy wood. The screen is then com- 
pleted by hanging over the bent wire, cloth or manilla 
paper 30 x 40 cm. The lower edge of the screen should 
be a little below the stage of the microscope and the 
tipper edge high enough to screen the eyes of the ob- 
server. 
§ io2. Shading the Objedt.—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 in the 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. //.] 
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. 
§ 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,” i. 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 be touched with the fingers, for that will 
soil them. 
The glass of which the lenses are made is quite soft, consequently it 
is necessary that only soft, clean 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. II. 
§ io8. 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. 
If 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 1 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 eve-screen if necessary (Fig. 
59, 60) ; and divide the labor between the two eyes, i. e.. use one eye 
for observing the image awhile and then the other. In the beginning CH. //.] 
LIGHTING AND FOCUSING. 
59 
it is not advisable to look into the microscope continuously for more 
than half an hour at a time. One 
never should work with the micro- 
scope after the eyes feel fatigued. 
After one becomes accustomed to mi- 
croscopic observation he can work for 
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. 
Fig. 59. — Ward's Eye-Shade. 
Fig. 6o. Double Eye-Shade. This is 
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 
readily over the tube of the microscope. 
This is then covered on both sides with 
velveteen and a central slit (s) made in 
the cloth. This admits the tube of the 
microscope and holds the screen in posi- 
tion. It may readily be pulled from side to side and thus serves for either eye, or 
for the use of the eyes alternately. 
§ no. 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. 
[Cl/. II. 
§ ill. 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 that 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 instruments that have 
been carefully tested and found to conform to the standard. This would be done 
as a matter of business prudence on tlieir part, but it is believed by the writer that 
microscope makers are artists first and takejan 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. 
The 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 and when using the fine adjustment. There should be no sw’aying of 
the image with either if the light is central (§ 73). 
I 113. Testing the Optical Parts.—As stated in the beginning, this can be done 
satisfactorily only by an expert judge. It would be of very great advantage to the 
student if he could have the help of such a person. In no case is 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). 
TESTING THE MICROSCOPE. CH. //.] 
L A BORA TOR Y 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. in.) and 3 
nun. 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. An illuminator or substage condenser, and an Abbe 
camera lucida are also necessities, and a micro-spectroscope and a micro-polarizer 
are very desirable. 
Even in case all the optical parts cannot be obtained in the beginning, it is wise 
to secure a stand upon which all may be used when they are finally secured. 
As to the objectives. The best that can be afforded should be obtained. Cer- 
tainly at the present, the apochromatics stand at the head, although the best achro- 
matic objectives approach them very closely. 
\ 115. 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 adjustments for focusing should be two,—a coarse adjustment or rapid move- 
ment with rack and pinion, and a fine adjustment by means of a micrometer screw. 
Bo h 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. 
\ 116. 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 motion. 
It is a modification of the mechanical stage of Tolies. The modification consists 62 
LABORATORY MICROSCOPES. 
\_CH. II. 
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, 
Reitz 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, (IV,, pp. 25-30). 
§ 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, i e., a V shaped thread, sides of thread inclined to an 
angle of 550 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 microscope], not less than o 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 considerable difference in the “Society Screws,” and 
the object of the society in providing a universal screw was partly defeated. 
In 1884 the American Microscopical Society appointed Mr. Edward Bausch and 
Prof. William A Rogers upon a committee to correspond with the Royal 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 w7ere 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 ; i860, pp. 
103-104. (All to be found in Quar. Jour. Micr. Sci., o. s., vols. VI, VII and VIII). 
Proc. Amer. Micr. Soc., 1884, p. 274; 18S6, 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. CM. II.] 
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. 
\ 118. Marker tor Preparations. (Figs. 61-66).—This instrument consists cf 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. Atner, Micr. Soc., 1894, pp. 112-118). 
\ 119. 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 eyelash (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. II. 
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 
amoeba. 
Figs. 61-63. Sectional Views of the two Forms of the Marker. 
Fig. 61. The simplest form of marker. It consists of the part SS with the 
milled edge {M). This part bears the Society or objective screw for attaching the 
marker to the microscope. R. Rotating part of the marker. This bears the eccen- 
tric brush (B) at its lower end. The brush is on a wire ( W). This wire is eccen- 
tric. and may be made more or less so by bending the wire. The central dotted 
line coincides with the axis of the microscope. The revolving part is connected 
with the “ Society Screw ” by the small screw (5). 
Fig. 62. 55, R, and B. All parts same as with Fig. 6/, except that the brush 
is carried by a sliding cylinder the end view being indicated in Fig. 63. 
64. 
65- 
Figs. 64, 65, 66. Specimens Showing the Use of the Marker. 
66. 
In Fig. 64. a section of a series is marked to indicate that this section shows some- 
thing especially well. In Fig. 65 some blood corpuscles showing ingested carbon 
very satisfactorily are surrounded by a minute ring, and in Fig. 66 the lines of a 
micrometer are ringed to facilitate finding the lines. CH. //.] 
L A BORA TOR Y MICRO SCOPES. 
65 
Fig. 67. Krauss' 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. This is of great 
advantage and facilitates the chang- 
ing of objectives, as one can see what 
objective is coming into place with- 
out trouble. 
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 is never doubt as to 
the position of each from the record. 
Fig. 68. The Tolies Mayall mechanical stage as constructed by Leitz. It is shown in 
position on the stage of the microscope: it is fastened to the stage by a pin and screw 
near the pillar. 
Fig. 69. The Tolies Mayall mechanical stage made by the Bausch & Lomb Optical 
Company. It is separated from the microscope. It is 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 is still used by them 
Figs. 68-69. The Tolles-Mayall Mechanical Stage (<j 116) 66 
LABOR A TOR Y MICROSCOPES. 
[CH. II. 
Fig. 70. Zeiss' Microscope /a with Mechanical Stage. This figure, from Zeiss' 
Catalog No. 30, represents the Continental Model of Microscope in its most per- 
fect 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. 
IV. Screw for moving the stage forward and backward. CH. //.] 
LABOR A TOR Y MICROSCOPES. 
67 
Fig. 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 40 years. (See Carpenter-Dallinger, 
p. 172). 
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 it 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 
[CII. II. 
LABORATORY MICROSCOPES. 
Fig. 72. Natcliet & Fils' Medium Microscope No. 4 with Movable Stage. The 
French microscopes set the fashion for the Continent of Europe, and the Conti- 
nental or Hartnack Model with the horse shoe base has extended to all lands, but 
see Carpenter Dallinger, p. 20S. CH. II.] 
LABOR A TOR Y MICROSCOPES. 
69 
Fig. 73. The BB Microscope of the Bausch & Lomb Optical Co*. 70 
LA BOR A TOR Y MICROSCOPES. 
\CH. II. 
Fig. 74. Reichert's Stand III B, after Specifications by Richards & Co., N. Y. CH. //.] 
LABOR A TORY MICROSCOPES. 
71 
He. 75. Queen & Company's Microscope of the Continental Pattern, No. II. 72 
LABOR A TORY MICROSCOPES. 
{CM. II. 
Fig. 76. Leitz’ Microscope, lb. CH. //.] 
LABORATORY MICROSCOPES. 
73 
Fig. 77. Ross Eclipse Microscope. The heavy circular base rotates to give 
firmness upon inclination. 74 
LABOR A TOR Y MICROSCOPES. 
[CH. II. 
Fig. 78. The A A Microscope of the Bausch & Lomb Optical Cowith slid- 
ing tube instead of rack and pinion. This microscope is also furnished with rack 
and pinion. CH. //.] 
LABOR A TOR Y MICROSCOPES. 
75 
Fig. 79. Beck's Star Microscope. LA BOR A TOR Y MICROSCOPES. 
[CH. II. 
Fig. 8o. Zentmayer's Clinical Microscope. This is 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. 
Fig. 8i. Zentmayer's Microscope, No. V. CH. //.] 
LABOR A TOR Y MICROSCOPES. 
77 
Fig. 82. Leitz' Demonstration Microscope. 7his is designed for class demon- 
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. 
Fig. 83. Leitz’ Microscope, 
No. IV, with sliding tube for 
coarse adjustment, and no joint 
for inclination. This micro- 
scope must always be used in the 
vertical position. Microscopes 
of this form are not so much the 
fashion as they were a few years 
ago. If any instrument re- 
quires 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 rack and pinio7i for the 
coarse adjustment {Fig. 76). 78 
LABORATORY MICROSCOPES. 
[CH. II. 
Pig. 84. Queen & Company's Acme Microscope, No. 4. CH. //.] 
LABOR A TOR Y MICROSCOPES. 
79 
Fig. 85. McIntosh Scientific Microscope, No. 2. CHAPTER III. 
INTERPRETATION OF APPEARANCES. 
APPARATUS AND MATERIAL FOR CHAPTER III. 
A laboratory, compound microscope (g 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 (g 127-130). Solid glass 
rod, and glass tube (J 135-137) ; Collodion (g 137) ; Carmine, India ink, or lamp 
black (g 139-141) ; Frog, castor oil and micro-polariscope (g 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. Ill ] 
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 anyobject from the visual appearances which it presents to him when 
examined in the various modes now specified will necessarily depend in 
a great degree upon his previous experience in microscopic observation 
and upon his knowledge of the class of bodies to which the particular 
specimen may belong. Not only are observations of any kind liable to 
certain fallacies arising out of the previous notions which the observer 
may entertain in regard to the constitution of the objects or the nature 
of the actions to which his attention is directed, but even the most 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 hast}' 
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 
ourselves, or may be proved fallacious at some future time, perhaps even 
by our own more extended and careful researches. The suspension of 
the judgment whenever there seems room for doubt is a lesson inculcated 
by all those philosophers who have gained the highest repute for prac- 
tical wisdom ; and it is one which the microscopist cannot too soon learn 
or too constantly practice.” 82 
INTERPRETATION OF APPEARANCES. 
[ CH. III. 
For these experiments no condenser is to be used except where specifi- 
cally indicated. 
§ 121. Dust or Cloudiness on the Ocular.—Employ the 16 nun. 
( fs 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). Eight the field wrell 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 wa}\ 
§ 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 use a clean slide, and the image may be made 
clear by elevating the tube slightly. The obscurity produced in this 
way is like that caused by clouding the front-lens of the 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 objedt. 
The general rule is that objects highest up come into focus last in 
focusing up, first in focusing down. 
Fig. 86. Letters mounted in stairs to show 
the order of coming into focus. 
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 letters are mounted. 
§ 124- Objedts 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. III. ] 
INTERPRETATION OF APPEARANCES. 
small piece of glass about as thick as a slide. The second letter is 
placed upon this and covered in like manner. The third letter is placed 
upon the second thick cover and covered with an ordinary cover-glass. 
The letters should be as near together as possible, but not over-lapping. 
Employ the same ocular and objective as above (§ 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 
so on. Focus down, and the top letter will first appear, then the mid- 
dle one, etc. The relative position of objects is determined exactly in 
this way in practical work. 
For example, if one has a micrometer ruled on a cover-glass 15-25 
hundredths 111m. thick, it is not easy to determine with the naked eye 
which is the ruled surface. But if one puts the micrometer under a 
microscope and uses a 3 mm. in.) objective, it is easily 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 Objecfts.—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 Objedts having Curved Outlines.—The sue- 84 
INTERPRETATION OF APPEARANCES. 
[CH. III. 
cess of these experiments will depend entirely upon the care and skill 
used in preparing the objects, in lighting, and in focusing. 
Employ a 3 mm. in.) or higher objective and a high ocular for all 
the experiments. It may be necessary to shade the object (§ 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. 
Fig. 87. Diagram showing 
how to place a cover glass upon 
an object with fine forceps. 
§ 128. Air Bubbles with Central Illumination.—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 
1 mm. in diameter, get it into the center of the field, and if the light is 
central the air bubble will appear with a wide, dark, circular margin 
and a small bright center. If the bright spot is not in the center, 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- CH. III.] 
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 last in focusing up. 
Fig. 88. Very small Globule of Oil (O) and an Air Bubble (A) 
seen by Oblique Light. The arrow indicates the direction of the 
light rays. 
A 
§ 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. 
o 
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 conies into 
focus first and that the central spot is smaller than that of the oil globule. 
Then, of course, the black ring will be wider in the air bubble than in 
the oil globule. Make the light oblique. The bright spot in the air 
bubble will move away from the mirror while that in the oil globule will 
move toward it. See Fig. 88.* 
§ 134. Air and Oil by Reflected Light.—Cover the diaphragm or 
mirror so that no transmitted light f§ 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.f 
§ 135. Distinctness of Outline.—111 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. 2\), hence the bright spot really moves toward the mirror for air, and 
away from it for oil. 
f 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. 0/% 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- 
pearances with objectives of small and of large aperture. 86 
INTERPRETATION OF APPEARANCES. 
[CH. III. 
line as both differ greatly in refractive power from the medium which 
surrounds them, the oil being more refractive than the mucilage and 
the air less. (Figs. 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. Tight with central light. The fragment will be outlined 
by a dark band. Put a drop of water at the edge of the cover-glass. 
It will run in and immerse the fragment. The outline will remain dis- 
tinct, but the dark band will be somewhat narrower. Remove the cover- 
glass, wipe it dry, and wipe the fragment and slide dry also. Put a 
drop of 50°]c 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 baud, with a wide, dark band on 
each side. 
The one ip glycerin will show a much wider bright central band, with 
the dark borders correspondingly narrow (Fig. 89 b). The dark contour 
depends also on the numerical aperture of the objective—being wider 
with low apertures. This can be readily understood when it is 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. 
Fig. 89. Solid glass rod showing the ap- 
pearance when viewed with transmitted, 
central lie;ht, and with an objective of medi- 
um aperture. 
a. Mounted in air. 
b. Mounted in 50 per cent, glycerin. 
If the glass rod or any other object were mounted in a medium of the 
same color and refractive power, it could not be distinguished from the 
medium.* 
* 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. ///.] 
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 Refradfive.—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), it 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 3 mm. 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). 
Fig. 90. Solid glass rod coated with col- 
lodion to show a double contour. Toward 
one end the collodion had gathered in a fusi- 
form drop. 
§ 138. Optical Sedtion.—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 is in focus. The upper 
part of the object which is out of focus obscures the image but slightly. 
By changing the position of the objective or object, a different plane will 
be in focus and a different optical section obtained. The most 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. III. 
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. (fi 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 quite slow. 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 3 mm. ( 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'sis 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. Ill,] 
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 been offered. See Carpenter-Dallinger, p. 373 ; Beale, p. 
195 5 Jevons, in Quart. Jour. Science, n. s., Vol. VIII (1878), p. 167. 
In 1894 Meade Bache published a paper in the Proc. Amer. Philos. Soc., 
Vol. XXXIII, pp. 163-167, entitled “The Secret of the Brownian 
Movement.” This paper is suggestive if not 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 Uutersuch- 
ungeu fiber des Herren Robert Brown Entdeckung lebender, selbst im 
Feuer unzerstorbarer Theilchen in alien 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. Add a drop of 
water, or of water containing a little gum arabic. Rub the white mass 
around in the drop of liquid and soon the liquid will have a milky ap- 
pearance. Remove the white mass, place a cover-glass 011 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- 90 
INTERPRETATION OF APPEARANCES. 
[Cl/. III. 
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 
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 wrork. 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. 
I11 the last will be found a regular museum of objects. 
For figures (photo-micrographs, etc.) of the various forms of starch, 
see Bulletin No. 13 of the Chemical Division of the U. S. Department 
of Agriculture. For Hair and Wool, see Bulletin of the National Asso- CH. III1 
INTERPRETATION OF APPEARANCES. 
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. Micr. 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, etc., in order to obtain a fairly accurate and 
satisfactory knowledge of it. so much the more is it necessary not to be 
satisfied in microscopical observation until every means of investigation 
and verification has been called into service, and then of the image that 
falls upon the retina, only such details will be noted as the brain behind 
the eye is ready to appreciate. 
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 
|ths ; Block for magnifier and compound microscope (§ 147, 151) ; Dividers ($ 147, 
151) ; Stage micrometer ($ 150) ; Wollaston camera lucida (\ 151) ; Ocular screw- 
micrometer (Fig. 100) ; Micrometer ocular (Figs. 98-99). Abbe camera lucida 
(Fig. 96). 
§ i45- 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 mi- 
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 40 mm. and 
the object magnified, 2 mm., the amplification must be 40 -f- 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.—111 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 mm., then the magnification must be, 15 3 = 5, that is, CH. IV.] 
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 400 millimeters long and its image on the ground 
glass is 25 mm. long, the ratio is 25 -4- 400 = 11g-. That is, the image 
is 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 y^th. 
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 
(A3B3). To obtain the size of this virtual image place the tripod mag- 
nifier near the edge of a support of such a height that the distance from 
the upper surface of the 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 Aths. 
As -3- mm. 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, A3B3), 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 MAGNIFICATION AND MICROMETRY. 
[CH. IV. 
94 
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 h- 2 = 7Yz. That is, the image is 7y2 times as long or wide as 
the object. In this case the image is said to be magnified 7 *4 diame- 
ters, or 7 ]/?, 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, B3AS), and theobject 
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, Objecft 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 yy and millimeter, and when high pow- 
ers are to be used the lines should be very fin:. It is of advantage to 
have the coarser lines filled with graphite (plumbago), especially when 
low powers are to be used. If one has 
an uncovered micrometer the lines may 
be very readily filled by rubbing some of 
the plumbago on the surface with the end 
of a cork ; the superfluous plumbago may 
be removed by using a clean dry cloth or 
a piece of lens paper. After the lines are 
filled and the plumbago wiped from the 
surface, the slide should be examined and if it is found satisfactory, i. 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 
Fig. 91. Diagram of a stage 
micrometer, with a ring on the 
lines to facilitate finding them. 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 millimeters at 
which to measure the image is very readily determined (Fig. 92, § 153). 
Employ the 16 mm. (2/s in.) objective and a 50111m. (2 in., A. or 
No. 1) ocular and stage micrometer as object. For this power the 
mm. spaces of the micrometer should be used as object. Focus sharply, 
and make the tube of the microscope horizontal, by bending the flexible 
Fig. 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. 
Axis, Axis. Axial rays from the 
microscope and from the drawing sur- 
face (Ch. V). 
Camera Lucida. A section of the 
quadrangular prism showing the 
course of the rays in the prism from 
the microscope to the eye. As the rays 
are twice reflected, they have the same 
relation on entering the eye that they 
would have by looking directly into the 
ocular. 
A B. The lateral rays from the mi- 
croscope and their projection on the 
drawing surface. 
C D. Rays from the drawing sur- 
face to the eye. 
A D, A' D'. Overlapping portion of the two fields, where both the microscopic 
image and the drawing surface, peticil, 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. Eook 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 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 9f millimeters. If now the object is yyths 
of a millimeter and the magnified image is 9f millimeters, the magnifi- 
cation (which is the ratio between size of object and image) must be 
9} tv = 47- That is, the magnification is 47 diameters, or 47 times 
linear. If the fractional numbers in the above example trouble the stu- 
Fig. 93. 
Fig. 9}. 
Figs. 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, i. e., bring ihe object and objective nearer together when 
the tube is lengthened. CH. I Vi] 
MAGNIFICATION AND MICROMETRY. 
97 
dent, both may be reduced to the same denomination, thus : If the size 
of the image is found to be 9f mm. this number may be reduced to 
tenths mm., so it will be of the same denomination as the object. In 9 
mm. there are 90 tenths, and in f there are 4 tenths, then the whole 
length of the image is 90 -f 4 = 94 tenths of a millimeter. The object 
is 2 tenths of a millimeter, then there must have been a magnification 
of 94 -f- 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 and a 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, B3A3) seen with the high ocularis 
larger than the one seen with the low one. The real image (Fig. 21, 
A1!!1) remains nearly the same, and would be just the same if positive, 
par-focal oculars (§ 34, 68, note) were used. 
