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OSMANIA UNIVERSITY LIBRARY
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Author
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This book should -oe returned <m or before the date last marked below,
THE STUDENT'S MANUAL OF
MICROSCOPIC TECHNIQUE
THE
STUDENT'S MANUAL
OF
MICROSCOPIC TECHNIQUE
WITH
INSTRUCTIONS
FOR
PHOTOMICROGRAPHY
BY
J. CARROLL TOBIAS
LONDON
CHAPMAN & HALL, LTD.
IT HKNRIKTTA STKKKT, W.C.i>
i () 3 6
COPYRIGHT, 1936, UY
AMERICAN PHOTOGRAPHIC PUBLISHING Co.
Made and printed in the United States of America
by the Plimpton Press, Norwood, Massachusetts
Preface
In compiling this volume on Microscopic Technique
the author has directed his efforts toward producing a
book that will fill a real need in its particular field. There
are a number of excellent books on the market dealing
with the revelations of the microscope, but there are very
few that tell the beginning student what to do with ma-
terial to bring it into proper condition for microscopical
examination. Practically no material can be studied to
advantage in its natural state, but it must be prepared
by a series of chemical or physical treatments to bring it
into condition for study.
The instructions given here are adequate for the use
even of the student who is just starting out upon his
excursions into the world of microscopical things, while
comprehensive enough for rather advanced students.
Every effort has been made to include the most modern
methods of preparing a large variety of materials, at the
same time phrasing the matter in such a way that it can
be easily understood. The bibliography mentions a num-
ber of advanced textbooks to which the student may re-
fer for more detailed information than the scope of this
work permits. The main thought back of this book is to
include a large variety of materials and objects from
which the student may learn the correct methods of
preparation before going into fields that are entirely new
to him.
The working methods described here have been tested
in the author's laboratory, using the equipment described
The writer can well remember his own flounderings
in this field before he learned correct procedure. When
he undertook his first examinations with the micro-
scope he was living in a small town with a very limited
library. The local public school principal, who had
given him his microscope, took an active interest, but
was handicapped because he did not know very much
about the subject. What he did know was freely given
and gratefully received, but had there been such a book
as this available then it would have been of inestimable
value.
Microscopy is a most absorbing hobby, with so many
ramifications that it never becomes boresome. Unless the
student is possessed of more than the usual amount of
perseverance, however, he is very apt to become dis-
couraged in a short time, due to failure to perceive in his
subjects the things the books say are there. Most of the
material published today concerning microscopy is re-
liable, and the mounts may be depended upon to reveal
the described features if they are properly prepared.
If this book helps the beginning student to improve
his technique to the point where he can prepare work-
manlike slides; if it gives him inspiration to continue his
studies and if it gives him greater enjoyment of his
hobby through increased knowledge of how to do the
right thing in the right way, the purpose of the book
will be served.
Contents
PREFACE v
CONTENTS vii
ILLUSTRATIONS xi
INTRODUCTION xv
CHAPTER I THE MICROSCOPE Optics of the Microscope
Care of the Microscope Focusing the Microscope
Setting up the Microscope i
CHAPTER II MICROSCOPIC OBJECTS FROM WATER Sources
of Material Collecting Net Classifying Material
Desmids Diatoms Rhizopods Amoeba Difflugia
Infusoria Carchesium Vorticellac Stentor
Paramecium Volvox Globator Dipping Tube
Live Cage Depression Slide Killing Agents Pre-
paring Slides Anaesthetizing Aquatic Organisms
Temporary Staining 14
CHAPTER III KILLING, FIXING AND PRESERVING Fixing
Agents Methyl Alcohol Acetic Acid Carney's
Fluid Carnoy and Le Brun's Fluid Formalin-
Acetic-Alcohol Karpenchenko's Fluid Mercuric
Chloride Gilson's Fluid Zcnker's Fluid Worces-
ter's Fluid Picric Acid Bouin's Fluid Alcoholic
Bouin's Fluid Kleinenberg's Fluid 33
CHAPTER TV DISSOCIATION Dissecting Needles Teas-
ing Staining Clearing Maceration Macerating
Agents Ranvier's One-Third Alcohol Solution
Gage's Formalin Solution Vegetable Tissues
Mayer's Albumin Fixative Corrosion 43
CHAPTER V SECTION CUTTING Microtome Sectioning
Dehydration Clearing Paraffin Infiltration Im-
beddingSection Cutting Freehand Knife for Sec-
tion Cutting Preparing the Slides Stretching
Coplin Jars Removal of Paraffin Coating with Cel-
loidin Transferring to Stain Clearing Mounting
Section Cutting by Freezing Infiltrating Mass
viii CONTENTS
Dry Ite Ethyl Chloride Handling Loose Sections
Wright's Method 52
CHAPTER VI STAINING Nuclear Stains Plasma Stains
Haematoxylin Carmine Safranin Eosin
Alum-Haematoxylin Iron-Haematoxylin Progres-
sive Staining Regressive Staining Counter Staining
Dehydration Preparing Standard Alum-Haema-
toxylin Grenadier's Alcoholic Borax-Carmine Ran-
vier's Picro-Carmine Eosin Y Indulin Malachite
Green Aniline Blue 79
CHAPTER VII PREPARING AND MOUNTING HARD OBJECTS
Grinding Sections of Bone Decalcified Bone De-
calcifying Agents Sections of Teeth Cutting with
the Emery Saw Grinding Sections of Coal Cutting
Sections of Coal Grinding Friable Objects ... 93
CHAPTER VIII PREPARATION OF ANIMAL MATERIAL Ar-
thropoda Hexapoda Hemiptera Arachnida
Vermes Diptcra Lepidoptera Hymenoptera Or-
thoptera Neuroptera Coleoptera Stages of Insect
Development An Interesting Experiment Reason
for Graded Series of Alcohols Imbedding in Glycerin
Jelly Preparing Insect Larvae for Sectioning Dis-
section Dissecting Microscopes Tools for Dissection
Physiological Salt Solution Preparation of Insects
Mounting Insect Specimens 103
CHAPTER IX PREPARING VEGETABLE SPECIMENS Stem
Groups Bundles Rhizomes Coniferous Leaves
Pollens Germinating Seeds 127
CHAPTER X THE POLARIZING MICROSCOPE Nicol Prisms
Point of Extinction Polarized Light Polarizer
Objects and Methods of Preparation Crystals of Me-
tallic Silver Starches Raphides Silicious Skele-
tons Leaf Cuticles Leaf Scales and Leaf Hairs
Textile Fibers Radula of Snails Hairs Horny
Tissues 139
CHAPTER XI ACCESSORIES Dissecting Microscope Dis-
secting Needles Dissecting Knives Forceps Shears
Section Lifter Lamps Slides Spirit Lamp Bal-
sam Bottle Microtome Paraffin Oven Brown Uni-
versity Oven Erlenmeyer Flask Turntable Po-
larizer How to Make Nicol Prisms Sawing the
Crystal Polishing the Prisms 152
CONTENTS ix
CHAPTER XII PHOTOMICROGRAPHY Good Slide Prepara-
tion Essential Record Sheet for Exposures The
Camera Adjusting the Apparatus How Critical Il-
lumination is Obtained The Absorption Band
Light Propagation in the Form of Waves Wave-length
Measured in Angstrom Units Residual Colors
Chart of Residuals How to Get Background Contrast
Object Contrast Filters Photographing by Ob-
lique Light Calculating the Exposure 'Fable of
Numerical Aperture Factors Table of Magnification
Factors How to Expose a Test Negative Using a
Brownie Box Camera for Photomicrography Photo-
micrographs without the Eyepiece Photographing
Opaque Objects Lighting Opaque Objects Stereo-
scopic Photomicrography 172
BIBLIOGRAPHY 206
INDEX 207
Illustrations
FIG.
1. DIAGRAM OF THE LIGHT PATH THROUGH A MICROSCOPE 2
2. LEITZ DISSECTING MICROSCOPE 3
3. BAUSCH & LOMB AMATEUR MICROSCOPE. X75 TO X3oo 3
4. WOLLENSAK MICROSCOPE. X235 3
5. GOERZ LOMARA MICROSCOPE 3
6. LEITZ SPX MICROSCOPE 5
7. BAUSCH 8c LOMB AMATEUR MICROSCOPE, MAGNIFYING
POWER X/150 5
8. BAUSCH & LOMB STANDARD MICROSCOPE STAND FOR STU-
DENT USE 5
9. ZEISS M44() MICROSCOPE 5
10. ZEISS M436 MICROSCOPE 7
1 1 . LEITZ O MICROSCOPE 7
12. ZEISS M445 MICROSCOPE 7
13. SPENCER 30 MICROSCOPE 7
14. ZEISS M3i7~4 MICROSCOPE 9
15. LEITZ ABM MICROSCOPE 9
16. SPENCER 5 MICROSCOPE 9
17. BAUSCH & LOMB LABORATORY RESEARCH MODEL MICRO-
SCOPE 9
18. LEITZ BI-D MICROSCOPE 11
19. GROUP OF DESMIDS FROM AN AQUARIUM CULTURE. Xfioo 17
20. DESMIDS. Two SPECIES OF THE GENUS MICRASTERIAS.
Xi8r, 18
21. A GROUP OF DIATOMS. X35 18
22. DIATOMS. X78 18
23. GROUP OF ARRANGED DIATOMS. X6o 19
24. LIVING DIATOMS FROM A WATER TROUGH. Xi8o . . 19
25. A DIATOM, TRICERATRUM. Xa65 19
26. A GROUP OF RHIZOPODS. APP. X3oo 21
27. THREE FAMILIAR INFUSORIA FOUND IN POND WATER . . 22
28. CARCHESIUM. A FIXED FORM OF INFUSORIA .... 24
29. DIAGRAMMATIC VIEWS OF LIVE BOXES 28
xii ILL USTRA TIONS
FIG.