Lengthen the tube of the microscope 50-60 111m. 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 
l 152, the magnification is sometimes increased by the use of an amplifier, that is 
a diverging lens or combination placed between the objective and ocular and serv- 
ing to give the image-forming rays from the objective an increased divergence. 
An effective form of this accessory was made by Tolies, 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. 98 
MAGNIFICA TION AND MICRO ME TR Y. 
[CM. IV. 
Fig. 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 
eye. (a) The size at 25 cm.; 
(b) at35 cm., (§ 153). 
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- 
Fig. 96. Sectional view of 
the Abbe Camera Lucida to 
show that in measuring the 
standard distance of 250 
millimeters, one must meas- 
ure along the axis from the 
point P, at the left of the 
prism, to the mirror, and 
from the mirror to the 
drawing surface. For a 
full explanation of this 
camera lucida, see next 
chapter, (Figs. 102,106). 
Fig. 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. IV.] 
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.— The following table should be filled out by each 
student. In using it for Micrometry and Drawing it is necessary to keep 
clearly in mind the exact conditions tinder 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. 
Objective. 
Tube 
in 
Tube 
out 
MM. 
Tube 
in 
Tube 
out 
MM. 
Ocular Micrometer 
Valuation. 
TUBE IN. OUT MM. 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
• 
X 
X 
X 
X 
Simple Microscope. 
X 100 
MAGNIFICATION AND MICROMETRY. 
f CH. IV. 
micrometry. 
§ 155. Micrometry is the determination of the size of objects by the 
aid of a microscope. 
micrometry with the simpee 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 mm. or 0.001 mm.) or 
one millionth of a meter (nnmmr or 0.000001 meter) as the unit. He 
named this unit micro-millimeter and designated it mmm. In 1869, 
Listing (Carl’s Repetorium fur Experimental-Physik, Bd, X, p. 5) 
favored the thousandth of a millimeter as unit and introduced the name 
Mikron or micrum. In English it is most often written Micron, plural 
micra or microns, pronunciation Mic'rou or Mic'rbu. By universal con- 
sent the sign or abbreviation used to designate it is the Greek /x. Adopt- CH. IV.] 
MAGNIFICATION AND MICROMETRY. 
101 
ing this unit and sign, one would express five thousandths of a milli- 
meter (x-jnnj- or o.oo5ths mm.) thus, 5/x.* 
§ 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 
msy 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 by dividing the size of the image by the magnifi- 
cation of the microscope.—For example, employ the 3 mm. (fs in.) ob- 
jective, 25 mm. (1 in.) 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 diameters (§ 145), then the actual length of this long axis of 
the corpuscle is 18 mm. -f- 400 = .045 mm. or 45 /x (§ 157). 
Fig. 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 micro 
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, 
and by original investigators, and is, in the opinion of the writer, the best term to 
employ. Jour. Roy. Micr. Soc., 1888, p. 502 ; Nature, Vol. XXXVII (1888), p. 388. MAGNIFICATION AND MICROMETRY. 
[<CH. IV. 
102 
§ 160. Micrometry by the use of a Stage Micrometer and a Camera 
Lticida.—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). 
OCUEAR MICROMETER. 
§ 161. Ocular Micrometer, Eye-Piece Micrometer.—This, asthe 
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 yyth or 
mm. apart, and every fifth line should be longer and heavier to facili- 
tate counting. If the micrometer is ruled in squares (net-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 (i. e., whether positive or negative) at the level at 
which the real image is formed by the objective, and the image appears 
to be immediately upon or under the ocular micrometer, and hence the 
number of spaces on the ocular micrometer required to measure the real 
image may be read off directly. This 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 (z. 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 is to do much meas- 
ureing it is almost a necessity to have an ocular micrometer with screw 
for moving the scale, (Figs. 98-99). 
Fig. 98. 
Fig. 99. 
Figs 9S-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. In 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. (fi in.) objec- 
tive. Place the stage micrometer under the microscope for an object, 
and put the ocular micrometer in position, either through a slit in the 
ocular, or remove 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 the}7 are not, 104 
MAGNIFICATION AND MICROMETRY. 
[CH. IV. 
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. 
Fig. i 00. Ocular Screw Micrometer with 
compensation ocular 6. The upper figure 
shows a sectional view of the ocular and the 
screw for moving the micrometer at the right. 
At the left is shown a clamping screw to 
fasten the ocular to the upper part of the mi- 
croscope tube. Below is a face view, showing 
the graduation on the wheel. An ocular mi- 
crometer like this is in 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- 
er its real image) regardless of the minuteness 
of the object. (Zeiss’ Catalog, No. jo). 
§ 164. Make 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. To do this it 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 TVth and 
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 millimeter on the stage micrometer, obviously one 
space on the ocular micrometer would include only one-tenth as much, 
or yyth mm. -4- 10 = y^¥ nun. That is, each space on the ocular mi- 
crometer would include yy-g- of a millimeter on the stage micrometer, 
or yyytli millimeter of length of any object under the microscope, 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 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 -yyyth mm. X 8 = Tfy mm. or 80 p. (§ 157). 
§ 165. Varying the Ocular Micrometer Valuation.—Any change 
in the objective, the ocular or the tube-length of the microscope, that 
is to say, any change in the size of the real image, produces a corre- 
sponding change in the ocular micrometer valuation (§ 152, 161). 
§ 166. Micrometry with the Ocular Micrometer.—Use the 3 mm. 
( }i in.) obj ective 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 
each 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 b}' 
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 method 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 x2fftlis millimeter of the 
stage micrometer, then obviously one space on the ocular micrometer would in- 
clude |th of mm. or 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, 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 = fths, or 0.6 mm. 106 
MAGNIFICATION AND MICROMETRY. 
[CH. IV. 
£ 167. 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 adjustment is closed as for thick cover glasses than when 
open, as for thin ones. This variation in the magnification of the objective pro- 
duces a corresponding change in the magnification of the entire microscope and 
the ocular micrometer valuation—therefore it is necessary to determine the mag- 
nification and ocular micrometer valuation for each position of the adjusting collar. 
While the principles of micrometry are simple, it is very difficult to get the 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 T mm., on the stage micrometer, 
then evidently it would require 5 -4- = 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 -f- 25 
= f or 0.6 mm. This method is really exactly like the one in (A), for dividing by 
25 is the same as multiplying by 5th. 
(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 and mm. and the ocular micrometer is ruled in 
millimeters and TVth mm. Taking T2<yth nim. on the stage micrometer as object, 
as in the other cases, suppose it requires 10 of the mm. spaces or 1 mm. to 
measure the real image, then the real image must be magnified = 5 diame- 
ters, that is, the real image is five times as great in length as the object, and the 
size of an object maybe 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 mm. divisions = f$ mm. 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- 5 = § mm., the same result as in the other cases. 
In comparing these methods it will be seen that in the first two (A and B) the 
ocular micrometer may be simply ruled with equidistant lines without regard to 
the absolute size in millimeters or inches of the spaces. In the last method the 
ocular micrometer must have its spaces some known division of a millimeter or 
inch. In the first two methods only one standard of measure is required, viz., the 
stage micrometer ; in the last method two standards must be used,—a stage mi- 
crometer and an ocular micrometer. Of course, the ocular micrometer in the first 
two cases must have the lines equidistant as well as in the last case, but ruling lines 
equidistant 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 shill 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, one may measure from the inside of one border- 
ing line and from the outside of the other. If the lines are of equal width this is 
as accurate as measuring from the center of the lines. Evidently it would not be 
right to measure from either the inside or the outside of both lines (Fig. 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., J890, pp. 97, 173. 
Fig. 10 r. The appearance of the coarse 
stage and of the fine ocular micrometer 
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 mi- 
crometer lines. 
(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 (s.m.). 
A 
B 
Fig. ioi. 
As to the limit of accuracy in micrometry, one who has justly earned the right 
to speak with authority, expresses himself as follows : “ I assume that 0.211 is the 
limit of precision in microscopic measures, beyond which it is impossible to go 
with certainty.''’ W. A. Rogers, Proc. Amer. Soc Micrs., 1883, p. 198. 
In comparing the methods of micrometry with the compound microscope, given 
above (158, 159, 160, 166), the one given in \ 158 is impracticable, that given in 
§ 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 \ 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. If the ocular micrometer has some quite narrow spaces, and others consid- 
erably larger, one can nearly always manage to exactly include the object by some 
two lines. The ocular screw-micrometer (Fig. ioo) 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-Degal Journal, Vol. XII. For the change in magnification due to a change 
in the adjustment of adjustable objectives, see Jour. Roy. Micr. Soc., 1880, p. 702 ; 
Amer. Monthly Micr. Jour., 1880, p. 67. 
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 MATERIAE 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 drawing 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. I11 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 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. 110 
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. 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. 
A B. Marginal rays of the field on the 
drawing surface, a b. Sectional view of 
the silvered surface on the upper of the tri- 
angular prisms composing the cubical 
ffl K prism (P). The silvered surf,ace is shown 
as incomplete in the center, thus giving passage to the rays from the microscope. 
Foot. Foot or base of the microscope. 
G. Smoked glass seen in section. It is placed between the mirror and the prism 
to reduce the light from the drawing surface. 
Mirror. The mirror of the camera lucida. A quadrant (Q) has been added to 
indicate the angle of inclination of the mirror, which in this case is 450. 
Ocular. Ocular of the microscope over which the prism of the camera lucida is 
placed. 
P, F. Drawing pencil and the cubical prism over the ocular. 
Fig. 103. Geometrical figure showing the angles made by the axial ray with the 
drawing surface and the mirror. 
A B. The drawing surface. 
Fig. 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). 
Fig. 103. 
Fig. 104. 
Fig. 102. CH. V.] 
DR A WING WITH THE MICROSCOPE. 
111 
designed to be used on the microscope in a 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.—In order that the picture drawn 
by the aid of a camera lucida ?nay not be distorted, it is necessary that the 
axial ray from the image on the drawing surface shall be at right angles 
to the drawing surface (Figs. 102, 105, 106). 
$171. Wollaston’s Camera Lucida.—This is a quadrangular prism of glass put 
in the path of the rays from the microscope, and it serves to change the direction 
of the axial ray 90 degrees. In using it the microscope is made horizontal, and the 
rays from the microscope enter one-half of the pupil while rays from the drawing 
surface enter the other half of the pupil. As seen in the figure (Fig. 105), the fields 
partly overlap, and where they do so overlap, pencil or dividers and microscopic 
image can be seen together. 
In drawing or using the dividers with the Wollaston camera lucida it is necessary 
to have the field of the microscope and the drawing surface about equally lighted. 
If the drawing surface is too brilliantly lighted the pencil or dividers may be seen 
very clearly, but the microscopic image will be obscure. On the other hand, if the 
field of the microscope has too much light the microscopic image will be very defi- 
nite, but the pencil or dividers will not be visible. It is necessary, as with the 
Abbe camera lucida (§ 173), to have the Wollaston prism properly arranged with 
reference to the axis of the microscope and the eye-point. If it is not, one will be 
unable to see the image well, and may be entirely unable to see the pencil and the 
image at the same time. Again, as rays from the microscope and from the draw- 
ing surface must enter independent parts of the pupil of the same eye, one must 
hold the eye so that the pupil is partly over the camera lucida and partly over the 
drawing surface. One can tell the proper position by trial. This is not a very sat- 
isfactory camera to draw with, but it is a very good form to measure the vertical 
distance of 250 mm. at which the drawing surface should be placed when determin- 
ing magnification ($ 153). 
Fig. 105. Wollaston's Camera Lu- 
cida, showing the rays from the mi- 
croscope and from the drawing sur- 
face, and the position of the pupil of 
the eye. 
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 center 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 
DR A WING WITH THE MICROSCOPE. 
[CH. V. 
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. Tight from the drawing 
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 if on 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, Teitz, and the Bausch & Tomb 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, i. -e., it is above or below the eye-point. 
§ 174. Arrangement of the Mirror and the Drawing Surface.— CH. V,] 
DR A WING 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 450, 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 450 
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 350 (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 350, then the 
axial ray must take an angle of 1 ro° 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 450. Practically the field for drawing 
can always be made free of the stage of the microscope, at 450, at 40°, 
or at 350. In the first case (450 mirror) the drawing surface should 
be horizontal, in the second case (40° mirror) the drawing surface 
should be elevated io°, and in the third case (350 mirror) the drawing 
board should be elevated 20° toward the microscope. Furthermore it 
is necessary in using an elevated drawing board to have the mirror bar 
project directly laterally so that the edges of the mirror will be in 
planes parallel with the edges of the drawing board, otherwise there 
will be front to back distortion, although the elevation of the drawing 
board would avoid right to left distortion. If one has a micrometer 
ruled in squares (net micrometer) the distortion produced by not hav- 
ing the axial ray at right angles with the drawing surface may be very 
strikingly shown. For example, set the mirror at 350 and use a hori- 
zontal drawing board. With a pencil make dots at the corners of some 
of the squares, and then with a straight edge connect the dots. The 
figures will be considerably longer from right to left than from front to 
back. Circles in the object would appear as ellipses in the drawings, 
the major axis being from right to left. 
The angle of the mirror may be determined with a protractor, but 
that is troublesome. It is much more satisfactory to have a quadrant 
attached to the mirror and an indicator on the projecting arm of the 
mirror. If the quadrant is graduated throughout its entire extent, or 
preferably at three points, 45 °, 40° and 35 one can set the mirror at a 114 
DR A WING WITH THE MICROSCOPE, 
\CH. V. 
Fig. 108. 
Fig. 107. 
Fig. 106. 
Fig 109. 
Figs. 106-109. Abbe Camera Lucida in position to avoid distortion. 
Fig. 106. The Abbe Camera Lucida with the mirror at 33°. 
Axis, Axis. Axial ray from the microscope and from the drawing surface. 
A B. Drawing; surface raised toward the microscope 20°. 
Foot. The foot or base of the microscope. 
Mirror with quadrant ( Q). The mirror is seen to be at an angle of 33°. 
Ocular. Ocular of the microscope. 
P, P. Drawing pencil, and the cubical prism over the ocular. 
IV. Wedge to support the drawing board. 
Fig. 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 33° must make an angle of i/o° with a horizontal drawing board. The 
board must then be elevated toward the microscope 20° in order that the axial ray 
may be perpendicular to it, and thus fulfill the requirements necessary to avoid dis- 
tortion {l 170, 174). 
Fig. 108. Upper view of the prism of the camera lucida. A considerable portion 
of the face of the prism is covered, and the opening in the silvered surface appears 
oval. 
Fig. 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 430, 40°, or 33°, 
only those angles are shown. CH. V.] 
DR A WING WITH THE MICROSCOPE. 
115 
known angle in a moment, then the drawing board can be hinged and 
the elevation of io° 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 io° 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 perfectly adapted to use with an inclined as with a vertical micro- 
scope. All that is requisite is to be sure that the fundamental law is 
observed regarding the axial ray of the image and the drawing surface, 
viz., that they should be at right angles. This is very easily accom- 
plished as follows : The drawing board is raised toward the microscope 
twice as many degrees as the mirror is depressed below 450 (§ 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. no). 
Fig. i 10. 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. hi 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 
DR A WING WITH THE MICROSCOPE. 
[ClI. 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 light 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 image 
of the microscopic object is very distinct and the pencil is invisible or 
very indistinct. If the drawing surface is too brilliantly lighted the 
pencil can be seen clearly, but the microscopic image will be very ob- 
scure. 
Fig. iii. 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- 
ror 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. V.-\ 
DR A WING WITH THE MICROSCOPE. 
117 
microscope may be placed in a very strong light and the drawing board 
shaded or the light may be concentrated upon the object by means of 
a concave mirror or a bull’s eye condenser (Fig. 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 011 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. hi. 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, 
iforth nun. 
Fig. 112. Showing the method of indicating the scale at which a drazuing 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 millimeter, then the enlargement would be 
25 —r- = 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.-] 
DR A WING 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 lias 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. 819, 1882, p. 402, 1883, pp. 283, 560, 1S84, p. 115, 1886, p. 516, 1888, pp. 113, 809, 
798; Zeit. wiss. Mikroskopie, 1884, pp. 1-21, 1889, p. 367, 1893, pp. 289-295. 
Here is described an excellent apparatus made by Winkel. 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 MATERIAE REQUIRED FOR THIS CHAPTER. 
Compound microscope ; Micro-spectroscope ($ 179) ; Watch-glasses and small 
vials, slides and covers ($ 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. 
§ 179- 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, i. e., 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 a spring. It may be swung free of the ocular. In connection with 
the ocular is the slit mechanism and a prism for reflecting horizontal rays verti- 
cally for the purpose of obtaining a comparison spectrum ($ 192). Finally near 
the top is a lateral tube with mirror for the purpose of projecting an Angstrom 
scale of wave lengths upon the spectrum (§ 193, Figs. 113-114). 
§ 180. Apparent Reversal of the Position of the Colors in a Diredt Vision Spec- 
troscope.—In accordance with the statements in § 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 POLAPISCOPE. 
Fig. 113. 
Longitudinal Section of 
the whole instrument. 
(yz Full Size.) 
Abbe's Micio spectroscope. 
Fig. i 14. 
Slit Mechanism separately. 
[Plan view, Full size.) 
“ The eye lens is adjustable so as to accurately focus on the slit situated between 
the lenses. The mechanism for contracting and expanding the slit is actuated by 
the screw F and causes the laminae to move symmetrically (Merz's movement). 
The slit may be made sufficiently wide so as to include the whole visual field. •The 
screw H serves to limit the length of the slit so as to completely fill the latter with 
the image of the object under investigation when the comparison prism is inserted. 
The comparison prism is provided with a lateral frame and clips to hold the 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 axial position by the spring catch L. 
A scale is projected on the spectrum by means of a scale tube and mirror attached 
to the prism casing. 1 he divisions of the scale indicate in decimals of a micron 
the wave length of the respective section of the spectrum. The screw P serves to 
adjust the scale relative to the spectrum. 
The instrument is inserted in the tube in place of the ordinary eye-piece and is 
clamped to the former by means of the screw M in such a position that the mirrors 
A and O, which respectively serve to illuminate the comparison 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 same is true in looking into the micro-spectroscope. The actual position of 
the different colors may be determined by placing some ground glass or some of 
the lens-paper near the prism and observing with the eye at the distance of distinct 
vision.* 
*The author wishes to acknowledge the aid rendered by Professor E. F. Nichols 
in giving the explanation offered in this section. 122 
MICRO SPECTROSCOPE AND POLARISCOPE. 
[CH. VI. 
Fig. i 15. 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. 
It frequently occurs that with a substance giving several absorption bands (e. 
g., chlorophyll) the density or thickness of the solution must be varied to show all 
the different bands clearly. 
Solar Spectrum.— With diffused day-light and a narrow slit the spectrum is not 
visible much beyond the fixed line B. In order to extend the visible spectrum in 
the red to the line A, one should use direct sunlight and a piece of ruby glass in 
place of the watch glass in Fig. iij. 
Sddium Spectrum.—The line spectrum (§ 182) of sodium obtained by lighting 
the microscope 7vith an alcohol flame in which some salt of sodium is glowing. 
With the micro-spectroscope the sodium line seen in the solar spectrum and with 
the incandescent sodium appears single, except tender very favorable circumstances 
(§ I93)- By using a comparison spectrum of day-light with the sodium spectrum 
the light and dark D-lines will be seen to be continuous as here shown. 
Permanganate of Potash.—This spectrum is characterized by the presence of five 
absorption bands in the middle of the spectrum and is best shown by using a T\, per 
cent, solution of permanganate in water in a watch glass as in Fig. iij. 
Met-hemoglobin.—The absorption spectrum of met-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 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 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 x\, per cent, aqueous 
solution of permanganate of potash or a few drops of hydrogen dioxid (H2OJ. 
Soon the bright red will change to a brownish color, when it may be examined. 
VARIOUS KINDS OF SPECTRA. 
By a spectrum is meant the colored bauds 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, V/,] 
MICRO-SPECTROSCOPE AND POLAR/SCOPE. 
123 
\ 181. Continuous Spedtrum.—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 Spedtrum.—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. 
\ 183. 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 ligl}t 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. 
$ 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-luminous. 
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. 
| 185. Angstrom 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-SPFCTROSCOPE AND POLARISCOPE. 
[CH. VI. 
Fig. 116. Absorption Spectrum of Oxy-Hemoglobin or arterial blood (/) and of 
Hemoglobin or venous blood (2). (From Gamgee and McMunn.) 
A, B, C, D, E, F, G, H. Some of the Principal Fraunhofer lines of the solar 
spectrum (g 183). 
.90, .80, .70, .60, .50, .4.0- Wave lengths in microns, as shown in Angstrom's 
scale (l 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 bauds 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. . 
$ 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, but a 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 be slightly, 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. 
\ 187. Complementary Spectra.—While it is believed that Angstrom’s law (§ 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 POLAR/SCOPE. 
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 F. 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. 
[CM. VI. 
Fig. 117. 
Fig. 118. 
Fig. i 19. 
Fig. 117 (1). Section of the tube and stage of the microscope with the spectral 
ocular or micro spectroscope in position. 
Amici Prism (? 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 (\ 189). 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 Angstrom scale and 
the lenses for projecting the image upon the upper face of the Amici prism, whence 
it is reflected upward to the eye with the different colored rays. At the right is a 
special mirror for lighting the scale. For arranging and focusing the scale, (see 
l 193)- 
Slit. — The linear opening between the knife edges Through the slit the light 
passes to the prism. It must be arranged parallel with the refracting edge of the 
prism, and of such a width that the Fraunhofer or Fixed Lines are very clearly 
and sharply defined when the eye-lens is properly focused ($ 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 
into 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- 
217). 
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 (§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 is to be investigated by polarized light. MICRO-SPECTROSCOPE AND POLARISCOPE. 
[CM. VI. 
128 
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 movable and may be 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-spectroscope 
the scale is in a separate tube near the top of the prism and at right 
angles to the prism-tube. A special mirror serves to light the scale, 
which is projected upon the spectrum by a lens in the scale-tube. 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 /a. 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 spectrum, and by a screw just 
under the scale-tube, but in the prism-tube, the proper point on the 
scale (o. 589 |U.) can be brought opposite the sodium band. All the scale 
will then give the wave lengths directly. Sometimes the scale is oblique 
to the spectrum. This may be remedied by turning the prism-tube 
slightly one way or the other. It may be due to the wrong position of 
the scale itself. If so, grasp the milled ring at the distal end of the 
scale-tube and, while looking into the spectroscope, rotate the tube until 
the lines of the scale are parallel with the Fraunhofer lines. It is 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 /x. The last would be indi- 
cated thus, A D = 0.5892 ix. 
§ 195. Lighting for the Micro-spedtroscope.—For opaque objects 
a strong light should be thrown on them either with a concave mirror 
or a condensing lens. For transparent objects the amount of the sub- 
stance and the depth of color must be considered. As a general rule it 
is well to use plenty of light, as that from an Abbe illuminator with a 
large opening in the diaphragm, or with the diaphragm entirely 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 spectruins should be about equally 130 
MICRO-SPECTROSCOPE AND POIARISCOPE. 
[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-spedtroscope.—If the 
material is of considerable bulk, a low objective (18 to 50 mm.) is to be 
preferred. This depends on the nature of the object under examina- 
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. 
§ 198. 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 homoeopathic vial, or in some glass tubing sealed at the end, 
then one can look lengthwise through the liquid and get the effect of a 
more concentrated solution. For minute bodies like crystals or blood 
corpuscles, one may proceed as described in the previous section. 
MICRO-SPECTROSCOPE—EXPERIMENTS. 
§ 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 Spedlrum of Permanganate of Potash.—Make 
a solution of permanganate of potash in water of such a strength that 
a stratum 3 or 4 mm. thick is transparent. Put this solution in a watch- 
glass with sloping sides, and put it under the microscope. Use a 50 mm. 
or 16 mm. objective, and use the full opening of the illuminator. Fight 
strongly. Took 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 Spedtrum 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- MICRO SPECTROSCOPE AND POLAR/SCOPE. 
[CM. VI. 
132 
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 homoeopathic 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 a little 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 to the 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 first 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, tlius reducing CH, VI,] 
MICRO-SPECTROSCOPE AND POLARISCOPE. 
133 
the oxy-hemoglobin to hemoglobin. This may be repeated 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 
yV 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 anaesthetizatiou, 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 bauds 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 
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 
MICROSPECTROSCOPE AND POLARISCOPE. 
[CH. VI. 
tion Spedtra.—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 Spedtra of Minerals.—As example take some 
monazite sand on a 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 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 satisfactorj’ 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. 
§ 20S. 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 POLAR/SCOPE. 
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-liemoglobin 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 
bauds 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 hitensities in the same parts of 
the spectrum which undergo analogous changes on the addition of the 
same reagent. ’ ’ 
REFERENCES TO THE MICRO-SPECTROSCOPE AND SPECTRUM ANALYSIS. 
The micro-spectroscope is playing an ever increasingly important role in the 
spectrum analysis of animal and vegetable pigments, and of colored mineral and 
chemical substances, therefore a somewhat extended reference to literature will be 
given. Full titles of the books and periodicals will be found in the Bibliography 
at the end. 
Angstrom, Recherches sur le spectre solaire, etc. Also various papers in period- 
icals. See Royal Soc’s Cat’l 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. II, p. 419. The larger works on Physi- 
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. 
I 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. 
$ 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 i. e., 
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 1800, where the corre- 
sponding faces are parallel and where light freely traverses the analyzer. There 
are also two crossing points of the Nicols, at 90° and 270°, where the light is extin- 
guished. In thedntermediate points there is a sort of twilight. 
\ 2ir. Putting the Polarizer and Analyzer in Position.—Swing the diaphragm 
carrier of the Abbe illuminator out from under the illuminator, remove the disk 
diaphragm or open widely the iris diaphragm and place the analyzer in the dia- 
phragm carrier, then swing it back under the illuminator. Remove the ocular, 
put the graduated ring on the top of the tube and then replace the ocular and put 
the analyzer over the ocular and ring. Arrange the graduated ring so that the indi- 
cator shall stand at o° when the field is lightest. This may be done by turning the 
tube down so that the objective is near the illuminator, then shading the stage so 
that none but polarized light shall enter the microscope. Rotate the analyzer until 
the lightest possible point is found, then rotate the graduated ring till the index 
stands at o°. The ring may then be clamped to the tube by the side screw for the 
purpose. Or, more easily, one may set the index at o°, clamp the ring to the 
microscope, then rotate the draw-tube of the microscope till the field is lightest. 
§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 in a 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. 
$ 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,—use 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 Objecfts 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). 
\ 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 anisotropic, uniaxial or biaxial ; (C) 
Pleochroism ; (D) The rotation of the plane of polarization, as with solutions of 
sugar, etc. ; (E) To aid in petrology and mineralogy; (F) To aid in the determi- 
nation of very minute quantities of crystallizable substances ; (G) For the 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). As an isotropic substance, put an ordinary 
glass slide under the microscope. The field will remain dark. As an 
example of a crystal belonging to the cubical system and hence iso- 
tropic, make a strong solution of common salt (sodium chloride Na Cl.), 
put a drop 011 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 MICRO-SPECTROSCOPE AND POIARISCOPE. 
[CH. VI 
138 
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. I11 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 be found to remain dark with crossed Nicols, 
others will shine continuously. If the crystals are in such a position 
that the light passes through them parallel with the optic 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 in a 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. As all 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- 
alyzer is rotated. A11 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 § 142) are uniaxial crystals. 
Borax crystals are bi-axial. CH. VI.~\ 
MICRO-SPECTROSCOPE AND POLARISCOPE. 
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 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 
§ 144 were all examined with polarized light an additional means of de- 
tecting them would be found. 
REFERENCES TO THE POEARISCOPE AND TO THE USE OF POEARIZED 
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-GEASSES ; MOUNTING ; ISOEATION, 
SECTIONING BY THE COLEODION 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 ($ 219-220) ; Cleaning mixtures for glass ($ 227); 
Alcohol and distilled or filtered water (§ 222) ; fine forceps for handling cover- 
glasses ($ 222-226) ; Old handkerchiefs or lens paper (£ 107, 223). Paper boxes for 
storing cover-glasses (§ 223, 225) ; Cover-glass measurer (Figs. 120-122) ; Mount- 
ing material,—Farrant’s solution, glycerin, glycerin-jelly and Canada balsam (§ 243, 
246) ; Centering card and card for serial sections ($ 236) ; Material for dissociation 
and for the paraffin and collodion method ($ 244) ; Material for paraffin and collo- 
dion sectioning (§ 250) ; Net-micrometer for arranging minute objects like diatoms 
(§ 3X7) ! babels (£ 309) ; Carbon ink for writing labels (§ 295) ; Writing diamond 
($ 295) ! Shellac cement ($ 316) ; Cabinet (# 296) ; Re-agents for experiments in 
micro-chemistry (§ 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 (1x3 inches). For rock sections, slides 25 x 45 mm. 
or 32 x 32 111m. are used ; for serial sections, slides 25 x 76 mm., 50x 75 
mm. or 37 x 87 mm. 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°]o or 75 °Jo 
alcohol, let them drain for a few moments on a clean towel or on blot- 
ting paper, and then wipe with a soft cloth. In handling the slides 
grasp them by their edges to avoid soiling the face of the slide. After 
the slides are cleaned they should be stored in a place as free as possible 
from dust. 
For 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 timeleaning 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, to millimeter 
(see table, § 27). It is better never to use a cover-glass over y 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 it is 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 ADD COVER-GLASSES. 
[CH, vn. 
(§ 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°jc to 75% alcohol. 
§223. Wiping the Cover-Glasses. — When read)' 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 the same. In wiping them one may proceed 
as for the small covers, but special care is necessary to avoid breakiug 
them. A safe and good way to clean the large covers is to take two CH. V//.] 
SLIDES AND COVED CLASSES. 
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 Tyy to T2-jy0y 
mm.—otherwise high objectives cannot be used in studying the prepa- 
rations. 
§ 225. Measuring the Thickness of Cover-Glasses.—It is of the 
greatest advantage to know the exact thickness of the cover-glass on an 
object; for, (a) 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 there 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, and 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 yyy mm. to y3yy 111m. 
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. 
Fig. 120. Micrometer Calipers (Brown and Sharpe). Pocket Calipers, gradu 
ated in inches or millimeters, and well adapted for measuring cover-glasses. 
It is then strongly recommended that every preparation shall be 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. 
[Clf. VII. 
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. 
Fig. i2i. Cover-Glass Measurer (Edward Bausch). 
The cover glass is placed in the notch between the two screws, and the drum is 
turned by the milled head at the right till the cover is in contact with the screws. 
The thickness is then indicated by the knife edge on the drum and may be read off 
directly in or T faffh inch. In other columns is given the proper tube- 
length for various unadjustable objectives (b A, and A 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 CM. VII. ] 
SLIDES AND CO FED GLASSES. 
145 
mm. may be put in the same box. For example, if one takes y 
mm. as a standard, covers varying yfy mm. on each side may be put 
into the same box. In this case the box would contain covers of y/y, 
1 4 15 lfi n 17 111111 
T0 0> TO O > T0T» ana TTT 111111 • 
Fig. 122. Zeiss Cover-Glass Meas- 
urer. With this the knife edge jaivs 
are opened by means of a lever, and 
the cover inserted. Ihe thickness 
may then be read off on the face as 
the pointer indicates the thickness in 
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 (K2 Cr2 07) - - - 200 grams. 
Water, distilled or ordinary ... 1000 cc. 
Sulphuric acid (H2S04) .... 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 chemically 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 (H2S04) ----- 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 146 
MOUNTING AND LABELING. 
[CM. vir. 
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 MICROSCOPICAE 
OBJECTS. 
§ 228. Mounting a Microscopical Objecft 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 is false economy to crowd them 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 in the serum. Some- 
times it is not easy or convenient to get the natural medium, then some 
liquid that has been found to serve in place of the natural medium is 
used. For many things, water with a little common salt (water 100 cc., 
common salt 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 
the cover is in place, if the preparation is to be studied 
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). 
Fig. 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. 
Fig. 124. Needle Holder (Queen & Co.). By means of the 
screw clavip or chuck at one end, the needle may be quickly 
changed. 
Fig. 123. CH. VII ] 
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 in air (§ 231) ; 
(B) In some medium miscible with zuater, as glycerin or glycerin jelly 
(§ 235) I (C) In some resinous medium like dammar or Canada balsam 
(§ 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 till 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- 
low cell and dry it (§ 233). Select a clean cover-glass slightly larger 
than the cell. Pour upon the cover a drop of a 10°Jo solution of sali- 
cylic acid in 95% alcohol. Tet 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. 
[c/i vii. 
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). 
Fig. 125. Turn-Table for sealing cover-glasses and making shallow mounting 
cells. (Queen & Co.) 
§ 233. Preparation of Mounting Cells.—(A) Thin 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 
ithere 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 
us too thick or too thin, or too much was on the brush. After a ring is 
itlius 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 drvs. 
(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 Objecfts 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-glass is not in contact with the cell wall at 
any point and the cell is shallow, there will be great danger of the fresh 
cement running into the cell and injuring or spoiling the preparation. 
When the cover-glass is properly sealed, the preparation is put in a 
safe place for the drying of the cement. It is advisable to add a fresh 
coat of cement occasionally. 
§ 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 glycerin or fixed by some fixing agent before being per- 
manently mounted in strong glycerin or either of the other media. 
I11 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. 
Fig. 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. 
[Cl/. VII. 
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 if a cell is used, 
enough to fill the cell. 
4. In putting on the cover-glass it is grasped with fine forceps and 
the under side breathed on to slightly moisten it so that the glycerin 
will adhere, then one edge of the cover is put on the cell or slide and 
the cover gradually lowered upon the object (Fig. 123). The cover 
is then gently pressed down. If a cell is used, a fresh coat of cement 
is added before mounting (§ 234.) 
Fig. 127. Slide and cover glass showing method of anch- 
oring a cover-glass with a glycerin preparation when no 
cell is used. A cover-glass so anchored is not liable to 
move when the cover is being sealed (§ 238). 
Fig. 128. Glass slide with cover-glass, a drop of reagent 
and a bit of absorbent paper to show method of irriga- 
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 Objecfts 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- VII.] 
MOUNTING AND LABELING. 
151 
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. 
c 
A 
B 
Fig. 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 
is inverted; S, slides on which are mounted preparations which are to be kept 
moist. These slides are seen endwise and rest upon a bench made by cementing 
short pieces of large glass tubing to a strip of glass of the desired length and width. 
B—Two 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 a?id raise it without da?iger 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 slide is put on a turn-table and 
a ring of the shellac cement added. (C) Balsam preparations may be 
sealed with shellac as soon as they are prepared, but it is better to allow 
them to dry for a few days. One should never use a cement for 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 parti}’’ or 
wholly ruin the preparation. Shellac is 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. VII. 
§ 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 transparency to enable 
one to use high enough powers for the demonstration of minute details. 
By using sufficient care almost any tissue may be mounted in a 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 (§ 243). 
§ 241. Order of Procedure in Mounting Objedts 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 011 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 (§ 238 C). 
§ 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 may 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 CM. VII. ] 
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- 
hydration,, 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 emplo}red for this. Plenty of it must be 
used to displace the last trace of water. The tissue may be soaked in a 
dish of the alcohol, or alcohol from a pipette may be poured upon it. 
Dehydration usually occurs in the thin objects to be mounted in balsam 
in 5 to 15 minutes. If a dish of alcohol is used it must not be used too 
many times, as it loses in strength. 
The second step is clearing. That is, some liquid which is miscible 
with the alcohol and also with the resinous medium is used. This 
liquid is highly refractive in most cases, and consequently this step is 
called clearing and the liquid a clearer. The clearer displaces the alco- 
hol, and renders the object more or less translucent. I11 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 
[CH. VII. 
ISOLATION OF ELEMENTS. 
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 one another. 
The chemical agents or solutions for isolating are, in general, the 
same as those used for hardening and fixing. But the solutions are 
only about one-tenth as strong as for fixing, and the action is very much 
shorter, that is, from one or two hours to as many days. In the weak 
solution the cell cement or connective tissue is softened so that the cells 
and fibers may be separated from one another, and at the same time the 
cells are preserved. In fixing and hardening, on the other hand, the 
cell cement, like the other parts of the tissue, are made firmer. It is 
better also to dilute the fixing agents with normal salt solution (§ 313) 
than merely with water. 
§ 245. Isolation by Means of Formaldehyde.—Formaldehyde in 
a t2t5-% 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 40% 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 011 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. 
§ 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. VII.} 
ISOLATION OF ELEMENTS. 
155 
§ 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. 
§ 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 
Fig. 130. Adjustable lens holder with universal joint. This is 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 ismoresuc- 156 
ISOLATION OF ELEMENTS. 
\_CH. VII. 
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 da)Ts 
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 
Fig. 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 specimens are to be 
stained. The specimens may be mounted in glycerin, glycerin jelly or 
balsam. Glycerin jelly is the most satisfactory, however. CH VII.] 
COLLODION SECTIONING. 
157 
THE PREPARATION OF SECTIONS OF TISSUES AND ORGANS. 
§ 250. At the present time there are three principal methods of ob- 
taining thin sections of tissues and organs for microscopic study. These 
methods are : The Collodion Method, the Paraffin Method, and the Freez- 
ing Method. 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 harm. 
§ 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 €/c alcohol added. I11 a day or two this is replaced by 
75°/c or 82% alcohol ; 82c/c 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. VII. 
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. VII.] 
COLLODION SECTIONING. 
159 
the chloroform is poured off and the carbol-xylene* clarifier (§ 302) 
added. In a 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 
proceed even if the clarification is incomplete.f 
Fig. 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°/c 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 (C8 HI0) is called xylol in German. In English, mem- 
bers of the hydrocarbon series have the termination “ene,” while members of the 
alcohol series terminate in “ol.” 
f 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. 
[C/I. VII. 
§ 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 enu 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/A to 
6/a 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.f 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. 
f 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 °/c 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. 
Fig. 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 somewffiat 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°fc or stronger alcohol, the sec- 
tions dehydrated in 95 °Jc 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 a moment. Then 
it is placed horizontally, and some of the stain placed on the sections 
with a pipette, or preferably, it is immersed in a jar of the stain ; in 
case of immersion, 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 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.* 
Fig. 135. 
Fig. 136. 
Fig 134. Waste Bowl zuith rack for supporting slides and a small funnel in 
which the slides stand while draining. This outfit is easily made by any tin smith. 
The rack is composed of two brass rods about 3 mm. in diameter. The bent end 
pieces are sheet brass. The funnel is made of tin, copper or brass. Either copper 
or brass is preferable to tin. A glass dish like that shown in Fig. 733 is better than 
a bowl, as it can be more readily and thoroughly cleaned. {Cut loaned by Wm. 
Wood & Co.). 
Fig. 135. Round glass aquarium. This glass vessel is better than the bowl for 
all the uses described for the bozvl. ( Whitall, Tatum & Co.) 
Fig. 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-glasses [\ 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% alcohol suc- 
cessively. Each percentage may vary, but the principle of a gradual passing from 
strong alcohol to water was advocated. On the other hand, I have found that the 
safest method is to plunge the slide directly into water from the 95% alcohol. The 
diffusion currents are almost or quite avoided in this way. There is no time for 
the alcohol and water to mix, the alcohol is washed away almost instantly by the 
flood of water. So in dehydrating after the use of watery stains, the slide is 
plunged quickly into a jar of 95% alcohol. The diffusion currents are avoided in 
the same way, for the water is removed by the flood of alcohol. This plan has 
been submitted to the severe test of laboratory work, and has proved itself perfectly 
satisfactory. CIL 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. 
r. Fixing and hardening the tissues 
(§ 252), 4 days or more. 