30. BAUSCH & LOME MINOT ROTARY AUTOMATIC MICROTOME 53
31. DIAGRAM OF PAPER IMBEDDING TRAY 62
32. CUTTING SECTIONS FREEHAND WITH A RAZOR .... 65
33. CUTTING SECTIONS BY HAND IN A WELL MICROTOME . . 67
34. DIAGRAM SHOWING ARRANGEMENT OF REAGENT JARS . . 71
35. FEMALE CYCLOPS. X5o 82
36. MITOSIS IN ONION ROOT TIP. Xgoo 84
37. CROSS SECTION THROUGH EYE OF A RAT. X50 ... 86
38. EMBRYONIC SEEDS IN OVARY OF HYACINTH. X25 . . 90
39. SPIRAL VESSELS FROM STEM OF RHUBARB 91
40. FORMING BONE IN THE FOOT OF A RAT. APP. Xioo . . 96
41. TURNTABLE FOR RINGING SLIDES AND TURNING CELLS . 98
42. SECTION THROUGH COAL. X5o 99
43. FRESH WATER CRUSTACEAN. X7o 104
44. FOOT OF A BEETLE. Xi8 105
45. WING OF A HOUSE FLY. Xig 105
46. Sy km mis surinainensis. X35 107
47. BAUSCH & LOMB GREENOUGH TYPE BINOCULAR MICRO-
SCOPE 115
48. COMPLETED SLIDE 126
49. CROSS SECTION OF TANSY STEM. Xao 128
50. CROSS SECTION OF OAK LEAF AT MIDRIB. X32 . . . 128
51. CRYSTALS OF POTASSIUM CHLORATE IN ORDINARY LIGHT 140
52. THE SAME CRYSTALS PHOTOGRAPHED IN POLARIZED LIGHT 141
53. BAUSCH & LOMB INEXPENSIVE DISSECTING MICROSCOPE.
*7-5 153
54. DISSECTING INSTRUMENTS 155
55. SMALL LAMP FOR VISUAL EXAMINATION OF MICROSCOPIC
SUBJECTS 157
56. HIGH POWER LAMP FOR PHOTOMICROGRAPHY . . . . 157
57. WELL MICROTOME FOR CUTTING SECTIONS . . . . 161
58. PARAFFIN OVEN FOR PARAFFIN IMBEDDING . . . . 163
59. TEST TUBE AND ERLENMEYER FLASK USED FOR IMBEDDING 165
60. NICOL PRISMS, SHOWING PLANE OF SECTION . . . . 168
61. RECORD SHEET FOR PHOTOMICROGRAPH EXPOSURES . . 173
62. HORIZONTAL SET-UP FOR PHOTOMICROGRAPHY . . . 174
63. VERTICAL SET-UP OF CAMERA AND MICROSCOPE . . . 175
64. DIAGRAM OF ABSORPTION BAND 182
ILLUSTRA TIONS xiii
FIG.
65. DIAGRAM OF RESIDUAL COLORS 183
66. FRESH WATER CRUSTACEAN PHOTOGRAPHED FOR CON-
TRAST 184
67. THE SAME CRUSTACEAN PHOTOGRAPHED FOR DETAIL . 185
68. BLUE COBALT MERCURIC THIOCVANATE CRYSTALS. Xi25 186
69. THE SAME CRYSTALS PHOTOGRAPHED ON " M " PLATE
WITH C FILTER 187
70. WHALEBONE SECTION 188
71. SECTION OF SKIN OF MAN. Xig 190
72. Sylvanus surinamensis PHOTOGRAPHED WITH DIAGONAL
LIGHTING 191
73. LEAD IODIDE PRECIPITATE. Xi2$ 192
74. FLEA FROM CAT 197
75. PHOTOMICROGRAPH OF SMOOTH PAPER SURFACE. Xioo . 198
76. EGGS OF Podisus spinosus. X75 199
77. EGGS OF Podisus spinosus. Xgo 200
78. HEAD OF SPIDER 201
79. TREE-HOPPER, Tclarnona Sf) 203
Introduction
The possessor of a microscope is wealthy beyond the
wildest dreams of avarice, for under the lens of his magi-
cal instrument he may visit kingdoms beyond the sight
of less fortunate individuals. He may peer into the in-
terior of insects and examine the various processes of nu-
trition, circulation and respiration. He may place upon
the microscope slide a drop of pond water and gaze in
ama/ement at the teeming life revealed there. He may
explore the inside of a twig or stem and marvel at the pre-
cise regularity with which nature has constructed it. He
may delve into the past by examining fossil remains that
give clues to the life that existed on this planet before the
age of man. In short, no object of nature can be ex-
amined under the microscope without revealing a sight
never dreamed of, beauties unsurpassed by anything to
which the unaided eye is accustomed, secrets of nature
impossible to comprehend by any other means.
The enjoyment of these beauties need not be expen-
sive. There are on the market a number of small micro-
scopes, really good instruments, that cost comparatively
little money. True, these microscopes are not extremely
powerful, nor need they be, for most of the work to be
dealt with in this book will require comparatively low
magnifications. Most beginners in microscopy make the
mistake of using powers that are too high and, as a result,
they comprehend very little of what they see. Magnifica-
tions of 50, 75, 100, 200, 350 and 500 times will an-
swer for most purposes. More to be sought in a micro-
xvi INTRODUCTION
scope than magnifying power is resolving power, which is
the ability of a lens to separate fine details and make them
visible. This is a variable quality which depends upon
several factors, as will be explained later. Of course, if
the student has the money to spend he should by all means
buy a professional microscope, for the various attach-
ments and accessories which may be secured for such an
instrument enormously increase its field of usefulness.
But he should not let the lack of such a microscope deter
him from the enjoyment to be found in the use of a less
pretentious instrument. The author's first work was
done with an old school microscope which was limited
to 1 50 diameters, but the optical system was good and it
provided endless hours of pleasant recreation.
In addition to the microscope, other apparatus and
equipment will be required. This is to be used in the
collection, preparation and mounting of subjects for ex-
amination, as well as for viewing and photographing
them. Most accessories can and should be made at home
by the student, not only to save money, but also because
the full enjoyment of any hobby comes only to him who
does as much of the incidental work as possible. Those
of us who really get pleasure out of our microscopes are
those who take up the work as a hobby. It is not a busi-
ness, hence we should not spend money on it as though
it were. Make as much of your equipment as your ability
and facilities permit. A hobby is an interest to which we
can turn for relaxation. It is the expression of a natural
desire to be doing something different from our regular
work. For genuine relaxation it must be something in-
teresting, and, to make it enduring, it must be active,
active as distinguished from static, such as merely collect-
ing things.