2. Dehydrating the object to be cut in 
95% or stronger alcohol (§ 253), 2- 
24 hours. 
3. Saturating the tissue in ether-alcohol 
(§ 254), 2-24 hours. 
4. Infiltrating with thin collodion 
(§ 255)> 2 hours to 2 days. 
5. Infiltrating in thick collodion (£ 256), 
5 hours to several days. 
6. Imbedding the tissue ($ 257), 15 to 
20 minutes. 
7. Hardening the collodion with chlo- 
roform (§ 258), 5-24 hours. 
8. Clarifying and further hardening the 
collodion with castor-xylene (§ 258), 
10-36 hours. 
9. Cutting the sections ($ 259), 10 min- 
utes to 2 hours. 
10. Transferring the sections to a slide 
with paper ($ 260), 1 minute. 
11. Fastening the sections to the slide 
with ether-alcohol (§ 261), 1 or 2 
minutes. 
12. Removing the oil from the sections 
with benzin and alcohol (§ 262), 3-5 
minutes, or 24 hours. 
13. Staining the sections with an alco- 
holic dye (| 263-264), 2 minutes to 
24 hours. 
14. Staining the sections with an aque- 
ous dye (§ 264), 2-10 minutes. 
15. Removing the superfluous dye by 
washing in water or alcohol ($ 263- 
264), 2-5 minutes. 
16. Staining with a general dye (§ 264), 
15-30 seconds. 
17. Washing with water or alcohol 
($ 263-264), 1 to 2 minutes. 
18. Dehydrating the sections in 95% al- 
cohol (§ 266), 5,min. to 24 hours. 
19. Clearing the sections (§ 266), 5 min. 
to 24 hours. 
20. Draining the sections, Jt-2 minutes. 
21. Mounting in Canada balsam (£ 266), 
1-2 minutes. 
22. Sealing the cover-glass ($ 238), 2 
minutes. 
23. Labeling the preparation (.§ 291), 2 
minutes. 
24. Cataloging the preparation (£ 294), 
5-10 minutes. 
§ 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. 
Fig. 137. Small spirit lamp modified into a balsam 
bottle, or a glycerin or glycerin-jelly bottle, or a bottle 
for homogeneous immersion liquid. For all of these 
purposes it should contain a glass rod as shown in the 
figure. By adding a small brush, it answers well for 
a shellac bottle also. 
\ 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. VII. ] 
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 °fo alcohol and add 82% alcohol. Leave this on the 
tissue at least 1 day. The tissue may remain indefinitely in 82 °]o 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.—Place 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 as in 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 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 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.—Prom 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. VII.] 
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. 
§ 279. Staining with an Aqueous Dye.—Wash away the alcohol 
h}' 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. 
§ 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 011 
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 
organ (§ 269), 4 days or more. 
2. Dehydrating the object to be cut in 
95% or stronger alcohol ($ 270), 5 
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. 
5. Imbedding in pure paraffin (§ 273), 
10 minutes. 
6. Cutting the sections (£ 274), 10 min- 
utes. 
7. Fastening the sections to a slide 
(3 275), 5 minutes. 
8. Removing the paraffin (§ 276), 10 
minutes to 24 hours. 
9. Washing in 95% alcohol to remove 
the benzin ($ 277), 2 minutes. 
10. Staining with an alcoholic or aque- 
ous dye (\ 278-279), 2 minutes to 24 
hours. 
11. Washing away the superfluous stain. 
(2 278-279). 
12. Staining with a general dye (§ 280), 
10 seconds to 10 minutes. 
13. Washing the sections with water or 
alcohol (£ 280), 3-5 minutes. 
14. Dehydrating the stained sections in 
95%, alcohol (2 281), 5 minutes to 
24 hours. 
15. Clearing the sections (§ 282), 5 min- 
utes to 24 hours. 
16. Mounting in balsam (§ 283), 2 to 5 
minutes. 
17. Sealing the cover-glass (§ 238), 2 
minutes. 
18. Labeling the preparation 291), 2 
minutes. 
19. Cataloging the preparation (§ 294), 
5 to 10 minutes. 
SERIAL SECTIONS. 
§ 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, in 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. VII. ] 
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) ina}r be 
made. Oblique sections are often very puzzling. 
With cylindrical objects, especially botanical specimens, one may cut 
tangential sections, i. e., sections at right angles to a radius, or parallel 
with the radii (radial sections), or transections, i. e., 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 
(Fig- 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, Tra?isectio?is, Fro?ital Sections, Sagittal Sections : 
(A) Transections, i. 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. I11 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, 
i. e., 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 sectio?is, i. <?., 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) Sagittal sections, i. e., 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 
towTard 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. 
Slide No. 1. 
Transections of a Diemyctylus 
Embryo. 
Sections 1-10. 
Total thickness of Sections, 1 mm. 
May 20, 1892. 
EEET END. 
12345 
6 7 8 9 10 
Fig. 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 (jVt to nun.) for 
serial sections, then the cover will not prevent the use of high powers. 
When the ordinary slides (25 X 76 mm., 1X3 inch) are used cover- 
glasses 23 X 55 mm. may be advantageously employed. 
The combined thickness of the sections 011 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 a slide 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 and a 
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 
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.) 
No. 475. S'' —— 
/J Secs. § n 
Striated Muscle ; transection of the 
Sartorius of the Cat. 
October 15, 1894. 172 
LABELING AND CATALOGING. 
[CH. VII. 
§ 293. Cataloging Preparations.—It is believed from personal ex- 
perience, and front 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- 
ing Microscopical Preparations : 
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. 
9. 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. 
A Catalog Card Written According to 
this Formula : 
Muscular Fibers. Cat. 
C. 15. 
Fibers 20 to 40 fi thick.. 
2. No. 475. (Drr. IX) Oct. r, 1891. S. 
H. G., Preparator. 
3. Tendinous and intra-muscular ter- 
minations of striated muscular fibers 
from the Sartorius of the cat (Felis do- 
mestica.) 
4. Cat eight months old, healthy and 
well nourished. Fasting and quiet for 
12 hours. 
5. Muscle pinned on cork with vas- 
elined pins and placed in 20 per cent, 
nitric acid immediately after death by 
chloroform. Left 36 hours in the acid ; 
temperature 20° C. In alum water ('/> 
sat. aq. sol.) 1 day. 
6. Fibers separated on the slide with 
needles. Oct. 3. 
7. Stained 5 minutes with Delsfield’s 
hematoxylin. 
8. Dehydrated with 95%. alcohol 5 
minutes, cleared 5 minutes with carbol- 
turpentine, mounted in xylene balsam ; 
sealed with shellac. 
9. 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). 
10. The nuclei or muscle corpuscles 
are very large and numerous ; many of 
the intra-muscular ends are branched. 
See S. P. Gage, Proc. Amer. Micr. Sci., 
1890, p. 132 ; Ref. Hand-book Med., Sci., 
Vol. V., p. 59. CH. VI/.] 
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. 
Fig. 136. Writing diamond for writing numbers and labels on glass slides, cut- 
ting cover-glasses, etc. (Queen & Co.). 
The author has found stiff cards, cm., like those used for cataloging 
books in public libraries, the most desirable form of catalog. A specimen that is 
for any cause discarded has its catalog card destroyed. New cards may then be 
added in alphabetical order as the preparations are made. In fact a catalog on 
cards has all the flexibility and advantages of the slip system of notes (see Wilder 
& Gage, p. 45). 
Some workers prefer a book catalog. Very excellent book catalogs have been 
devised by Ailing and by Ward (Jour. Roy. Micr. Soc., 18S7, pp. 173, 348 ; Amer. 
Monthly Micr. Jour., 1890, p. 91 ; Amer. Micr. Soc. Proc., 1887, p. 233). 
The fourth division has been added as there is coming to be a very strong belief, 
practically amounting to a certainty, that there is a different structural appearance 
in many if not all of the tissue elements depending upon the age of the animal, 
upon its condition of rest or fatigue ; and for the cells of the digestive organs, 
whether the animal is fasting or full fed. Indeed as physiological histology is 
recognized as the only true histology, there will be an effort to determine exact 
data concerning the animal from which the tissues are derived. (See Minot, Proc. 
Amer. Assoc. Adv. Science, 189"), pp. 271-2S9 ; Hodge, on nerve cells in rest and 
fatigue, Jour. Morph., vol. VII. (1892), pp. 95-168 ; Jour. Physiol., vol. XVII., 
pp. 129-134; Gage, The processes of life revealed by the microscope ; a plea for 
physiological histology, Proc. Amer. Micr. Soc., vol. XVII. (1895), pp. 3-29 ; Sci- 
ence. vol. II., Aug. 23, 1895, pp. 209-218). 
CABINET FOR MICROSCOPICAL PREPARATIONS. 
| 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 convenient where one has but a few 
slides. For a laboratory or for a private collection where the slides are numerous 
the following characters seem to the writer essential: 
(1) The cabinet should allow the slides to lie flat, and exclude dust and light. 
(2) Each slide or pair of slides should be in a separate compartment. At each 
end of the compartment should be a groove or bevel, so that upon depressing 
either end of the slide the other may be easily grasped (Fig. 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 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. 
Fig. 140.—A—.Part of a cabinet drawer seen 
from above. In compartment 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 is removed at one place to show that in 
sealing the cover-glass, the cement is put 
partly on the cover and partly on the slide. 
(§ 229> 234)- 
B.—This represents a section of the same 
part of the drawer. (a) Slide resting as in 
A. No. 96 The preparation is seen to be 
above a groove in the floor of the compart- 
ment. (b) 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) O11 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. 140. 
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. 
It is better to have the slides 
on which the drawers rest 
somewhat shorter, then the 
drawer front may be entire 
and 7iot 7iotched as here 
shown. (From Proc. Amer. 
Micr. Soc. 1883). CII. VII ] 
PREPARATION OF REAGENTS. 
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 50 cc. of this x gram of salicylate of soda 
is added to prevent putrefactive changes. Probably a small amount of formal- 
dehyde, say 1 cc. of the 40%, to 50 or 100 cc. 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 r part of water. 
\ 299. 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 t he solution is saturated after it has stood a few days. An easy w7ay 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. For a half saturated solution take 100 cc. of water and xco cc. of saturated 
alum water and mix the two. 
$ 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 a stove or on a steam radiator, and allowed to re- 
main until the balsam may be pow'dered when it is cold. This requires a long 
time, the time depending on the temperature and the thickness of the layer of 
balsam. By heating the natural balsam in a tin or agate vessel over a Bunsen 
burner or an alcohol 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 
too thin 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 a syrupy 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 a cone and cementing the paper where it overlaps, or wind- 
ing a string several times around the lower part. Such a funnel is best used in one 
of the rings for holding funnels. 176 
PREPARATION OF REAGENTS. 
[CH. VI[. 
The xylene is much the bast substance to use for thinning the balsam. Such 
xylene balsam, as it 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. 
| 301. Chloroform Paraffin.—This is made bv 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. If it is kept warm the chloro- 
form evaporates in 3 to 6 days, leaving pure paraffin. 
\ 302. Clarifier, Castor-Xylene Clarifier.—This is composed of castor oil I part 
and xylene* 3 parts. 
§303. Clearing Mixture ($ 266, 282). — (A). One of the most satisfactory and 
generally applicable clearers is carbol turpentine, made by mixing carbolic acid 
crystals {Acidum carbolicum. A. phenicum crysializatum) 40 cc. with rectified 
oil of turpentine (Oleum terebinthinae rectificatum) 6o cc. If the carbolic acid 
does not dissolve in the turpentine add 5 cc. of 95% alcohol, or increase the tur- 
pentine, thus : carbolic acid 30 cc., turpentine 70 cc. 
(B). Carbol-Xylene, Clearer.—Vasale recommends as a clearer, xylene 75 cc., 
carbolic acid (melted crystals) 25 cc. It is used in the same way as the preceding. 
§ 304. Collodion.—This is a solution of soluble cottonf or other form of pyroxy- 
lin in equal parts of sulphuric ether and 95% alcohol. Three solutions are used : 
*The hydrocarbon xylene (CgHI0) is called xylol in German. In English, 
members of the hydrocarbon series have the termination “ene” while members 
of the alcohol series terminate in “ol.” 
fThe substance used in preparing collodion goes by various names, soluble cot- 
ton or collodion cotton is perhaps best. This is cellulose nitrate, and consists of a 
mixture of cellulose tetranitrate C12 Hl6 (N03)4 06, and cellulose pentanitrate, 
H,. (N03)5 Os. Besides the names soluble and collodion cotton, it is called gun 
cotton and pyroxylin. Pyroxylin is the more general term and includes several 
of the cellulose nitrates. Celloidin is a patent preparation of pyroxylin, more ex- 
pensive than soluble cotton, but in no way superior to it for imbedding. 
Soluble cotton should be kept in the dark to avoid decomposition. After it is in 
solution this decomposition is not so liable to occur. The decomposition of the dry 
cotton gives rise to nitrous acid, and hence it is best to keep it in a box loosely 
covered so that the nitrous acid may escape. 
Cellulose nitrate is explosive under concussion and when heated to 150° centi- 
grade. In the air, the loose soluble cotton burns without explosion. It is said not 
to injure the hand if held upon it during ignition and that it does not fire gun- 
powder if burned upon it. So far as known to the writer, 110 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 cc. of 9.5% 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) 1 )/l% or thin collodion. To prepare this 1*4 grams of soluble cotton are 
added to 100 cc. of ether-alcohol ($ 306). 
(C) 3X%0 collodion or cementing collodion. To prepare it of a gram of 
soluble cotton is added to iod cc. of ether-alcoliol. 
As both ether and alcohol are very volatile it is necessary to keep the bottles 
containing them well corked. 
\ 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 i-ioth of a gram of dry eosin added. 
The eosin is used after the hematoxylin in most cases (§ 280), and, as it is in 
alcoholic solution, it may be washed off with 95% alcohol if the object is to be 
mounted in balsam. If it is to be mounted in glycerin or glycerin jelly, the excess 
of eosin should be washed away with distilled water. 
\ 306. 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. 
I 3°7- Warrant’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- 
bic is soaked for several days in the arsenic water, then the glycerin is added and 
carefully mixed with the dissolved or softened gum arabic. 
This medium retains air bubbles with great tenacity. It is much easier to avoid 
than to get rid of them in mounting. 
\ 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 x2<y% solution of formalde- 
hyde in normal salt solution (§ 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 isolatingcells see \ 245. (Gage 
Micr. Bulletin and Sci. News, vol. XII. (1895), pp. 4-5). 
\ 309. 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., w'atersocc., 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 last a 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 rid of. 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 I7S 
PREPARATION OF REAGENTS. 
[Cl/. VII. 
usually takes about half an hour. When the gelatin is softened, as may be readily 
determined by taking a little in the fingers, pour off the superfluous water and 
drain well to get rid of all the water that has not been imbibed by the gelatin. 
Warm the softened gelatin over a water bath and it will melt in the water it has 
absorbed. Add to the melted gelatin about 5 cc. of egg albumen, white of egg ; 
stir it in well and then heat the gelatin in the water bath for about half an hour. 
Do not heat above 750 or 8o° 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 (*. e., in a place where the gelatin remains melted) the 
air-bubbles will rise and disappear. 
In case the glycerin jelly remains fluid or semi-fluid at the ordinary temperature 
(i8°-2o° C.), the gelatin has either been transformed into meta-gelatin by too high a 
temperature or it contains too much water. The amount of water may be lessened 
by heating at a moderate temperature over a waterbatli in an open vessel. This 
is a very excellent mounting medium. Air-bubbles should be avoided in mounting 
as they do not disappear. 
\ 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 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 200 cc. After the mixture is cool, 4 grams of chloral hy- 
drate, and i?ffths 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 flistilled 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 w'ith 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). 
| 31 x. Liquid Gelatin.—Gelatin or clear glue, 75 to 100 grams. Commercial ch. vir :j 
PREPARA TION OF REAGENTS. 
179 
acetic acid (No. 8) ioocc., water ioocc., 95% alcohol 100 cc., glycerin 15 to30cc. 
Crush the glue and put it into a bottle with the acid, and set in a warm place, and 
shake occasionally. After three or more days add the other ingredients. This 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. 
\ 312. Nitric Acid Dissociator.—This is prepared by mixing 80 cc. of water with 
20 cc. of strong nitric acid. It is used mostly in dissolving the connective tissue of 
muscle and thus making it possible to separate the fibers. Alum water is used as a 
restrainer (§299 and 249). (Gage, Proc. Atner. 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, prevents this deleterious action, or greatly lessens it, 
hence the name of normal salt solution. It is a T%% solution of table salt (sodium 
chlorid) in water ; water 1000 cc., salt 6 grams, or water 100 cc., salt T gram. 
§ 314. Paraffin.—Paraffin 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 im- 
bedding paraffin. For chloroform paraffin, 4 parts of imbedding paraffin are mixed 
with one part of chloroform ($ 301, 272). 
§ 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 is a \% solution of picric 
acid in 50% alcohol). It acts quickly, in from one to three days. (§ 252, 269). 
(Proc. Amer. Micr. Soc., Vol. XII (1890), pp. 120-122). • 
$ 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 ($ 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, 
i. e., in a week or two in a warm place, or in less time if the bottle is frequently 180 
MICRO-CHE MIS TR Y. 
[CH. VII. 
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. 
$ 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 to dry. 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 (§ 232, 240). See Newcomer, Arner. Micr. Soc.’s Proc., 
i885, p. 128; see also E. H. Griffith and H. L. Smith, Amer. Jour, of Micros., iv, 
102, v, 87; Amer. Monthly Micr. Jour., i, 66, 107, 113. Cunningham, The Micro- 
scope, viii, 1888, p. 237. 
MICRO-CHEMISTRY AND CRYSTAREOGRAPHY—EXPERIMENTS. 
$ 318. 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 common 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.” As a rule, 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. VI/.] 
MICRO- CHEM IS TR Y. 
181 
in a tall glass vessel to deposit its sediment. Take a concentrated drop of this sed- 
iment and mount it on a slide under a cover-glass. Study the preparation with the 
microscope. Probably there will be an abundance of animal aud vegetable life as 
well as of solid sediment. Put a drop of dilute sulphuric acid (Acidum sulphuri- 
cum, i. <?., strong sulphuric acid 1 gram, water 9 grams) at the edge of the cover 
Fig. 139. Czapski's Ocular Iris-diaphragm with cross hairs 
for 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). 
A. 
B. 
and at the opposite edge a small piece of the lens paper (Fig. 128). The acid will 
gradually diffuse, aud 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 au 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. 
\ 321. 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. 
1. 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. I/. M. 
Dennis, of the Chemical Department. 182 
MICRO-CHE MISTR Y. 
[Cl/. VI/. 
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 011 the cover-glass, mount dry, or preferably fuse in 
balsam ($ 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 ($ 2x8). 
\ 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 ; Klement & Re- 
nard; Carpenter-Dallinger ; Hogg ; Behrens, Kossel und Scliiefferdecker ; Frey ; 
Dana, and other works on mineralogy ; Davis. CHAPTER VIII. 
PHOTO-MICROGRAPHY AND PHOTOGRAPHY WITH A 
VERTICAL, CAMERA.* ‘ 
APPARATUS AND MATERIAL FOR THIS CHAPTER. 
Compound microscope with achromatic condenser; Achromatic and apochro- 
maticobjec 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 photography, like printing frames, etc., etc. ; Photographic-objectives, 
one for large objects and one for small objects to be magnified from two to fifteen 
diameters. 