INTRODUCTION xvii
What could be a more active hobby than microscopy!
Embracing every phase of nature, material for examina-
tion is always present everywhere. New accessories may
be added from lime to time. If one line of study loses in
interest, there are others waiting to be investigated, each
revealing new aspects, each worthy of attention, arid all
marvelous beyond words.
To tell the student in the microscopic field where to
go for materials, what to do with them when he has them,
and what the magnifying power of the microscope reveals
to him, is the purpose of this book. It does not pretend to
be a complete text book of microscopy, but merely a guide
to set the wandering footsteps of the student on the right
path, so that he may proceed with greater intelligence
toward the selection of those branches of a broad subject
that seem to him most interesting. The principal rea-
son for the book is to describe the methods of photomi-
crography. Before a picture of a specimen may be made,
however, the slide must be prepared, so it is necessary to
describe the methods by which material is prepared for
the microscope. Also, in order to make the student fa-
miliar with the technique of photomicroscopy, a chapter
has been included which deals with the optics, care and
adjustment of the microscope, as well as a short discussion
of light, the theory of which, it is hoped, will clarify cer-
tain mistaken ideas about magnification.
CHAPTER I
The Microscope
OPTICS OF THE MICROSCOPE The microscope is an
optical instrument designed for the purpose of enlarging
details to such an extent that they may be clearly discerned
by the eye. It may be simple or compound, depending
upon whether it contains one or more lenses. A simple
microscope is usually found in the form of one double
convex lens and is commonly called a magnifying glass.
The compound microscope differs from this in that it has
several lenses, each magnifying the image of the other
until great enlargement is secured. It consists essentially
of one lens, the objective, close to the subject, which
forms an image which is in turn magnified by another
lens, the ocular or eyepiece. Reference to Fig. i will help
to make this clear.
In this diagram the objective O forms an image of the
specimen on the slide S, at the plane PI. This image, if
allowed to reach the eye, would be inverted and a real
image. Before it can be formed, however, the light rays
encounter the lower lens of the eyepiece B which, in com-
bination with the upper lens C, produces a magnified
virtual image at the plane P 2 , corresponding to the real
image P^ Thus the magnifying power is the product of
the separate magnifying powers of the two lens systems, or
that of the objective multiplied by that of the eyepiece.
Thus it would appear that any magnification desired,
however great, could be secured simply by increasing the
magnifying power of the two lens systems. It would seem
at first sight that there is no limit to the amount of detail
2 MICROSCOPIC TECHNIQUE
we could perceive, for could we not use another micro-
scope to magnify the image produced by the first and thus
secure unlimited magnification? Magnification yes, but
detail no, for, unfortunately, the amount of detail which
may be discerned is limited by optical laws. Mere mag-
nification of the subject does not enable us to see more
detail. This quality, known as resolving power, is de-
termined by the construction of the objective.
Fig. i. Diagram of the light path through a microscope.
An eyepiece with a magnification of ten times, that is,
one which magnifies the objective image ten times, will
give about all the detail that the objective is capable of
resolving. This limit of resolving power is fixed by the
nature of light itself. Light is not a continuous flow of
substance. It consists of definite waves of definite wave-
length. This gives to light, in a manner of speaking, a
certain structure which makes it impossible to see things
that are smaller than the structure of light itself. Re-
solving power may be defined as the distance by which
two small elements in an object must be separated in
Fig. 2. Lcitz Dissecting Mi-
croscope .
Fig. 3. Bausch & Lomb Ama-
teur Microscope. Xy5 to
4. Wollensak Micro-
scope, X235
Fig. 5. Goerz Lomara Micro-
scope.
4 MICROSCOPIC TECHNIQUE
order to be visible, and is a function of what is known tfs
the numerical aperture of the lens.
In microscopical writings the term numerical aperture
is abbreviated to N.A. 'The higher the N.A. the greater
the resolving power and the finer the detail which is re-
vealed. Numerical aperture is equal to the effective
aperture of the back lens of the objective divided by twice
the equivalent focus. Thus if a very narrow pencil of
light is used for illumination, the finest detail which may
be revealed by a microscope of sufficient magnification is
equal to ^L in which wl is the wavelength of the light
used for illumination. As the pencil of light becomes
wider, the resolving power is increased until a maximum
is reached when the whole aperture is filled with light.
In this case the resolving power is twice as great, as repre-
sented by the formula 2 -^ ] A - This same limit is reached
when a narrow pencil of light enters the lens as obliquely
as possible. The wavelength of light may be taken as one
half of i / 1,000 of a millimeter, or about i 750,000 of an
inch. If then we assume a lens in which the effective
aperture of the back lens is equal to the equivalent focus,
the lens will have an N.A. of 0.5. This lens can separate
lines which are 1/25,000 of an inch apart if the back lens
is filled with light, but if a narrow pencil is used the lines
must be only 1/12,500 of an inch apart to be resolved by
this objective. So we see that extremely high resolving
power requires objectives of wide numerical aperture, in
the order of i.o N.A., which will resolve 50,000 lines to
the inch. Use of such objectives calls for special equip-
ment and manipulation.
In using a microscope we look through a sheet of glass,
the cover glass over the specimen. While this is trans-
parent it may act also as a reflector if the light passing
Fig. 6. Leitz SPX Microscope. Fig ? B . ulsch & Lomb Ama .
teur Microscope. Magnifying
Power X45O
Fig. 8. Bausch 8c Lomb
Standard Microscope stand
for high school and college Fig. 9. Zeiss M446 Micro-
student use. scope.
6 MICROSCOPIC TECHNIQUE
through it strikes it at an angle greater than a certain
fixed angle. For the same reason, and more readily be-
cause of the black background provided by the inside of
the microscope tube, the lens of the objective may become
a reflector. In order to control this angle and provide a
definite path for the light to travel we equip the micro-
scope with a condenser (the substage condenser) placed
in the path of the light. Thus we may control the light
and regulate the amount so that it just fills the rear ele-
ment of the objective when it is examined with the eye-
piece removed from the tube. We also place a drop of
oil on the cover glass and immerse the objective in this.
The oil, having the same refractive index as the glass,
presents a homogeneous material through which the light
may travel in its own medium, thereby preventing the dis-
persion which would otherwise take place. Such lenses
are known as oil-immersion lenses and by their use nu-
merical apertures as high as 1.4 may be attained, which,
under the most favorable conditions, permit resolutions of
the order of 100,000 lines to the inch. These objectives
are available only on the most expensive professional
microscopes, so the beginner need not search for them as
accessory equipment to amateur instruments. This di-
gression into numerical aperture is included solely for
the fortunate possessors of more pretentious micro-
scopes in order to clear up the terms used in catalog de-
scriptions of such equipment.
CARE OF THE MICROSCOPE A microscope should be
treated with the same care as would be given to any other
optical instrument. While not in use it must be kept
clean, either by keeping it in the original box or under a
protecting cover of some sort. If the box is not used the
microscope may be protected from dust by placing it
Fig. 11. Leitz O Microscope.
Fig. 10. Zeiss IVL^G Micro-
scope.
Fig. 12. Zciss M445 Micro- Fig. 13. Spencer 30 Micro-
scope, scope.
8 MICROSCOPIC TECHNIQUE
under a bell jar or a large battery jar. Any chemical sup-
ply house can furnish a jar suitable for the purpose, or
possibly one may be found around the house which will
serve. In any event the microscope should not be allowed
to stand unprotected in the dusty atmosphere of the home
or workshop.
If the microscope has been neglected and has accumu-
lated dust, use a soft camels-hair brush and a piece of
chamois to clean it. Be extremely careful not to scratch
the lenses. If they are merely dusty, wipe the dust away
with the brush. If there are drops of water or chemical
on the lenses, breathe on them lightly to deposit a thin
film of condensed moisture and then remove this with
Japanese tissue. This is a soft, light, vegetable tissue
made in Japan which was first suggested for use by micro-
scopists by Prof. S. H. Gage of Cornell University. It is
universally used today by microscopists to clean the ob-
jectives after immersion in oil or water. Photographers
also use this tissue to clean photographic lenses and it
may be purchased from any photo-supply house. If the
objective lens should become soiled with a substance such
as Canada balsam, it should be cleaned immediately as
well as possible by simply wiping with a piece of dry tis-
sue to remove the greater part of the balsam, then finished
with a well-washed linen rag moistened with alcohol.
Note that the rag is to be only moist, not wet, and the
glass must be wiped at once with a dry part of the rag,
followed by the breath and Japanese tissue. If the eye-
piece of your microscope consists of two lenses do not take
them apart. This may be possible, but serves no good
purpose and affords a splendid opportunity for dust to
get into the tube and make all sorts of trouble. Keep
the lenses clean but do not overdo the cleaning. Optical
Fig. 14. Zeiss 1^317-4 Micro-
scope.
Leit/ ABM Micro-
scope.
16.
Spencer 5
scope.
Micro-
Fig. 17. Bausch & Lomb Lab-
oratory Research Model Mi-
croscope. Triple objective,
substage condenser and extra
large stage.
10 MICROSCOPIC TECHNIQUE
glass is soft and easily scratched and a scratched lens will
never reveal the full beauty of the preparations to be
examined.
While it may seem not worth mentioning, it is sur-
prising to learn that a great many people do not know
how to focus a microscope. The author has seen many
students look through the tube and focus down on the
subject. This is reprehensible. Never do it, for sooner
or later you are going to run the objective down too far
and break the cover glass and possibly ruin the slide, or
you may break the objective, which would be even more
regrettable. The proper way to bring an object into
focus is to focus up on it. To do this, look at the objec-
tive from the outside, at the same time lowering the tube
toward the specimen until you are sure it is closer than
is necessary to secure sharp focus. Then look into the
tube and draw the lens away from the subject until it is
in focus. A good rule to remember is this: always move
the tube down when you are looking at it and up when
you are looking through it. If the microscope is equipped
with both coarse and fine adjustments the tube may be
lowered with the coarse adjustment knob, then roughly
focused by raising the tube and finally getting critical
focus with the fine adjustment.
Never try to examine an object that is not in perfect
focus, for this causes eye strain. Be sure the focus is the
best possible by making slight adjustments with the fine
adjustment knob. Run the tube down a little distance
and if the image appears to be improved the first focus
was not correct. If there is no apparent change in appear-
ance the focus was probably just about right and so should
be restored.