$ 323. 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 
(1802). 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.” f 
* 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 mean difer- 
ent things. Thus : A photo-micrograph is a photograph of a small or microscopic 
object, usually made with a microscope and of sufficient size for observation with 
the unaided eye; while a micro-photograph is a small or microscopic photograph 
of an object, usually a large object, like a man or woman, and is designed to be 
looked at with a microscope. 
Dr. A. C. Mercer, in an article in the Proc. Amer. Micr. Soc., 1886, p. 131, says 
that Mr. George Shadbolt made this distinction. See the Liverpool and Manches- 
ter Photographic Journal (now British Journal of Photography), Aug. 15, 1858, p. 
203 ; also Sutton’s Photographic Notes, Vol. Ill, 1858, pp. 205-208. On p. 208 of 
the last, Shadbolt's word “Photomicrography” appears. Dr. Mercer puts the 
case very neatly as follows : “A photo micrograph is a macroscopic photograph of 
a microscopic object; a micro-photograph is a micro scopic photograph of a macro- 
scopic object.” See also Medical News, Jan. 27, 1894, p. 108. 
fin 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 
PHO T0- M1CROGRA PH Y. 
{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 is sometimes 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 
macro-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 isochromatic 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 achromatic 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 object 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 samples of the best work that has been produced. Among those who 
showed the possibilities of plioto-micrographs was Col. Woodward of the U. S. 
Army Medical Museum. The photo-micrographs made by him and exhibited at 
the Centennial Celebration at Philadelphia in 1876, serve still as models, 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 plioto-micrographs 
of histological objects have ever exceeded those made by Woodward, and most of 
them are vastly inferior. It is gratifying to state, however, that at the present 
time (1896) many original papers are partly or wholly illustrated by photo-micro- 
graphs, and no country has produced works with photo-tnicrographic 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 [Donne & 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. VII!.] 
PHO TO-MICROGRAPHY. 
185 
As the difficulties of photo-micrography are so much greater than of ordinary 
photography, the advice is almost universal that no one should try to learn photo- 
graphy and photo-micrography at the same time, but that one should learn the 
processes of photography by making portraits, landscapes, copying drawings, etc., 
and then when the principles are learned one can 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 tnicroscopist, in 
examining a section with high powers, has his finger on the fine adjustment screw 
and focuses up and down to bring different planes into view. He is in the habit of 
fixing his attention on the part of the field which is in focus and discarding the 
rest. But in a photograph the part of the field not in focus appears in a promi- 
nent way which mars the beauty of the picture.” 
APPARATUS FOR PHOTO-MICROGRAPHY. 
I 324. Camera.—For the best results with the least expenditure of time one of 
the cameras especially designed for photo-micrography is desirable but is not by 
any means 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 even 
from the best makers are not always correctly adjusted. By using a straight edge 
of some kind, one can measure the distance from the inside or ground side of the 
focusing screen to the surface of the frame. This should be done all around to see 
if the focusing screen is equally distant at all points from the surface of the 
frame. If it is not it should be made so. When the focusing screen has been ex- 
amined, an old plate, but one that is perfectly flat, should be put into the plate 
holder and the slide pulled out and the distance from the surface of the plate 
holder determined exactly as for the focusing screen. If the distance is not the 
same, the position of the focusing screen must be changed to correspond with that 
of the glass in the plate holder, for unless the sensitive surface occupies exactly 
the position of the focusing screen the picture will not be sharp no matter 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. i86 
PHO TO-MICROGRA PA Y. 
ICH. VIII. 
No. 7148 
WALMSLEY’S IMPROVED E. R. & C. PHOTO-MICROGRAPHIC CAMERA. 
Arranged for Work. 
Fig. 143. CH. VIII] 
PHO TO MICROGRAPHY. 
187 
Fig. 143. Walmsley's Improved Photo-Micrographic Camera. In this figure is 
shown an excellent form 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-mi- 
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. It is also arranged for making lantern slides. A 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 it works with great smoothness. “A groove 
is turned in the periphery of the fine adjustment screw, around which a small cord 
is 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 is 
fixed upo?i the image oti the screen, suffices to adjust the focus with the utmost 
exactness. If preferred, a rod running the entire length of the camera-bed, and 
terminating at the rear e?id 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 zvith the cord and weights." (From Mr. Walmsley.) 
I 325. Work Room.—It is almost self-evident that the camera must be in some 
place free from vibration. Frequently a basement room where the camera table 
may rest directly on the ground or on a pier is an excellent situation. Such a sit- 
uation is almost necessary for the best work with high powers. For those living 
in cities, a time must also be chosen when there are 110 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. 
\ 326. Arrangement and Position of the Camera and the Microscope.—For the 
greater number of plioto-micrograplis, 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 square of the power, and only inversely as the numerical 
aperture (§ 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 pow’er with a 
shorter bellows. (See Carpenter-Dallinger, pp. 318-319). 
\ 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 
PHO TO-MICROGRA PH Y. 
\CH. VIII. 
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. 
Fig. 144. 
Fig. 144. Leitz’ Photo - Micrographic Camera for Vertical Microscope (from 
Leitz' American House, New York). The camera may be raised or lowered to 
adapt it 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 focusing can be done with the hand on the fine adjust- 
ment, as in ordinary microscopical study. CH. V///.] 
PHO TO-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 he has good achromatic ob- 
jectives. From 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 Huvgenian 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 specimens.* 
Fig. [45. Projection Oculars with section re- 
moved to show the construction. Below are 
shown the upper end with graduated circle to 
indicate the amount of rotation found necessary 
to focus the diaphragm on the screen. No. 2, 
No. 4.. The numbers indicate the amount the 
ocular magnifies the image formed by the ob- 
jective as with the compensation oculars. 
{Zeiss' Catalog, No. 30). 
\ 329. 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 apocliromatics are practically free 
from it. Since the introduction of orthochroinatic 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. inch), 5 mm. (1 
to | inch) and 2 mm. (inch) homogeneous immersion of the Bausch & Lomb 
Optical Co.; Gundlach Optical Co.; Reitz; 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 
PHO TO MICROGRAPHY. 
[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 ortliochromatic plates are used 
the fault may not rest with the objective but with the plate holder and focusing 
Fig. 146. Walmsley's Autograph Photo-Micrographic Camera in a vertical po- 
sition. Compare Fig. 147 (From the Proc. Amer. Micr. Soc. Vol. XVII, 1895). CH. VIII.] 
PHO TO-MICROGRAPHY. 
191 
screen. They should be carefully tested to see if there is coincidence in position 
of the focusing screen and the sensitive film as described in (§ 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. If a 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 Steiuheil lens combination gives a more brilliant light and one also more 
nearly achromatic. For high power work all are agreed that nothing will take the 
place of an achromatic condenser. This may be simply an achromatic condenser, 
but preferably it should be an apochromatic condenser. Whatever the form of the 
condenser it should possess diaphragms so that the aperture of the condenser may 
be varied depending upon the aperture of the objective. For a long time, objec- 
tives have been used as achromatic condensers, and they are very satisfactory, 
although less convenient than a special condenser whose aperture is great enough 
for the highest powers and capable of being reduced by means of diaphragms to 
the capacity of the lower objectives. It should also be capable of accurate 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 difference 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 sensitive plate. This action of the 
longer waves (see under spectroscope \ 179-198), is greatly aided by the isocliro- 
matic or ortliochromatic 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 it is 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. Teaming 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- 
lin and then after drying, Canada balsam and a 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. 
§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. 
If one examines a series of photo-micrographs the chances are that the greater 
number will be of diatoms, plant sections or preparations of insects. That is, they 
are of objects having sharp details and definite outlines, so that contrast and defi- 192 
PHO TO-MICROGRAPH Y. 
[C/f. VIII. 
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 vegetable tissue. It is useless to expect to get a clear photo- 
graph of a section in which the details are seen with difficulty when studying it 
under the microscope in the ordinary way. 
Many sections which are unsatisfactory as wholes, may nevertheless have parts 
in which the structural details show with satisfactory clearness. In such a case the 
part of the section showing details satisfactorily should be surrounded by a delicate 
ring by means of a marker (see Figs. 61-66). If one’s preparations have been 
carefully studied and the special points in them thus indicated, they will be found 
far more valuable both for ordinary demonstration and for photography. The 
amount of time saved by marking one’s specimens can hardly be overestimated. 
The most satisfactory material for making the rings is shellac colored with 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. 
Fig. 147. Walms/ey's Autograph Photo micrographic Camera in a horizontal 
position. A microscope lamp and bull's-eye condenser are in position. Compare 
Fig. 148 in (Proc. Amer. Micr. Soc., Vol. XVII, 1895). 
$ 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 CH. VHP] 
PHO TO-MICROGRA PH Y. 
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 aud moderate power work. 
And even for 2 mm. homogeneous immersion objectives, the time of exposure is 
not excessive for many specimens (1'f to 3 minutes). This light is also cheapest 
and most available. 
Fig. 148. Vertical Photo-Micrographic 
Camera furnished by the Bausch & Lomb 
Optical Company. (From the 15th edition 
(/896) of their Catalog.) 
EXPERIMENTS IN PHOTO-MICROGRAPHY. 
§ 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 10 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, wdde angled, photographic objectives 
to give excellent results. It is necessary to have considerable length 
(i}4 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 194 
PHO TO-MICROGRAPH V. 
\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 
(z. 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 
90 seconds, depending 011 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 for a 
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. VIII.} 
PHO TO-MICROGRAPHY. 
195 
Fig. 149. Zeiss' Apochromatic, Projection Objective of 70 
mm. equivalent focus, for photo-micography. (Zeisss' Catalog. 
No. jo), This, and another of jj mm. focus, are designed for 
making pictures of moderate magnification. Usually rather 
large objects are photographed with them. The object may be il- 
luminated in the ordinary way. They are used without an ocular, 
like a photographic objective. The one of jj mm. is screwed 
into the tube of the microscope like an ordinary objective, but 
the one of 70 mm. here shown, is, by means of a conical 
adapter, screwed into the ocular end of the tube. 
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 it focuses the light upon the objective, not upon the 
object. The object is 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 to the 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 
Fig. 150. Focusing screen with clear center 
for the final adjustment with a focusing glass 
like that shown in Fig. 153 or 154. 
r. The ground surface of the focusing screen; 
it is translucent but not transparent. 
2. Central clear part of the screen made by 
cementing a cover-glass to the ground surface 
with Canada balsam. In the center is shown 
the pencil mark to hidicate 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 
PHO TO-MICROGRAPH Y. 
[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. 
Fig. 151. Objective of about go millimeters equivalent focus for 
making photo-micrographs at a magnification of 2 to 15 diameters. It was found 
to give the best results when used right end to as in ordinary photography. (From 
the Gundlach Optical Co.) 
Fig. 152. Zeiss' A11 as - 
tigmat Objective of about 
85 millimeters equivalent 
focus for photo-micro- 
graphs at a magnification 
of 2 to 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- 
isfactory 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 CII. VIII.] 
PHO TO-MICROGRA PH Y. 
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. A113’ good developer may be used. One can as a rule do no 
better than to follow the directions accompanying the plates used. 
The writer’s experience has been so satisfactory with Mr. Wahnsley’s 
developers that he desires to call attention to them. 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 Cramer’s instantaneous isochromatic plates, and the image com- 
menced to appear with Wahnsley’s developer in 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 
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. 
Fig. 153. Focusing Glass. “ It is 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 Gundtach 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 
PHO TO MICROGR A PHY. 
[ClI. VIII. 
carefully, however. This label should contain as far as possible, the 
following information : (i) 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. 
Fig. 154. Tripod magnifier. This serves fairly well 
as a focusing glass, but is inferior to the one show?i in 
Fig- 453- 
§ 339- Low Microscopic Objectives.—Mi- 
croscopic objectives of 35 111111. 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 Tomb 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 
photographic objectives (Figs. 151-152) and the apochromatic objec- 
tives of 35 and 70 mm. focus made by Zeiss. 
Fig. 155. Engraving glass to serve as 
a bull's-eye condenser. (From the 
Bausch and Lomb Optical Co.). 
§ 34°- Photo-Micrographs of 
20 to 50 Diameters.—For these, 
low objectives are used (35 mm. to 
20 mm. 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. ] 
PHO TO-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 drawr 
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. 
Fig. 156. Lens holder composed of several links and balls, thus giving the flexi- 
bility of a chain and enabling one to 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 is 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 
PIIO TO-MICROGRAPH Y. 
[CR. VIII. 
circle, i. e., 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. 
Fig. 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 get a 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. 
255, or the tripod magnifier {Fig. 13f) answers fairly. The Steinheil lenses are 
still better. 
3. Screen showing image of the flame 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 bv 
putting a smaller diaphragm over the eye lens. This would aid in 
making the field uniformly sharp. CH. VIII.} 
PHOTO-MICROGRAPHY. 
201 
§ 341- Photo-Micrographs at a Magnification of ioo 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. I11 the beginning one may try without a screen, and 
with different screens and compare 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. 
§ 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 
PHO T0-M1CROGRA PH 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. 
Fig. 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) is held. This rod is now 
graduated in centimeters and is a ready 
means of determining the length of the cam- 
era. M. Mirror of the microscope. L. The 
sleeve serving to make a light tight connection 
between the camera and microscope. O. The 
lower end of the camera. R. The upper end 
of the camera where the focusing screen and 
plate holder are situated. (From Zeiss' Photo- 
micrographic Catalog'). 
§ 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- Fig. 161. 
a 
Fig. 160. 
/ CH. VIII.} 
PHO TO-MICROGRAPHY. 
203 
Figs. 160-161. Fine tint, half-tone 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. 
/. Frontal section of the head of a large red Diemyctylus viridescens (red newt) 
at the level of the portae of the brain, magnified 10 diameters. Negative made 
with a Gundlach perigraphic objective of about go 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. 
Fig. 159. Rack for drying negatives. (From 
the Rochester Optical Co.) 
§ 347. Photo-Micrographs at a Mag- 
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 directions 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., 1 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. 204 
PHO TO-MICROGRAPHY. 
[ CH. VIII. 
Camera 
and 
Objective. 
Ocular. 
Magnifica- 
tion and 
Length of 
Bellows. 
Light, 
Condenser.: Hour, 
Date. 
Exposure 
and 
Remarks. 
Object. 
Stain. 
Color 
Screen. 
Plates. 
Developer. 
Remarks. 
Photogra- 
pher. 
Vertical 
(Zeiss’). 
X 4 
X 160 
iZNiSA„ ! daylight. 
35 sec. 
Silvered 
Hepatic 
Ligt. 
Neclurus. 
Silver 
and Hema- 
toxylin. 
None. 
Cramer’s 
Inst. Iso- 
chromatics 
Rodinal 
1-30 
Negative 
Good. 
Stotsen- 
burg. 
Zeiss’ 8 mm. 
Apochromatic 
Projection. 
Bellows, 
32 cm. 
Achro- ; 5 p. m., 
matic. May 16, ’95. 
Correct. 
■ 
I 348. RECORD TABLE FOR PHOTO-MICROGRAPHY. CH. V/II.] 
PHO TO MICROGR A PH Y. 
205 
PHOTOGRAPHING NATURAL HISTORY SPECIMENS WITH A VERTICAL 
CAMERA.* 
§ 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. In case 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 almost if not quite as sharp 
as those made in air. Even the corrugations on the scales of such 
fishes as the sucker (Catostomus teres) show with great clearness. 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- 
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 18S3 ; and in Science, Vol. 
III, pp. 443. 444- 206 
PHO TO-MICROGRAPH Y. 
[CH. VIII. 
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 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 : 
i°. The avoidance of distortion ; 2°. The adjustment of the camera to 
obtain an image of the desired size ; 30. Focusing ; 40. Fighting and 
centering the object; 50. Obtaining outlines for tracing upon the 
drawing-paper. ’ ’ 
“ i°. 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. ’ ’ 
“ 20. By placing the camera on a long table, and a scale of some kind 
against the wall, the exact position of the ground glass for various 
sizes may be determined once for all. 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. ’ ’ 
30. 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.} 
PHO TO-MICROGRAPHY. 
207 
Arrangement of a vertical or horizontal camera for making photographs of nat- 
ural history objects—sectional view. 
1. 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 arid 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 
desired. 
4. Ground glass. It is an advantage to have a clear space in the middle for 
accurate focusing, as for the photo-micrographic camera (Tig. 133). 
3. 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. 
9. Hinge on which the camera swings. 
10. Drawer in which are kept the objective and other accessories. 
11. 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. 
Fig. 162. 
4°. If the photographic prints are to be used solely for outlines, the 
well-known blue prints so much used in engineering and architecture [CH. VIII. 
208 
PHO TO-MICROGRA PH Y. 
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. 
§351. 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 CH. VII/.] 
PHO TO-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 Mier. 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. APPENDIX. 
Abbe’s Test-Plate ; The Apertometer ; Experimental Deteimination of the 
Equivalent Focus of the Objective and of the Ocular; Testing Homogeneous 
Fluid ; Preparation of Diagrams ; Drawings for Photo-Engraving. 
§ 352. For the sake of those who may have opportunity to use Abbe’s Test- 
Plate the following directions from Zeiss are appended : 
“ 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 
a slide. The thickness of each is written on the silver film. Groups of parallel 
lines are cut through the film and these are so coarsely ruled that they are easily 
resolved by the lowest powers, yet from the extreme thinness of the silver they 
form a very delicate test for objectives of even the highest power and widest 
aperture. To examine an objective of large aperture the plates are to be focused 
in succession observing each time the quality of the image in the center of the 
field and the variation produced by using alternately central and very oblique 
illumination. When the objective is perfectly corrected for spherical aberration 
for the particular thickness of cover-glass under examination, the contour of the 
lines in the center of the field will be perfectly sharp by oblique illumination 
without any nebulous doubling or indistinctness of the minute irregularities of the 
edges. If after exactly adjusting the objective for oblique light central illumina- 
tion is used no alteration of the adjustment should be necessary to showr the con- 
tours with equal sharpness.” 
“ If an objective fulfills these conditions with any one of the plates it is free 
from spherical aberration when used wdtli 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 overcorrection 
or to irregularity in the convergence of the rays.” 
“ The test of chromatic correction is based on the character of the color bands, 
which are visible by oblique illumination. With good correction the edges of the 
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- 
“ CARE ZEISS, JENA. ON THE METHOD OF USING ABBE’S TEST-PEATE. APPENDIX.] 
ABBE’S TEST-PLATE. 
211 
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 i8o° 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 4o°-5o°) 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 in employing the test as above described to 
have brilliant illumination and to use an eye-piece of high power.” 
“ When from practice the eye has learnt to recognize the finer differences in the 
quality of the contour images this method of investigation gives very trustworthy 
results. Differences in the thickness of cover-glasses of o.oi or 0.02 mm. can be 
recognized with objectives of 2 or 3 mm. focus.” 
“ With oblique illumination the light must always be thrown perpendicularly to 
the direction of the lines.” 
“ 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.” 
Fig. 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 maybe 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 Tolies, but it 
is carried out in a simpler manner ; it is shown in Fig. 165. As seen by this figure 
Fig. 165. Abbe Apertometer. 
it consists of a semi-circular plate of glass. Along the straight edge or chord the 
glass is beveled at45°, and near this straight edge is a small, perforated circle, the 
perforation being in the center of the circle. To nse the apertometer the micro- 
scope is placed in a vertical position, and the perforated circle is put under the mi- 
croscope and accurately focused. The circular edge of the apertometer is turned 
toward a window or plenty of artificial light so that the whole edge is lighted. 
When the objective is carefully focused on the perforated circle the draw-tube is 
removed and in its lower end is inserted the special objective which accompanies 
the apertometer. This objective and the ocular form a low power compound mi- 
croscope, and with it the back lens of the objective, whose 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 i8o° 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 the exact equivalent of the aperture of the objective. As the nu- 
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 tljat 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 lo designate it, the limit of aperture, 
for curious as the expression may appear we have found at times that the back 
lens of an objective is larger than the aperture of the objective requires. In that 
case the edges of the screen refuse to touch the periphery.” 