Another error of many students is the use of too much
THE MICROSCOPE n
light. More detail can be seen when the field is moder-
ately lighted than when the eye is blinded by a dazzling
glare. Just as the strong sun has a blinding effect when we
look into it, so the light projected through the micro-
scope affects the eye. Too much light is objectionable
because it may lead to serious eye injury. On the other
hand, do not go to the other extreme and try to work
with not enough light, for this is just as bad, since we are
forcing the eyes to work in semi-darkness. Keep the field
sufficiently lighted to be pleasant to the sight and no in-
jurious results will be felt. If your microscope is fitted
with a diaphragm, stop this down until there is just
enough light to see the object clearly. If the object is
thick or opaque, more light will be needed, in which case
open the diaphragm. If this accessory is not available, as
Fig. 18. Lei tz BI-D Microscope.
in the smaller microscopes, reduce the intensity of the
light by tilting the mirror, or by moving the illuminator
away from the microscope, by placing sheets of tissue
I* MICROSCOPIC TECHNIQUE
paper in the light path or by reducing the size of bulb
used.
SETTING UP THE MICROSCOPE When an object is to
be examined, the microscope is placed near a window, or,
if at night, near a lamp of some sort from which light may
be picked up by the mirror and reflected up through the
object. The tube is inclined at a convenient angle and
the mirror is manipulated until the light is reflected upon
the slide where it may be seen. The eye is then applied
to the eyepiece and the illumination examined. It will
probably be imperfect or unevenly distributed, in which
case the mirror must be adjusted until the entire field is
illuminated. Now remove the eyepiece from the tube (if
this is so arranged that it may be done without unscrew-
ing it) and examine the little spot of light which appears
upon the rear lens of the objective. If this spot is not in
the exact center of the lens, shift the mirror until it is,
for this is the position for the best illumination. The
secret of securing this condition lies in picking up the
light in the center of the mirror. If artificial light is
used, move the illuminator until the image of the bulb
or other ilium inant is in the exact center of the mirror,
then direct the light upwards through the stage opening
into the microscope. Now lower the tube, focus upward
until sharp focus is secured and the examination may pro-
ceed.
Many objects may be examined in a dry state simply by
placing them on a slide for viewing. Others, such as the
minute organisms found in pond water, may be examined
only in their natural state, for as yet no method has been
discovered or invented for mounting many of them.
Other subjects require preparation for mounting, either
by washing, slicing, decoloring, softening, staining or any
THE MICROSCOPE 13
one or combination of several processes. Indeed, the
preparation of the collected material is just as fascinating
as the examination of the prepared slides, and the student
should take pains at the very start to learn the correct ways
of preparing material for examination. This preparation
by the student is not only desirable, but really necessary
if he wishes to learn something of the wonders of nature.
Dealers' lists contain large numbers of slides ready made,
but for the student to purchase a slide containing the foot
of a fly is foolish and extravagant. If he lives in some in-
accessible place and has no idea what a slide is he might
buy one to show what is to be aimed at in preparation, but
for no other reason. Certain very rare slides may be pur-
chased but there are so many thousands of subjects wait-
ing to be taken home and mounted that he could fill his
days for a year and do no more than scratch the surface.
Methods of preparation and mounting will be described
later in the text. The student may begin at once his work
of collecting a library of microscopic material.
CHAPTER II
Microscopic Objects from Water
The main sources of microscopic material may be di-
vided into three classes, namely, the waters of ponds, lakes
and streams, animal life, and vegetable life. These, with
their various side branches, afford an endless variety of
material. One of the most interesting of these groups is
the first, ordinary water that has collected in a small or a
large body, either still or moving. Here are to be found
microscopic creatures by the million, in endless variety,
some animal, others vegetable. Hours of study and ob-
servation may be expended upon one drop of water from
a pail into which a handful of straw has been thrown and
left to stand for several weeks. Should this source not be
prolific enough to suit the fancy, take a small jar and run
it along the trunk of a tree submerged in a lake. Put a
drop of this water on a slide, magnify it fifty times and
you will see creatures tumbling over one another, crea-
tures you never thought existed. If you are still not satis-
fied, take the small jar again and gently scrape it along
the bottom of a shallow portion of a lake or stream. Be
very careful not to collect too much mud, but let it just
skim along the surface. If there are any aquatic plants
growing there, scrape their stems and the under sides of
the leaves with your collecting bottle. Put a drop of this
collection on your slide and be prepared for a shock. You
have now collected an entirely new lot of animalcules,
thousands of them, and yet you have seen but a very small
portion of what you may find. Almost every drop of
water you examine will contain new forms of life. These
14
MICROSCOPIC OBJECTS FROM WATER 15
forms vary with the seasons, the location and the character
of the water. Certain forms live only in fresh water,
others in salt water, still others may be found only in
places where there is a mixture of the two, such as at the
mouth of rivers.
Since water is such a prolific source of microscopic life
many beginners turn to it for their first material, so we
will describe a few collecting tools which will make the
work easier and more interesting.
While a bottle or jar may be used for collecting micro-
scopic material from shallow pools and streams, it is not
the most desirable device because it permits the collection
of too much water along with the organisms. We there-
fore make a collecting net which permits the water to
drain a\vay while the aquatic life is concentrated in the
bottle at the apex of the net.
To the end of a broom handle three or four feet long
attach a ring made of heavy galvanized iron wire about
eight inches in diameter. This may be fastened by drill-
ing a hole in the end of the wood and inserting the twisted
ends of the loop, or it may be wired to the handle with
thin copper wire. To this loop fasten a conical net of
muslin about twelve inches long. The apex of this cone
should be hemmed to prevent fraying. Into the hole
left in the apex insert a small wicle-mouth bottle of about
four ounces capacity, holding it with stout rubber bands.
This permits removal of the bottle when it is filled. To
use the net simply drag it through the water, skimming
the bottom, scraping the stems and leaves of aquatic
plants, scraping the trunks of submerged trees, piling or
anything else that may be in the water. When the small
bottle is filled with the concentrated collection it may be
emptied into a larger bottle containing a quantity of
i6 MICROSCOPIC TECHNIQUE
water. In this way samples may be taken from a number
of places, thereby increasing the number of species of ani-
mal and vegetable life collected.
One drop of this water examined under the microscope
will reveal an enormous quantity of living things. These
need air to support life and should not be confined in a
tightly closed jar or bottle. The forms of life are tiny but
there are thousands of them and they use up air at a sur-
prising rate, so leave a generous amount of air in the con-
tainer in which you carry home your collection. These
specimens must also be fed if they are to be kept alive for
future study. This is not difficult to do as any aquarium
affords an excellent breeding place. The aquarium must
be kept for just this purpose and should not contain fish.
Let the student look up the construction of a balanced
aquarium and build one along the lines suggested. In-
troduced into this aquarium the microscopic animals will
live and flourish and be always at hand when wanted for
study.
Now to get back to the examination of the material
gathered with the collecting net. This will contain a
variety of things, all new to the student. It is difficult to
say just what has been collected since each locality pro-
duces its own species, and many that are found abun-
dantly in one place are entirely absent in another. How-
ever, it is fairly safe to say that the greater portion of the
forms present will consist of diatoms, desrnicls and algae.
There will doubtless be some Rhizopods, a good many
Infusoria, probably some worms, numerous Rotifera and
some Polyzoa.
To classify all of these forms would be impossible in
the space allotted to this portion of microscopic examina-
tion, so with a few descriptions and illustrations, we will
MICROSCOPIC OBJECTS FROM WATER 17
pass on to the methods of examining and preparing cer-
tain of the subjects for examination.
! rtCV 1 ailfc , _ - ...
?J^Bmfete
m>
.l^Pka,^^
f - : '**Kiik
Fig. 19. A group of desmids from an
aquarium culture. X6oo
The desmids and the diatoms are two closely related
groups of aquatic plants. Some difficulty will be experi-
enced in distinguishing one from the other, but after a
little study the differences will become apparent.
Desmids are usually found in the sweetest and freshest
water. Salty or brackish water contains none at all, while
diatoms flourish there as well as in a mill-pond. Living
desmids are always green, diatoms are always brown.
There are other means of identification, such as flatten-
ing the soft cell wall of a desmid by pressing the cover
glass down against the slide, and rupturing the cell wall.
The green coloring matter (chlorophyl) and the color-
less protoplasm that fills the cell may then be forced out.
The cell wall of a diatom is hard and brittle, being com-
posed of silicate. The cover glass may be pressed down
on a diatom until the glass breaks without flattening or
Fig. 20. Desmids. Two species of the Genus Micrasterias. XiS
Slides by J. M. Furber. Photographs by Irving L. Shaw.
Fig. 21. A group of diatoms.
After Beavis.
Fig. 22. Diatoms. Type-
slide by Chr. Michelson,
Odense, Denmark. X78.
Photograph by Irving L.
Shaw.
Fig. 23. Group of arranged diatoms. X6o.
Photograph by Irving L. Shaw.
Fig. 24. Living diatoms from
a water trough. Xi8o. Slide
and photograph by Irving L.
Shaw.
Fig. 25. A Diatom, Tricer-
atrum. Slide by P. Klarsen,
Odensc, Denmark. X265.
Photograph by Irving L.
Shaw.
20 MICROSCOPIC TECHNIQUE
changing its shape. It may roll over and change its posi-
tion, presenting an aspect quite different from that first
seen, but it will probably roll back again and appear as it
did at first. If the cell wall is fractured the break will not
be irregular or of the appearance presented by a soft wall
when broken, but will display the characteristic fracture
of a hard, glass-like substance.
Both desmids and diatoms have the power of locomo-
tion, frequently moving from place to place. When
mixed with mud, as they probably will be when collected
with a net, desmids slowly work themselves free and rise
to the surface where they collect in a green scum or line
at the side of the vessel nearest the window, whence they
may be taken for examination. Diatoms have a similar
power of motion, but they usually move more rapidly.
Under the microscope the desmids may be seen moving
slowly and sedately across the field in a straight line. The
diatoms start across the field, get half way, then stop and
retreat or go in an entirely different direction. They
always seem to have important business to do and to be in
a tremendous hurry to get it done. Thus an object that
may seem to be either a diatom or a desmid is not a diatom
if it moves slowly, nor is it a desmid if it darts around the
field like a humming bird.
Rhizopods, while not a large group, are interesting be-
cause they are lowest in the scale of animal life. Some are
entirely without body protection of any kind, having
neither skeleton nor shell, the body consisting merely of
a soft jelly like mass of protoplasm. Yet they move about,
live and reproduce their kind. The most familiar mem-
ber of this family is the amoeba. This animal moves by
protruding a portion of its body to form a sort of arm,
then elongating the entire body and finally contracting
MICROSCOPIC OBJECTS FROM WATER 21
forward until it has resumed its former shape. This pro-
trusion may take place from any part of the body, for it
has no organs of locomotion as we understand them.