In determining the aperture of homogeneous immersion objectives the proper 
immersion fluid should be used as in ordinary observation. So, also, with glycerin 
or water immersion objectives. APPENDIX.] 
TESTER POP 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 T. 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., 18S5, 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 (§ 46-47). 
$ 355 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, r.oo N. A. (Fig. 41) : 
TESTER AND LIQUID IN THE 
CONCAVITY. 
SIZE OF THE 
FIELD. 
ELEVATION OF THE TUBE NEC- 
ESSARY TO RESTORE THE 
FOCUS. 
No tester used 
Whole thickness of the tester at 
1.825 mm. . 
Standard position. 
one end, not over the cavity . . 
1.85 mm. . . 
No change of focus. 
Tester with air in the cavity . . . 
.6 mm. . . 
Tube raised 6 mm. 
Tester with water. 
1.075 mm. . 
Tester with 95% alcohol .... 
1.15 111m. . . 
kerosene .... 
1.4 mm. . . 
2 111m. 
Gundlach Opt. Co’s 
horn, liquid 
Rausch & Tomb Opt. Co.’s honi. 
1.825 mm. . 
.16% mm. 
liquid 
1 825 mm. . 
mm- 
Leitz’ hom. liquid 
1.825 mm. . 
tA mm. 
Zeiss’ hom. liquid 
1.825 mm. . 214 
DETERMINATION OF EQUIVALENT FOCUS. 
{APPENDIX. 
It will be seen by glancing at the above table that whenever the liquid in the 
tester is of lower index than glass, that the concavity with the liquid acts as a 
concave lens, or in other words like an amplifier (§ 152), and the field is smaller 
than when no 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, i. e., 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 oe 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. 
\ 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 and focus carefully. Make the lines of the two micrometers 
parallel (Fig. 101). 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 -fa mm. and that it required 10 spaces on the ocular micrometer 
to enclose 2 spaces on the stage micrometer, evidently then 5 spaces would cover 
one. The image, A'B1 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 (x2ff mm.), that of the image directly 
measured ({§ mm.), the distance from the lens to the image also determined in the 
beginning, there remains to be found the distance between the objective and the 
object, which will represent approximately the equivalent focus. The general 
formula is, Object, O : Image, I : : equivalent focus, F : 250. Supplying the 
known values, 0 = x2ff, I = x§ then T%m : 1 mm : :F : 250 whence F — 50mm. 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 (A1 B', 
Fig. 21) unaffected by the ocular. It may be determined experimentally exactly APPENDIX.-] 
PREPARA TION OF DIAGRAMS. 
215 
as described in \ 357. For example, the image of the object (T2ff mm.) measured 
by the ocular micrometer, at a distance of 250 mm. is Jj} mm., i. e., 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 230 mm. by the equivalent focus in millimeters. Thus the 
initial magnification of a 5 mm. objective is — 50 ; of a 3 mm., 2|o =83.3 ; of 
a 2 mm., = 125, etc. 
| 359 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 this case. 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 \ 357, 358, then 
by the aid of Wollaston’s camera lucida the magnification of the whole microscope 
is obtained, as described in \ 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 a 50 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. Now if the ocular multiplies the real 
image 10 fold it has the same multiplying power as a simple lens of 25 mm. focus 
for, using the same formula as before ; o = 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. 1037 to 1049 °f Carpenter-Dallinger for 
mathematical formulae ; also Daniell, Physics for medical students; Czapski, 
Theorie der optischen Instrumente; Dippell, Nageli und Schwendener, Zimmer- 
man n. 
PREPARATION OF DIAGRAMS 
§ 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 a fact, 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 
/JAM WINGS FOR PHOTO-ENGRA VING. 
IAPPENDIX. 
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 a sponge. 
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 back 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 Coude crayons. The Cond£ cray- 
ons are better for lettering, however. 
It requires only about 12 hours for the fixative to dn . 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 it is 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 
§ 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. 
\ 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 § 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. 
\ 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. 
| 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, APPENDIX. ] 
DP A WINGS FOR PHO TO-ENGRA VING. 
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 x/% is sufficient ; in 
the latter, x/3 or even is desirable. The most generally useful reduction is found 
to be y3*. 
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 io x 16 cm., and the reduction is ]/3, then the drawing should be made 
Yz larger than the plate ; that is, 15 x 24 cm. and ]/3 reduction will produce the ex- 
act size needed. Then the enlarged 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. 
\ 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. For a large natural history object, a blue print ($ 349) or a tracing made 
from a camera (§ 350) is obtained ; or for a microscopic object, a camera lucida or 
embryograph drawing is made ($ 176, 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 i\ory 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 (£ 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. If a 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 a shaded 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 done 
by dotting the surface with a pen (stipple) or by lines. It is much easier to pro- 
duce acceptable results by stippling, because a slight erasure may be made or a 
deeper shadow obtained by adding a few carefully placed dots. This method is 
well adapted for showing the structure of histological specimens. 
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. 
$ 367. Lettering. — A half-tone 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 be 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 reduc'ion determined upon is chosen. 
Numbers of the following size have been found convenient for numbering the 
separate figures of a plate for '/$ reduction ; 
60, 61, 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 
l/3 reduction, APPENDIX.] 
DR A WINGS FOR PHOTO-ENG RA VING. 
219 
R. be L. g. t. Filament of Necturus C. Leucocytes, 
For y% reduction the following are large enough, 
Meten. mtc. mtp. mtpr. 12345. 
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 by 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 
arc of a circle, by snipping the slip on which it is printed with the scissors and 
curving it after it is moistened. 
$ 36S. 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. 
§ 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 and thus making it impossible to add clear cut lines with the 
pen. 
\ 370. 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 
any 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. 
F'or 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 Society's Catalog of 
Scientific Papers, and in the bibliographical references given in special papers. A full list of peri- 
odicals may also be found in Vol. XVI of the Index Catalog. 
BOOKS. 
Adams, G.—Micrographia illustrata, or the microscope explained, etc. Illustrated. 4th edi- 
tion, London, 1771. Also Essays, 1787. 
Angstrom.—Recherches sur le spectre solaire, spectre normal du soleil. Upsala, 1868. 
Anthony, Wm. A., and Brackett, C. F.—Elementary text-book of physics. 7th ed. Pp. 527, 165 
Fig. New York, 1891. 
Barker.—Physics. Advanced course. Pp. 902, 380 Fig New York, 1892. 
Bausch, E-—Manipulation of the microscope. Pp. 95, illustrated. Rochester, 1891. 
Beale, L- S.—How to work with the microscope. 5th ed. Pp. 518, illustrated. London, 1880. 
Structure and methods. 
Beauregard, H., et Galippe, V.—Guide de l’61£ve et du praticien pour les travaux pratiques de 
micographie, comprenant la technique et les applications du microscope a l’histologie v£g6tale, 
a la physiologie, a la clinique, a la hygiene et a la medicine legale. 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., uud Schiefferdecker, P.—Das Mikroskop uud 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 Encyc. 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, E- C.—Guide to photo-micrography. 2d ed. Illustrated. London, 1892. 
Carnoy, J. B., Le Chanoine.—La Biologie Cellulaire ; Etude compar6e de la cellule dans les deux 
rdgnes. Illustrated (incomplete). Paris, 1884. Structure and methods. 
Carpenter, W. B.—The microscope and its revelations. 6th ed. Pp. 882, illustrated. London, 
and Philadelphia, 1881. Methods and structure. 
Carpenter-Dallinger.—The microscope and its revelations, by the 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. London and Philadelphia, 
1891. BIBLI OCR A PH Y. 
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. Part I. The microscope and instructions 
for its use. Part II. Microscopic objects, how prepared and mounted. Illustrated. 2d edition. 
London, 1895. 
Czapski, Dr. Siegfried.—Theorie der optischen Instrumente nach Abbe. Illustrated. Bres- 
lau, 1893. 
Dana, J. D.—A system of mineralogy. Illustrated. 6th ed. New York, 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 fiber die Ursachen der Anisotropie organischer Substanzen. 
Leipzig, 1882. Large number of references. 
Ellenberger, W.—Handbuch der vergleichenden Histologie und Physiologie der Haussauge- 
thiere. Berlin, 1884 +. 
Fol, H.—Lehrbuch der vergleichenden mikroskopischen Anatomie, init Einschluss der ver- 
gleicheuden Histologie und Histogenie. Illustrated (incomplete). Leipzig, 1884. Methods and 
structure. 
Foster, Frank P.—An illustrated encyclopcedic medical dictionary, being a dictionary of the 
technical terms used by writers on medicine and the collateral sciences in the Latin, English, 
French and German languages. Illustrated, four quarto volumes. 1888-1893. 
Fraenkel und Pfeiffer.—Atlas der Bacterien-Kunde. Berlin, 1889 +. 
Francotte, P.—Manuel de technique microscopique. Pp. 433, no Fig. Brussels, 1886. 
Francotte, P.—Microphotographie appliqu6e & l’histologie, l’anatomie compare 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. Einffihrung 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 
BIBLIOGRA PH V. 
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. Ann 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 natfirlichemundkfiustlichem 
Lichte. Figs, and Plates. Pp. 245. Berlin, 1888. 
King, J.—The microscopist’s companion. A popular manual of practical microscopy. Illus" 
trated. Cincinnati, 1859. 
Kldment and Renard.—Reactions microchemiques 4 cristaux et leur application eu 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. 
Dee, A. B.—The microtomist’s vade-mecum. A hand-book of the methods of microscopic 
anatomy. 4th ed. Philadelphia, 1896. 
Lehmann, C. G.—Physiological chemistry. 2 vols. Pp. 648+547, illustrated. Philadelphia, 
1855- 
Lehmann, O.—Molekularphysik mit besonderer Beriicksichtiguug mikroskopischer Unter- 
suchungen und Auleitung zu solchen, sowie eiuem Anhang fiber 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. 
MacMunn,-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. E. 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.-Dee., 1885. History of the micro- 
scope, and figures of many of the forms used at various times. 
Moitessier, A.—La photograpliie appliqude aux recherches micrographiques. Paris, 1S66. 
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, L.—Traits technique d’histologie. Pp. 1109, illustrated. Paris, 1875-1888. Structure 
and methods. Also German translation 1888. 
Reeves, J. E.—A hand-book of medical microscopy, including chapters on bacteriology, neo- 
plasms and urinary examination. Illustrated. Philadelphia, 1894. 
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.—Trait6 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 use of 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 Angstrom’s and Kirchhoff’s maps. London, 1885. 
Science Lectures at South Kensington. 2 vols. Pp. 290 and 344, illustrated. One lecture on 
microscopes and one on polarized light. London, 1878-1879. 
Sedgwick and Wilson.—General biology. 2d ed. Illustrated. New York, 1895. 
Seiler, C.—Compendium of microscopical technology. A guide to physicians and students in 
the preparation of histological and pathological specimens. Pp. 130, illustrated. New York, 1881. 
Silliman, Benj., Jr.—Principles of physics, or natural philosophy. 2d ed., rewritten. Pp. 710, 
722 illustrations. New York and Chicago, i860. • 
Starr, Allen M., with the cooperation of Oliver S. Strong and Edward Learning.—An Atlas of 
nerve cells. Columbia University Press, New York, 1896. The atlas consists of text, diagrams, 
and some of the best photo-micrographs that have ever been published. 
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 Practicuin. Anleitung zum Selbststudium der mikroskopis- 
cheti Botanik, fur 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 Win. 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 photographic appliqufie a l’histoire naturelle. Pp. 225, illustrated. Paris, 1884. 
Valentin, G.—Die Untersuchung der Pflanzen und der Thiergewebe in polarisirtem Licht. 
Leipzig, 1861. 224 
BIBLIOGRA PHY. 
Vierordt.—Die quantitative Spectralanalyse in ihrer Anwendung auf Physiologie, 1876. 
Vogel, Conrad.—Practical pocket-book of photography. Pp. 202, Figs. London, 1893. 
Vogel, H. W.—Practische Spectralanalyse irdischer stoffe ; Anleituug zur Benutzung der Spec- 
tralapparate in der qualitativen und quantitativen chemische Analyse organischer und unorgan- 
ischer Korper. 2d ed. Figs. Berlin, 1889. 
Wethered, M.—Medical microscopy. Pp. 406, Figs. London 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. 2d ed. New York and Chicago, 
1886. 
Wilson, Edmund B., with the cooperation of Edward Learning.—An atlas of fertilization and 
ICaryokiuesis. 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, no date. Upwards of 400 
figures of pretty objects for the microscope, also brief descriptions and directions for preparation. 
Wormly, T. 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. 4th ed. Pp. 434, 252 Fig. Philadelphia, 1880. 
Zeiss. R.—Special-catalog fiber Apparate ffir Mikro-photographie. Jena. Instructions for 
using the apochromatic objecthes 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, Washington, D. C., 18804-. 
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 microscopical society, Proceedings. 1878+. 
Anatomischer Anzeiger. Centralblatt fur die gesammte wissenschaftliche Anatomie. Amt- 
liches Organ der anatomischen Gesellschaft. Herausgegeben von Dr. Karl Bardeleben. Jena, 
18864-. Besides articles relating to the microscope or histology, a full record of current anatomi- 
cal literature is given. 
Annales de la soci£t£ beige de microscopie. Bruxelles, 1874+. 
Archiv fur microscopische Anatomie. Illustrated. Bonn, 1865+. 
Centralblatt fur Physiologie. Unter Mitwirkung der physiologischen Gesellschaft zu Berlin 
Herausgegeben von S. Exner und J. Gad. Leipzig und Wien. 1887+. Brief extracts of papers 
having a physiological bearing. Full bibliography of current literature. 
English mechanic. London, 18664-. 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, 18904-. 
Journal of anatomy and physiology. Illustrated. London and Cambridge, 18674-. 
Journal de micrographie. Illustrated. Paris, 1877-1892. 
Journal of microscopy and natural science. London, 18854-. 
♦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. 
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 trail - 
sactions of the state microscopical society of Illinois. Illustrated. Chicago, 1872-1873. 
The Microscope. Illustrated. Washington, D. C., 1881 + . 
Microscopical bulletin and science news. Illustrated. Philadelphia, 1883+. The editor, Ed- 
ward Pennock introduced the term “ par-focal” for oculars (see vol. iii, p 31). 
Monthly microscopical journal. Illustrated. London, 1869-1877. 
Nature. Illustrated. London, 1869J-. 
The Observer. Portland, Conn., 1890+. 
Philosophical Transactions of the Royal Society of London. Illustrated. London, 1665+. 
Proceedings of the American microscopical society, 18784-. 
Proceedings of the Royal Society. London, 1854+, 
Quarterly journal of microscopical science. Illustrated. London, 1853+. 
Science Record. Boston, 1883-4. 
Zeitschrift fur Instrumeutenkunde. 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 apd Annuals of Photog- 
raphy. Dec. i, 1896. Just Published 
Sixth Edition of 
THE 
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 FIGUREvS IN THE TEXT. 
PRICE $1.50 POST PAID. 
ITHACA, N. Y. 
Comstock Publishing Co. 
1896. 1 his new edition of Professor Gage’s work on the Microscope remains in 
general plan like that of former editions, that is, definitions and discussions are 
followed by laboratory experiments in which the student makes the demonstra- 
tions for himself. 
The figures have been increased from 103 to J65. In matter it has grown from 
165 to 237 pages. This increase is due to additions in the text of previous 
editions and to some wholly new matter upon methods of isolation and of 
sectioning by the collodion and by the paraffin methods. 
The preparation of permanent drawings for lecture purposes and for publication 
is so important both for teacher and pupil, that considerable space is give to 
these subjects. 
It is believed that the work as it now appears will aid both pupil and teacher 
still more efficiently than previous editions. All of the directions and methods 
have been so repeatedly put to the practical test of the laboratory, that they are 
believed to be correct, convenient, and applicable to the work of the experienced 
and of those just learning microscopical manipulation. 
I11 spite of the increase in figures and in text the price remains the same as 
for previous editions ($1.50 post paid). CONTENTS. 
CHAPTER I. 
PAGE. 
$ i- 55—The Microscope and its Parts—Demonstration of the Function 
of each Part. Figures of Laboratory Microscopes, 1-32 
CHAPTER II. 
£ 56-119—Lighting and Focusing, Manipulation of Dry, Adjustable, and 
Immersion Objectives; Care of the Microscope and of the 
Eyes 33-79 
CHAPTER III. 
§ 120-144—Interpretation of the Appearances under the Microscope, . . 80- 91 
CHAPTER IV. 
$ 145-167—Magnification of the Microscope; Micrometry, 92-108 
CHAPTER V. 
« 
$ 168-178—Drawing with the Microscope, 109-119 
CHAPTER VI. 
$ 179-218—Micro-spectroscope and Micro-polariscope ; Use and Applica- 
tion, 120-139 
CHAPTER VII. 
$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. 
$ 322-351—Photo-micrography and the Photography of Natural History 
Specimens in a Horizontal Position with a Vertical Camera, . 183-209 
APPENDIX. 
$ 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- 
ing 213-219 
BOOKS AND PERIODICALS 220-225 
INDEX, 227-237 Press Comments on the Third and Fourth Editions. 
From the American Monthly Microscopical Journal, Dec , 1891: 
“ It is up to date, and the section devoted to homogeneous immersion objectives, 
the substage illuminator, the camera lucida, the micro-spectroscope and the micro- 
polariscope will be especially welcome to workers. . . . Altogether it 
is one of those books that the student and worker in microscopy cannot afford to 
be without.” 
“ If the owner of his first instrument will carefully read this book, and follow 
all the experiments described by the author, he will find that he has within his 
reach almost a royal road to the proper understanding of the microscope and of its 
manipulation.” 
From the Microscope, Dec., 1891. 
From the Buffalo Medical and Surgical Journal, Jan., 1892. 
‘‘To the student or physician who wishes to study the microscope, we can 
heartily recommend this work and feel assured that knowledge thus gained will 
be of inestimable value in pursuing the study of histology, pathology and bac- 
teriology.” 
From the Medical News, April 30, 1892. 
“As indicated, this book has been written* for laboratory students. It is thor- 
oughly practical in scope and will prove a valuable adjunct in teaching the prin- 
ciples that govern the employment of the microscope as well as in demonstrating 
the application of these principles. There is but one way of acquiring a knowledge 
of the working as well as skill in the use of the microscope, and that is by per- 
severing application. The acquisition of such knowledge and skill may be ren- 
dered easy by intelligent direction. It is for this that the book under review com- 
mends itself. It is essentially a student’s book from which the most good is to be 
derived by performing the various manipulations that it directs. It admirably 
fulfills the object for which it was written.” 
From the Boston Medical and Surgical Journal, Jan. 26, 1893. 
“ We know of no other work so well calculated to give a beginner a clear and 
sufficient insight into the principles involved in microscopical observation and in 
the proper care of the instrument and specimens. 
From the Journal of the Royal Microscopical Society, June, 1892. 
“ Tliis is one of the most practical books yet presented to the laboratory student.” 
Front Nature, Sept. 8, 1892. 
(Review by Rev. W. H. Dallinger, editor of the last edition of Carpenter. The 
Microscope and its Revelations.) “ In short, this treatise lays the foundation for 
a thorough microscopical training, entirely adapted to the wants of medical 
students.” INDEX. 
A 
Abbe apertometer, 212. 
Abbe camera lucida, in; arrangement 
of, 112; drawing with, 115-118; fig- 
ures, 98, no-, 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. 
Aerial image, 30, 31. 
Air bubbles, 84, 85 ; with central and 
oblique illumination, 84, 85 ; air and j 
oil, distinguished optically, 85 ; by j 
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, 191. 
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. 
Artificial illumination, 36, 46, 48 ; for 
photo-micrography, 200. 
Autograph, photo-micrographic camera, 
190-192. 
Avoidance of diffusion currents, 162 ; of 
distortion, in. 
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 
INDEX. 
Benzin, removing of from sections, 166. 
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, 88. 
Body of microscope, Frontispiece. 
Bottle for balsam, glycerin or shellac, 164. 
Bowl, waste, 162. 
Bread crumbs, examination of, 90. 
Bromide paper, 208. 
Brownian movement, 88. 
Brunswick black, removal from lenses,58. 
Bubble, air, 84-85. 
Bull’s-eye, 49, 191, 200; engraving, glass 
for, 198. 
Burning point, 6 ; finding of, 30. 
Butterfly scales, 90. 
c 
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, 1x4, 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, no 111, 114 ; Wollaston’s, 95, 
111. 
Canada balsam, 175 ; mounting in, 163, 
167 ; preparation of, 175 ; removal 
from lenses, 58; removal from slides, 
141. 
Carbol-xyletie, 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. 
Cells, deep, thin, 148 ; isolated, prepara- 
tion of, 155 ; mounting, 148 ; stain- 
| i»g. 155- 
j 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 ol 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, paraffin, 176; infiltrating 
with, 165 ; saturating with, 165. 
j Chromatic aberration, 4 ; correction, 12, 
13 ; correction, test lor, 210. 
1 Chemical focus, 13 ; rays, 13. 
Clarifier, 176; castor-xv lene 176. 
Cleaning back lens of objective, 58 ; ho- 
mogeneous objectives, 56 ; mixtures 
for glass, 145 ; slides and cover- 
glasses, 140-142 ; water immersion 
objectives, 55. 
Clearer, clearing, 153, 164, 167, 176. 
Clearing mixture, preparation of, 176. 
I Clinical microscope, 76. 
I Clothes moth, examination of scales, 90. 
Cloudiness of objective and ocular, how 
to determine, 82 ; removal, 58. 
I 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. 
j Color correction, 13 ; images, 51, 56 ; law 
of, 124; production of, 138; screens, 
191. 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. 
Comparison prism, 127, 12S ; 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 ; apocliromatic, 40, 
191 ; bull’s-eye, 49, 198, 200; center- 
ing, 41, 45 ; illuminating cone with, 
44 ; mirror for, 46 ; non-achromatic, INDEX. 
229 
44; numerical aperture of, 43-44; 
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 spectium, 123. 
Contoured, doubly, 87. 
Converging lens, 3, 5 ; lens-system, 10. 
Convex lenses, 3, 5. 
Corn starch, examination of, 90. 
Correction, chromatic, or color, 13, 210 ; 
cover-glass, 15, 52-53; cover, tube- 
length for, 53-54; over and under, 
r3- 
Cost of microscope, 63. 
Cotton, collodion, 176; examination of, 
90; gun, 176; soluble, 176. 
Cover-glass, or covering glass, 141 ; ab- 
erration by, 51 ; adjustment, spe 
cific directions, 53 ; adjustment for, 
in pho o micrography, 201 ; adjust- 
ment and tube-length, 13, 14, 53 ; 
anchoring, 150; cleaning, 141, 142; 
correction, 52, 53 ; efftct 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 011. S4, 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, ) 66. 
D 
Dammar, for fixing crayon drawings, 
216 ; removal from lenses, 58. 
Dark-ground illumination, 36, 47, 48; 
with Abhe illuminator, 48 ; with 
mirror. 48 
Daylight, lighting with, 34. 
Defining power, 21. 
Dehydration, 157, 162, 167. 
Demonstration microscope, 77. 
Depolarizing crystals, 138. 
Designation of oculars, 26 ; of wave 
length, 129. 
Determination of field of microscope, 29 ; 
magnification, 92,94, 214; of sedi- 
ment in water, 180; of working dis- 
tance, 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, 44 ; with condenser, 44. 
Diffraction, grating, 122 ; illusory ap- 
pearances due to, 91. 
Diffusion currents, avoidance of, 162. 
D.reet, 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, in. 
Diverging It 11s, 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 lncida, 114- 
115 ; board for Abbe camera lucida, 
115-116; distortion avoidance of, in; 
embryograph for, t 19 ; with micro- 
scope, 109; photographic camera 
for, 119; for photo engraving, 216; 
scale and enlargement of, 118 ; tran- 
ferring, 217. 
Dry objectives, ji, 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, 
90 ; on objectives and oculars, how 
to determine, 82 ; removal, 58*. 
Dye, general, staining with, 167; aque- 
ous, 16 r, 167 ; alcoholic, 161, 167. 230 
INDEX. 
E 
Eccentric diaphragm, 42, 48. 
Embryograph, 119. 
Engraving glass for bull’s eye condenser, 
198. 
Erect image, 1. 
Equivalent focal length, 11,214; focus of 
objectives, 11, 26, 214; focus of ocu- 
lar, 26, 214. 
Elements, histological, isolation of, 154. 
Eosin, 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- 
mal hairs, potato, rice, scales of but- 
terflies and moths, wheat, 90. 
Experiments, Abbe illuminator, 46 ; with 
adjustableand 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 ;withmicro- 
poliriscope, 137 ; in mounting, 146- 
161 ; photo-micrography, 193; simple 
microscope, 6. 
Exposure of photographic plates, 194, 
199, 200, 203. 
Extraordinary ray of polarize 1 light, 136 
Eye and microscope, 1, 6, 10, 31. 
Eyes, careof, 5S; muscse volitantes of, 90 
Eye-lens of the ocular, 22. 
Eye-piece, 22 ; micrometer; ro2. 
Eye-point, 7, 22, no ; 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, 90. 
Fibers, examination of, 90. 
Field, 27 ; with camera lucida, 95 ; i 11 u 
initiation of, 44, 49 ; with orthoscopie 
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 micVometer ocular, 23 ; ocular mi- 
crometer, 103. 
Filter paper, Japanese, 57. 
Filters, light, 191. 
Filtering balsam, etc., paper funnel for, ; 
175- 
Fine adjustment, Frontispiece ; testing, 
60. 
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, ir. 
j Focus, 6 ; always up, 38 ; actinic, 189 ; 
chemical, 13 ; conjugate, 3 ; of ob- 
jectives, equivalent, n, 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 photomicrography, 
19I, 199, 2or ; screen for photomi- 
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- 
lation, 154. 
Formula for cataloging, 172. 
Fraunhofer lines, 123. 
Front combination or lens of objective, 
10. 11. 
Frontal sections, 170. 
Function of objective, 29 30 ; 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, 
140. 
GUie, 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 from lenses, 58. 
Goniometer ocular, 23. 
Graduation of adjusting collar, 53. 
Grating, diffraction, 122. 
Ground glass, preparation of, 29. 
Gun cotton, 176. INDEX. 
231 
H 
Hairs, examination of, 90. 
Half-tones from photo-micrographs, 208, 
216. 
Hardening collodion, 158; tissue, 157, 
164. 
Heliostat, 209. 
Hematoxylin, 178. 
Hemoglobin spectrum, 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 ; 
for laboratory microscope, 61 ; light 
utilized, 18, 21 ; numerical aperture, 
21. 
Homogeneous liquid, 12, 213 ; tester for, 
55. 213. 
Huygeuiau ocular, 22, 24, 31. 
I-J 
Illuminating, cone, aperture of, 43, 44 ; 
for condenser, 44; objective, 14 ; 
power, 21, 22. 
Illumination, for Abbe camera lucida, 
116; artificial, 36, 48; artificial for 
photo-micrography, 194; centering 
image of source of, 41-45 ; central 
with air and oil, 84, 85 ; dark-ground, 
36, 47, 48 ; daylight, 34 ; of entire 
field, 49 ; lamp for, 48 ; methods of, 
34, 47; for micro-polariscope, 137 ; 
for micro-spectroscope, 129 ; oblique, 
with air and oil, 84, 85 ; for photo- 
micrography, 192 ; for Wollaston’s 
camera lucida, m. 
Illuminator, 39-40; Abbe, 44, 47 ; Abbe, 
axial and oblique light, 36 ; Abbe, 
experiments, 46 ; Abbe, mirror and 
light for, 46; achromatic, 40; cen- 
tering and arrangement, 45 ; immer- 
sion. 45. See also condenser. 
Image, actinic, 184; aerial, 30, 31 ; cen- 
tering i. of source of illumination, 
42 ; color, 49, 51, 56 ; with dry apo- 
chromatic and water immersion ob- 
jectives, 13 ; erect, 1 ; inverted, 7 ; 
inverted, real of objective, 29 ; of 
flame, 43 ; formation of, 3 ; geomet- 
rical construction of, 5 ; and object, 
size and position, 5, 11, 96 ; real, 5, 
10, 22, 29-31, 92 ; refraction, 49, 55 ; 
retinal, 6, 10, 31 ; swaying of, 46 ; 
virtual i. and standard distance at 
which measured, 6, 31, 92. 
Image-power of objectives, 19. 
Imbedding, 158, 165. 
Immersion fluid, 55, 213; illuminator, 
45 J 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 ; for 
drawing, 216. 
Interpretation of appearances under the 
microscope, 80-91. 
Inverted image, 7, 29. 
Iris diaphragm, 40, 181. 
Irrigating with reagents, 150. 
Isochromatic plates, 194, 197. 
Isolation, 154 ; with formaldehyde, 154 ; 
nitric acid, 155. 
Isotropic, 137. 
Japanese filter or tissue paper, 57, 160. 
Jelly, glycerin, 177. 
Jena glass, 63. 
Jurisprudence, micrometry in, 108. 
L 
Labels and catalogs, ink for 173 ; prep- 
aration of, 179. 
Labeling microscopical preparations, 
171 ; photographic negative, 197 ; 
serial sections,, 171. 
Laboratory compound microscope, 61. 
Lamp, microscope, 48 ; spirit, 164. 
Lamp-light, 36. 
Law of color, 124. 
Lens, concave, 3 ; converging, 3 ; con- 
vex, 3 ; eye, 22 ; field, 22, 26 ; holder, 
8, !55» 156, 199 ; paper, 57 ; system, 
10. 
Lens-systems, 10. 
Letters in stairs, 82 ; for photo-engrav- 
ing, 218. 
Lettering oculars, 26. 
Light, with Abbe illuminator, 46; ace- 
tylene, 36, 48 ; artificial, 46 ; axial, 
35> 39 ! axial with Abbe illuminator, 
46 ; direct, 34 ; central, 35, 39 ; filters, 
191 ; incident, 34; with mirror, 39; 
oblique, 35, 39 ; oblique with Abbe 
illuminator, 46 ; lamp, 36 ; for pho- 
to-micrography, 192 ; polarized, 136 ; 
reflected incident or direct, 34 ; trans- 
mitted, 35 ; utilized with different ob- 
jectives, 13 ; wave length of, 129. 
Lighting, 34 ; for Abbe camera lucida, 
115 ; artificial, 48 ; experiments, 36 ; 
and focusing, 33, 36 ; for micro- 
polariscope, 137 ; for micro-spectro- 232 
INDEX. 
scope, 129 ; with a mirror, 36 ; with 
daylight, 34; for photo-micrography, 
192. 
Line spectrum, 123. 
Linen, examination of, 90. 
Liquid, currents in, 88; homogeneous, 
55, 213. 
M 
Macro-photography, 184. 
Magnification of compensating oculars, 
26 ; effect of adjusting objective, io5 ; 
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, 118; 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, 
88. 
Magnifier, tripod, 7, 198. 
Marker for preparations, 63, 64. 
Marking objects, 63, 64, 94, 101 ; nega- 
tives, 197 ; objectives, 65. 
Material and apparatus, 1, 33, 80, 92, 120, 
140, 175-181, 183, 210, 216. 
Measure, unit of, in micrometry, 100 ; of 
wavelength, 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, 
57 ; 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; filar m. ocular, 103; filling 
lines of, 94 ; net, 113 ; lines, arrange- 
ment of ocular and stage, 107 ; object 
or objective, 94 ; ocular or eye-piece, 
102, 103 ; ocular, micrometry with, 
105 ; 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, 
10S ; limit of accuracy in, 107 ; with 
ocular micrometer, 105 ; with simple 
microscope, ioo; remarks on, 106 ; 
unit of measure in, ioo. 
Micro-millimeter, joi. 
Micron, ioo ; 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, 1, 6, 10; field of, 27, 29 ; focus- 
ing, 31 ; magnification, 92 ; lor photo- 
micrography, 187; polarizing, 136; 
price of, 61, 63 ; putting an object 
under, 27 ; screen, 56. 
Microscope compound, definition, 7 ; 
drawing with, no; figures, 10, 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 ; 
micrometry with, 100 ; optic axis of, 
to ; optical parts of, 10, 61 ; polariz- 
ing, pedesis with, 89; varying magni- 
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; 
magnification of, 93 ; micrometry 
w-ith, 100 ; working distance of, 33. 
Microscopic objective, 10 ; objective low-, 
attached to camera, 198; objects, 
drawing, 109 ; ocular, 22 ; slides or 
slips, 140. 
Microscopical preparations, cabinet for, 
174 ; cataloging, 172; labeling, 171 ; 
mounting, 146-170; tube-length, 14, 
*5- 
Microscopy and photography, 183. 
Micro-spectroscope, 120-121 ; adjusting, 
125 ; experiments, 131 ; focusing, 
130 ; focusing the slit, 125 ; for labo- 
microscope, 6r ; lighting for, 
129 ; objectives to use with, 130 ; re- 
versal, apparent, of colors in, 120 ; 
slit, mechanism of, 121, 125. 
Micrum, 100. 
Mikron, 100. 
Minerals, colored, absorption spectra of, 
134- 
Minute objects, arrangement of, 180. INDEX. 
233 
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, 15 r. 
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, 1S0; permanent, 147 ; in resin- 
ous media, 152 ; in resinous 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, 90 
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 
dr\ ing, 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. 
Normal salt solution, 179. 
Nose-piece, 9, 27 ; marking objectives 
011, 65. 
Numerical aperture of condenser, 43 ; ob- 
jectives, 17, 212 ; table of, 20; reso 
lution and, 21. 
o 
Object, determination of form, 83 ; hav- 
ing plane or irregular outlines, rela- 
tive position in a microscopical prep- 
aration, 82 ; and image, size of, 5, 11, 
96 ; marking parts of, 63, 64 ; mi- 
crometer, 94; mounting, 146; put- 
ting under microscope, 27 ; shading, 
56 ; suitable for photo-micrograpliy, 
191 ; transparent with curved out- 
lines, relative position in microscopic 
preparations, 83. 
Objective, 7, jo ; 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, 11 ; dry, 11, 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, experi- 
ments, 51, 55 ; illuminating, 14 ; im- 
age, power of, 19; immersion, 12 ; 
index of refraction of medium in 
front of, 17, iS ; inverted, real image 
of, 29 ; for laboratory microscope, 61 ; 
lettering, it ; light utilized with, j8 ; 
low, attached directly to camera, 198; 
low, focusing with, 36 ; magnifica- 
tion of, 214; marking, by Krauss’ 
method, 65 ; to use 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 ; 11011-adjustable, 13 ; 11011-ad- 
justable, thickness of cover-glass for, 
table, 15 ; numbering, 11 ; numeri- 
cal aperture, 21, 22, 212 ; oil immer- 
sion, 12 ; panto-chromatic, 13 ; para- 
chromatic, 13 ; perigraphic, 196; for 
photo micrography, 189; projection, 
14, '95 1 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 i experiments, 51 ; work- 
ing distance of, 11, 33, 38. 
Oblique light, 35, 39 ; with Abbe illumi- 
nator, 46 ; experiments, 39, 46 ; with 
a mirror, 39. 
Ocular, 22 ; achromatic, 23 ; Airy’s, 23 ; 
aplanatic, 12, 23 ; binocular, 23 ; 
cloudiness, how to determine and re- 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- 
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, 
23 ; holosteric, 23 ; Huygenian, 22- 
24, 31 ; iris diaphragm, 18r ; 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 rnagni 
fication of, 99 ; micrometer, valua- 
tion of, 162 ; micrometer, varying 
valuation, 105 ; micrometer, ways of 
using, 105 ; micrometric, 23 ; micro- 
scopic, 22, 23 ; negative, 22, 23 ; num- 
bering, 26 ; orthoscopic, 23, 28 ; par- 
focal, 16, 24, 37 ; periscopic, 24, 28 ; 
for photo-micrography, 25, 189; pho- 
to-micrography with and without o., 
198, 199 ; pointer, 63 ; positive, 22 ; 
projection, 25, 189 ; projection, des- 
ignation of, 189 ; projection, use of 
in photo-micrography, 199, 202 ; put- 
ting in position and removing, 27 ; 
Ramsden’s, 24 ; screw micrometer, 
26, 103 ; searching, 24, 25 ; shallow, 
24 ; solid, 24 ; spectral, 24, 106 ; spec- 
troscopic, 24, 120; staurosc<~pic, 24; 
stereoscopic, 24 ; table of field of, 28 ; 
w'orking, 25. 
Oil, and air, appearances and distinguish- 
ing optically, 85 ; removal, 58 ; re- 
moval from sections, 161. 
Oil-globules, with central illumination, 
84 ; with oblique illumination, 85. 
Oil-immersion objectives, 12. 
Optic axis, 2, 5 ; of condenser, or illumi- 
nator, 35 ; of microscope. 6. 
Optical center, 2, 3; combination. 104, 
105 ; focus, 13 ; parts of compound 
microscope, 10, 61 ; parts of micro- 
scope, care of, and testing, 57, 60 ; 
section, 87. 
Order of procedure in mounting objects, 
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 
Ordinary ray with polarizer, 136. 
Orthochromatic plates, 191. 
Orthoscopic ocular, field with, 28. 
Outline, distinctness of, 85. 
Over-correction, 5. 
Oxy-hemoglobin, spectrum of, 122, 132. 
P 
Pantachromatic objective, 13. 
Paper, aristotype, 208 ; bibulous, filter, 
lens, or Japanese, 57, 160 ; bromide, 
208 ; for cleaning oculars and object- 
ives, 57, 160; funnel, 175 ; Usago, 
160. 
Parachromatic objective, 13. 
Paraffin, 179; chloroform, 176; chloro- 
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. 
Pedesis, 88; compared with currents, 
88 ; with polarizing microscope, 89 ; 
proof of reality of, 89. 
Penetrating power, 21, 22. 
Penetration of objective, 21. 
Perigraphic objective, 196. 
Periscopic ocular, field with, 28. 
Permanent mounting, 147 ; preparations 
of isolated cells, 155. 
Permanganate of potash, absorption spec- 
trum of, 122, 13T. 
Picric-alcohol, 179. 
Pin hole diaphragm, 45. 
Pipette, 16 r. 
Photo engraving, 216, 219 ; drawing for, 
216-219 ; lettering for, 218. 
Photographic negatives, marking, 197 ; 
objectives, 196 ; for photo microgra- 
phy, 196. 
Photography, 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. 
Photogravures from photo-micrographs, 
208. 
Photo micrograph, 183; developers for, 
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, 
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 ; 
distinguished from micro-photogra- 
pfiy, 183 ; experiments, 193 ; expos- 
ure for, 193, 194, 199, 200, 203 ; fo- 
cusing for, 195 ; focusing screen for, 
j95 J lighting, 192, 193, 198, 203 ; ob- 
jectives and oculars tor, 14, 189, 195 ; 
vertical camera with, 205 ; visual and 
actinic foci in, 189 ; with long and 
short bellows, 187 ; with and without 
ocular, 189-199; record table for, 204. 
Physiological histology, 173. 
Plane mirror, use of, 36. 
Plates, exposure of, 193, 194, 199, 200, 
203 ; gelatino-bromule, 184 ; isochro- 
matic, or orthochromatic, 189, 191, 
197. 
Pleochroistn, 138 
Pleurosigina angulatum, 39 
Point, axial, 16 ; burning, 6. 
Polariscope, 127, 136. 
Polarized light, extraordinary and ordi- 
dinary ray of, 136. 
Polarizer and analyzer, putting in posi- 
tion, 136. 
Polarizing microscope, pedesis with, 89. 
Position of objects or parts of same ob- 
ject, 82. 
Positive oculars, 11, 22. 
Power of microscope, 92 ; illuminating, 
penetrating, resolving, 2r; of object- 
ive, 19, 215 ; of ocular, 26, 215. 
Preparation of Canada balsam, Farrant’s 
solution, glycerin, glycerin jelly, etc. 
175-179- 
Preparation of clearing mixture, liquid 
gelatin and shellac cement, 175-179. 
Preparations, cataloging, 171, 172; cabi 1 
net for, 173-174 ; labeling, 171 ; per- 
manent, of isolated cells, 155 ; stor- 
ing, 171- 
Preparation of diagrams, 215 ; of ground 
glass, 29 ; vials, 159. 
Price of American and foreign micro- 
scope, 61, 63. 
Principal focus, 3, 5 ; focal distance, 3, 
30; optic axis, 2, 5. 
Prism of Abbe camera lucida, m ; Am- ; 
ici, 126; comparison, 127, 128; dis- ' 
persing, 126 ; Nicol, 136 ; and slit of 
micro-spectroscope, mutual arrange- 
ment, 125; of Wollaston’s camera 
lucida, 111. 
Prints and mechanical printing of photo 
micrographs, 208. 
Projection objective, 14, 195 ; ocular, 25, 
26, 189 ; designation of, 189 ; in pho- 
to micrography, 199, 202. 
Putting on cover-glass, 146 ; an object 
under microscope, 27 ; an objective 
and ocular in position, 26, 27. 
Pyroxylin, 176. 
Q-R 
Quadrant for camera lucida, 113. 
Quinine, Herapatk’s method of determin- 
ing minute quantities of, 181. 
Rack for drying negatives, 203. 
Ratio, ocular micrometer, 106. 
Reagents for fixing, 175 ; irrigation with, 
150 ; for mounting, 175. 
Real image, 5, io, 22, 29-31 ; magnifica- 
tion, 92. 
Record table for photo-micrography, 204. 
Reflected light, 34. 
Refraction, 50 ; images, 49, 55 ; index of, 
50 ; of medium in front of objective, 
20. 
Refractive, doubly, 137; highly, 87 ; 
si”gly, 137- 
Relative position of objects, 82. 
Resinous media, mounting objects in, 
order of procedure, by drying or 
desiccation, 152 ; by a series of dis- 
placements, 153. 
Resolution and numerical aperture, 21. 
Resolving power, 20. 
Retinal image, 6, 10. 
Revolver, 27. 
Revolving nose-piece, marking objec- 
tives on, 65. 
Rice, examination of, 90. 
s 
Sagittal sections, 170. 
Salicylic acid, crystallization, 48. 
Salt solution, normal, 179. 
Scale of drawing, 118 ; of wave lengths, 
128. 
Scales of butterflies and moths, examin- 
ation of, 90. 
Screen, color, 191 ; focusing s. for photo- 
micrography, 194, 195 ; of ground 
glass, 29 ; for microscope, 56. 
Screw, society, 62 ; micrometer, 26, 104. 
Sealing cover glass, 148. 
Searching ocular, 25. 
Secondary axis, 3. 
Section, optical, 87. 
Sections, arrangement of tissue for, 168 ; 
clearing, 167; cutting, 160, 166; 
fastening to slide, 160, 166; frontal, 
170; removing benzin, oil and 
paraffin from, 166, 16r ; sagittal, 170 ; 
serial, 168-17r 1 staining, 161, 167 ; 
transferring, 160. 
Sediment in water determination of 
character, 180. 
Selenite plate for polariscope, 138. 
Semi-apochrotnatic objective, 13. 
Serial sections, 168 ; arranging and label- 
ing, 169, 171 ; determining thickness INDEX. 
of, 170 ; stage for, 65 ; thickness of 
cover-glass for, 170. 
Shellac cement, preparation of, 179; re- 
moval from lenses, 58. 
Sight, injury or improvement in micro- 
scopic work, 59. 
.Significance of aperture, 20. 
Silk, examination of, 90. 
Simple microscope, see under micro- 
scope, 1. 
Slides, 140; cleaning, 140. 
Slips, 140. 
Slit mechanism of micro-spectroscope, 
121. 
Society screw, 62. 
Sodium, lines and spectrum, 122, 123. 
Solar spectrum or s. of sunlight, 122. 
.Soluble cotton, 176. 
Solution, alum, 175 ; Farrant’s, 177. 
Spectral, colors, 123; ocular. 120, 126. 
Spectroscope, direct vision, 120. 
Spectroscopic ocular, 24, 1 20. 
.Spectrum, 122 ; absorption, 123 ; amount 
of material necessary and its proper 
manipulation, 130 ; analysis, 135 ; 
Angstrom and Stokes’ law of, 113 ; 
banded, not given by all colored 
objects, 133 ; of blood, 131 ; of car- 
bon monoxide bemaglobin, 133 ; of 
carmine solution, 133 ; of colored 
minerals, 134; of colorless bodies, 
134; comparison, 128; complemen- 
tary, 124 ; continuous, 123; double, 
128; incandescence, 123; line, 123; 
met-hemaglobin, 133 ; monazite 
sand, 134 ; nitrate of uranium, 135 ; 
oxy-hemaglobin, 132; permanganate 
of potash, 131 ; single-handed of he- 
maglobin, 132; sodium, 122, 123; so- 
lar, 122, 123 ; two-banded of oxy-he- 
maglobin, 132. 
Spherical aberration, 4; test for, 210. 
Stage, 61 ; mechanical, 6r, 65, 66 ; mi- 
crometer, 94 ; for serial sections, 65. 
Stain, alcoholic, 161; aqueous, 161. 
Staining cells, 155 ; sections, 16r, 167. 
Stand, of microscope, 61 ; for laboratory 
microscope, 61. 
Standard distance (250 mm.) at which 
the virtual image is measured, 97. 
Starch, examination of, 90. 
Stokes and Angstrom’s law of absorption 
spectra, 123. 
Storing preparations, 171. 
Substage, 67. 
Substances for crystallography, 181. 
Sulplional with polarizer, 139. 
Sulphuric ether, 177. 
Swaying of image, 46. 
System, back, front, intermediate, of 
lenses, 10. 
T 
Table, for immersion fluid, 213 ; of mag- 
nification and valuation of ocular 
micrometer, 99 ; of tube-length and 
thickness of cover-glasses, 15 ; 
natural sines, third page of cover ; 
of weights and measures, second 
page of cover; of numerical aper- 
ture, 20; record, for photo-microg- 
raphy, 204 ; size of fields, 28 ; of val- 
uations of ocular micrometer, 99. 
Temporary mounting, 146. 
Terminology of objectives, it. 
Test of chromatic and spherical aberra- 
tion, 210. 
Tester, cover-glass, 144, 145 ; for homo- 
geneous liquids, 55. 
Testing a camera, 185; ink, 216; a mi- 
croscope and its parts, 60. 
Test-plate, Abbe’s, method of using, 210. 
Textile fibers, examination of, 90. 
Thickness of cover glass for non-adjust- 
able objectives, table, 15 ; of serial 
sections, 170. 
Tissues, arranging for sections, 168 ; fix- 
ing or hardening, 157, 164. 
Tolles-Mayall mechanical stage, 65. 
Transections, 169. 
Transfering drawings, 217 ; sections, 160. 
Transmitted light, 35. 
Transparent objects having curved out- 
lines, relative position in microscop- 
ic preparations, 83. 
Triplet, achromatic, 7. 
Tripod, 7 ; as focusing glass, T9S. 
Tube of microscope, Frontispiece. 
Tube-length, 14-16, 54; for cover-glass 
adjustment, 53, 54; importance of, 
53, 54 ; microscopical, 14, 15 ; of 
various opticians, table, 15 ; and op- 
tical combinations, 104. 
Turn-table, 148. 
u—v—w-x 
Unadjustable objectives, 13. 
Under-correction, 5. 
Unit of measures, in micrometry, 100 ; 
of wave length, 129. 
Uranium nitrate spectrum of, 135. 
Usago paper, 160. 
Valuation of ocular micrometer, 102, 
103 ; table, 99 
Variable objective, 13. 
Varying magnification of compound mi- 
croscope, 97. 
Varying ocular micrometer valuation, 
105. 
Velocity under microscope, 88. INDEX. 
237 
Vertical camera, 188, 190, 192, 193, 202, 
207 
Vials for preparation 159 
Virtual image, 5, 6, 10, 31 ; standard dis- 
tance at which measured, 97. 
Vision, double or binocular, 92, 93. 
Ward’s eye shade, 59. 
Waste bowl, 162. 
Water immersion objective, 17, 19, 55; 
light utilized, 18; numerical aper- 
ture, 20 
Water, for immersion objectives, 55 ; re- 
moval, 58 ; solid sediment in, 180. 
Wave length, designation of, 129 ; scale 
of, 128. 
Weights and measures, see 2d p. of cover. 
Wollaston’s camera lucida, 95, in. 
Work-room for plioto-micrograpliy, 187. 
Work-table, position, etc., 59. 
Working distance of microscope or ob- 
jective, 11, 33 ; determination of, 38 ; 
oculars, 25. 
Writing diamond, 173. 
Xylene, 159, 176; balsam, 175, 176. 
Xylol, German form of xylene, 159, 176. TABIyE OF NATURAE SINES. 
Minutes. 
Degrees 
AND 
Quarter Degrees up to 90°. 
i'o. 00029 
0.01745 
16°, 
0.27564 
3i°, 
0.51504 
46°, 
0.71934 
6i°, 
0 87462 
76°, 
0.97030 
2 0.00058 
I®, 15 
0.02181 
16°, 15' 
0.27983 
3i°,15 
0.51877 
46°, 15' 
0.72236 
6i°,i5 
0 87673 
76°, 15 
o.97i34 
3 0.00087 
1,30 
0 02618 
16,30 
0 28402 
3D30 
0.52250 
46,30 
0.72537 
61,30 
0.87882 
76,30 
0.97237 
4 0.00116 
i,45 
0.03054 
16,45 
0 28820 
3i,45 
0.52621 
46,45 
0.72837 
6i,45 
0.88089 
76,45 
0.97338 
5 0.00145 
2 
0 03490 
17 
0.29237 
32 
0.52992 
47 
0 73135 
62 
0.88295 
77 
0-97437 
6 0.00175 
2,15 
0.03926 
17.15 
0.29654 
32,15 
0 53361 
47,15 
o.73432 
62,15 
0.88499 
77D5 
0-97534 
7 0.00204 
2,3° 
0.04362 
17,30 
0.30071 
32,30 
0.53730 
47,30 
0.73728 
62,30 
0.88701 
77,30 
0.97630 
8 0.00233 
2,45 
0.04798 
'7,45 
0.30486 
32,45 
0.54097 
47,45 
0.74022 
62,45 
0.88902 
77,45 
0.97723 
9 0.00262 
3 
0 05234 
18 
0.30902 
33 
0.54464 
48 
0 74314 
63 
0.89101 
78 
0.97815 
10 0.00291 
3,15 
0.05669 
18,15 
°-3i3i6 
33.15 
0.54829 
48,15 
0.74606 
63,15 
0.89298 
78,15 
0.97905 
11 0.00320 
3,30 
0 06105 
18.30 
0.31730 
33,30 
0-55'94 
48,30 
0.74896 
63,30 
0.89493 
78,30 
0.97992 
12 0.00349 
3-45 
0 06540 
18,45 
0.32144 
33,45 
0.55557 
48,45 
0.75'84 
63,45 
0.89687 
78,45 
0.98079 
13 0.00378 
4 
0 06976 
'9 
0 32557 
34 
o.559i9 
49 
o.7547i 
64 
0.89879 
79 
0.98163 
14 0.00407 
4,i5 
0.07411 
19,15 
0.32969 
34,15 
0.56280 
49. >5 
0.75756 
64.15 
0.90070 
79-'5 
0.98245 
15 0.00436 
4,30 
0.07846 
J9,3o 
0 3338i 
34.30 
0.56641 
49,30 
0.76041 
64,30 
0.90259 
79,30 
0.98325 
16 0.00465 
4,45 
0.08281 
19,45 
0 33792 
34.45 
0.57000 
49.45 
0.76323 
64.45 
0.90446 
79,45 
0.98404 
17 0.00495 
5 
0.08716 
20 
0.54202 
35 
0.57358 
50 
0.76604 
65 
0.90631 
80 
0.98481 
18 0.0)524 
5,15 
0.09150 
20,15 
0.34612 
35 15 
0.57715 
50,15 
0.76884 
65.15 
0.90814 
80.15 
0.98556 
190.00553 
5.30 
0.09585 
20,30 
0.35021 
35 30 
0 58070 
50,30 
0.77162 
65.30 
0 90996 
80.30 
0.98629 
20 0.00582 
5.45 
0.10019 
20,45 
0.35429 
35,45 
0.58425 
50,45 
0.77439 
65 45 
0.91176 
80,45 
0.98700 
21 0.00611 
6 
0 10451 
21 
0.35837 
36 
0.58779 
5i 
o.777i5 
66 
0.91.355 
81 
0.98769 
22 0.00640 
6.15 
0 10887 
21,15 
0.36244 
36,15 
0.591,31 
5I.I5 
0.77988 
66 15 
o.9i53i 
81,15 
0.98836 
23 0.00669 
6,30 
0.11320 
21,30 
0.36650 
36,30 
0.59482 
51.30 
0.78261 
66,30 
0.91706 
81,30 
0.98902 
24 0.00698 
6,45 
0.11754 
21.45 
0.37056 
36,45 
0.59812 
51.45 
0.78532 
66.45 
0.9187Q 
8i,45 
0.98965 
25 0.00727 
7 
0.12187 
22 
0.37461 
37 
o.6or82 
52 
0.78801 
67 
0.92050 
82 
0.99027 
26 0.00756 
7.15 
0.12620 
22,15 
0.37865 
37,15 
0.60529 
52,15 
0.79069 
67,15 
0.92220 
82,15 
0.99087 
27 0.00785 
7,30 
0 13053 
22.30 
0.38268 
37.30 
0.60876 
52,30 
0 79335 
67,-0 
0.92388 
82,30 
0.99144 
28 0.00814 
7,45 
0.13485 
22,45 
0.38671 
[37-45 
0.61222 
52,45 
0.79600 
67,45 
0.92554 
82,45 
0.99200 
29 0.00844 
8 
0.13917 
23 
0 39073 
38 
0.61566 
53 
0.79864 
68 
0.92718 
83 
0.99255 
30 0.00873 
8,15 
0 14349 
23,15 
0.39474 
38.15 
0.61909 
53,15 
0.80125 
68,15 
0.92881 
83,15 
0.99307 
31 0.00902 
8.30 
0.14781 
23,30 
0.39875 
38 30 
0 62251 
53.30 
0.80386 
68,30 
0.93042 
83,30 
0-99357 
32 0.00931 
8,45 
0.15212 
23,45 
0.40275 
38,45 
0.62592 
53.45 
0.80644 
68,45 
0.93201 
83,45 
0.99406 
33 0.00960 
9 
0.15643 
24 
0.40674 
39 
0.62932 
54 
0.80902 
69 
0.93358 
84 
0-99452 
34 0.00989 
9, r5 
0.16074 
24,'5 
0.41072 
39,15 
0.63271 
54,15 
0.81157 
69.15 
o.935r4 
84,15 
0.99497 
35 0.01018 
9-30 
0.16505 
24.30 
0.41469 
39 30 
0.63608 
54,30 
0.81412 
69.30 
0.93667 
84,30 
o- 99540 
36 0 01047 
9 45 
0.16935 
24,45 
0.41866 
39 45 
0.63944 
54.45 
0.81664 
69,45 
0.93819 
84.45 
0.99580 
37 0.01076 
io 
0.17365 
25 
0.42262 
40 
0 64279 
55 
0.81915 
70 
0 93969 
85 
o- 99619 
38 0.01105 
*0,15 
0.17794 
25,15 
0.42657 
4o,i5 
0.64612 
55.15 
0.82165 
70.15 
0.94118 
85,15 
0 99657 
390.01134 
io 30 
0.18224 
25,30 
0.4305' 
40.30 
0.64945 
55 30 
0.82413 
70,30 
0.94264 
85.30 
0 99692 
40 0.01164 
Io,45 
0.18652 
2.5,45 
0.43445 
40,45 
0.65276 
55.45 
0.82659 
70,45 
0.94409 
85,45 
0.99725 
41 0.01193 
11 
0 19081 
26 
0.43837 
4i 
0.65606 56 
0.82904 
7i 
0 94552 
86 
0.99756 
42 0.01222 
11,15 
0 19509 
26,15 
0.44229 
4i,i5 
0.65935 
56,'5 
0 83147 
7I.I5 
0.94693 
86,15 
0.99786 
43 0.01251 
n.30 
0.19937 
26,30 
0 44620 
41.30 
0 66262 
56,30 
0.83389 
71.30 
0.94832 
86,3° 
0.99813 
44 0.01280 
'i,45 
0.20364 
26,45 
0 45010 
41.45 
U. 66588 
56,45 
0.8^629 
71.45 
0.94970 
86 45 
0.99839 
45 0.01309 
12 
0.20791 
27 
0-45399 
42 
0.66913 
57 
0.83867 
72 
0.95106 
87 
0 99863 
46 0.01338 
12,15 
0.21218 
27D5 
0 45787 
42,15 
0.67237 
57,15 
0.84104 
72,15 
0.95240 
87,15 
0.99885 
47 0.01367 
12,30 
0.21644 
27.30 
0.46175 
42 30 
0.67559 
57,3° 
0.84339 
72.30 
0.95372 
87,30 
0.99905 
480.01396 
12,45 
0.22070 
27.45 
0.46561 
42,45 
0 678S0 
57,45 
0.84573 
72,45 
0.95502 
87.45 
0.99923 
490.01425 
13 
0.22495 
28 
0.46947 
43 
0.68200 
58 
0.84805 
73 
Q. 95630 
88 
0 99939 
50 0.01454 
13,'5 
0.22920 
28,15 
0 47332 
43.'5 
0.68518 
58,15 
0.85035 
73,15 
0-95757 
88,15 
0-99953 
5r 0.01483 
13,30 
0.23345 
28,30 
0 47716 
43,30 
0.68835 
58,30 
0.85264 
73.30 
O.95882 
88,30 
0.99966 
520.01513 
13,45 
0.23769 
28,45 
0.48099 
43-45 
0.69151 
58.45 
0.85491 
73,45 
0.96005 
88,45 
0 99976 
530.01542 
14 
0.24192 
29 
0.48481 
44 
0.69466 
59 
0.85717 
74 
O 96126 
89 
0 99985 
54 0.01571 
I4J5 
0.24615 
29 '5 
0.48862 
44.15 
0 69779 
59.15 
0.85941 
74D5 
O 96246 
89D5 
0.99991 
55 0.01600 
14,30 
0.25038 
29,30 
0.49242 
44 30 
0.70091 
59,30 
0.86163 
74,30 
O.96363 
89,30 
0.99996 
560.01629 
14,45 
0.25460 
29.45 
0 49622 
44,45 
0 70401 
59,45 
0.86584 
74,45 
0.96479 
89.45 
0-99999 
57 0.01658 
15 
0.25882 
30 
0.50000 
45 
0.707II 
60 
0.86603 
75 
0.96593 
90 
1.00000 
580.01687 
I5,i5 
0.26303 
30,15 
0.50377 
45.15 
0.71019 
60,15 
0.86820 
75,1.5 
O.96705 
590.01716 
15,30 
0.26724 
30,30 
050754 
45,30 
0.71.325 
60,30 
0.87036 
75.30 
O.96815 
60 0.01745 
15,45 
0.27144 30,45 
0 51129 
45.45 
0.71630 
60 45 
0.87250 
75,45 
0.96923 
Compiled from Prof. G. IV. Jones' Logarithmic Tables.