When the animal feeds it literally surrounds its food.
Having no stomach, digestive organs or alimentary canal,
it simply wraps itself about the food particle and some-
how digests and converts it into protoplasm. It repro-
duces asexually, that is, by a sort of budding. A pro-
tuberance shows itself which gradually grows until it
Fig. 26. A group of rhizopods.
From a drawing by the author.
Approximately
attains some size, then parts from the parent and goes
away to live its own life.
Others of the rhizopods are slightly more advanced,
especially the members of the genus Difflugia which
build for themselves shells of sand grains, cemented to-
gether in a perfectly regular form with each grain fitted
into its place. If sand is scarce it will use diatom shells
to construct its own shell, often taking those which are
longer than itself, attaching them lengthwise, side by side
22
MICROSCOPIC TECHNIQUE
and parallel to one another. Still another form, the most
beautiful of all the fresh water rhizopods, lifts itself on a
long stem and surrounds its body with a hollow latticed
sphere through the openings of which the pseudopods
(false feet) are extended in search of food. Any small
animal or vegetable substance supplies the rhi/opods with
food. Diatoms, desmids, Infusoria or anything small
enough to be seized is grasped by a pseudopodium, sur-
rounded by protoplasm and digested.
Fig. 27. Three familiar Infusoria found
in pond water. From a drawing by the
author.
The Infusoria group contains many of the microscopic
objects familiar to us from high school study of biology,
such as Vorticella, Stentor and Paramecium. The group
derives its name from the fact that many of the individ-
uals were first discovered in infusions, that is, water in
which animal or vegetable matter had been steeped and
was decaying. Since their first discovery the animals
MICROSCOPIC OBJECTS FROM WATER 23
have been found in great variety and abundance in even
the sweetest water, although they abound in incredible
numbers in stagnant pools.
The best way to procure specimens is to place the dip-
net under the mass of aquatic plants upon which they are
found and lift the mass with the net. If the plant is lifted
from the water, the water draining away will carry with
it many of the specimens, which are thus lost. A bottle
may be placed under the plant and the leaves and stems
scraped with the bottle rim, when the Infusoria will be
carried into the bottle. Some of them are free-swimming,
others are permanently attached to water plants, while a
third group builds shells like those of the rhizopods. The
free-swimming varieties may be easily collected and
placed upon the slide for examination, while those per-
manently attached may be found only by cutting away
a part of the plant and examining it. Some of the most
interesting types are firmly adherent to the water plant
Utricularia and to other plants with finely divided leaves,
every part of which should be searched with the micro-
scope, especially the forks of the leaves.
Those that build cases secrete a sticky substance to
which extraneous floating matter is pretty sure to ad-
here, building up a protective covering which surrounds
the animal. These cases or loricae are built of material
carried toward the animal by motion set up in the water
by organs with which it is provided. These organs serve
to create a current of water which carries food to the
animal, for being immobile it cannot move about in
search of food.
One very beautiful form of Infusorium representing
the fixed type is the species of Carchesium, illustrated
in Fig. 28. The single stem, which is attached to some
24 MICROSCOPIC TECHNIQUE
submerged object, divides at the summit into a large
number of branches, each one bearing at its end a tiny
bell-shaped Infusorium, while numerous other individu-
als are disposed along the stems on branchlets of their
Fig. 28. Carchcsium. A fixed form of
Infusoria. From a drawing by the
author.
own. Many of the fixed Infusoria are equipped with
muscles that enable them to contract when alarmed, of
which Carchesium is a good example. Running through
the stem and all of the branches is a cord-like muscular
thread which contracts when the animal is stimulated,
pulling the entire colony, which is colorless and may con-
tain as many as a hundred individuals, toward the point
of attachment. This contractile property is elective, for
one branch at a time may be affected without disturb-
ing the others, or the entire colony may contract at once.
All Infusoria are provided with cilia (hairs) or flagella
(lash or thread-like appendages) , which serve two pur-
MICROSCOPIC OBJECTS FROM WATER 25
poses. They provide a means of locomotion, and by their
vibration create a current of water which moves food par-
ticles toward the animal. Carchesium is an example of
the ciliated type in which the front or large end of the
bell is surrounded by a wreath of cilia visible under a
high power. When the creature contracts these vibratile
hairs fold together and the animal appears like a tiny ball.
Another interesting group of Infusoria are the Vorti-
cellae. There are about seventy species known, but we
will describe only one, since the microscopist will have no
difficulty in recognizing the genus once he has seen one
member. They belong to the class of fixed Infusoria,
are very common, scarcely a leaf or twig of any aquatic
plant being without at least one of them, and most of
them are colorless, or nearly so. Green ones do occur,
Vorticella smaragdina being one example, but most of
them are entirely devoid of color. Individuals are in-
visible to the naked eye, but magnifications of Xs5O will
reveal them clearly. The body is somewhat bell shaped
and is carried at the apex of a contractile stem. The front
or rim is wreathed with cilia, for the same purpose as was
explained in connection with Carchesium.
While individual Vorticellae are invisible, their multi-
plication in colonial groups is sometimes so rapid during
the summer that the entire colony breaks away from its
mooring and goes floating away. These groups look like
small spots of saliva or mucus floating on the water or
attached to the leaves and stems of plants. When such a
spot is discovered and touched with a needle-point, it
seems to grow smaller or disappear almost entirely. Pick
the plant on which it is found and place it in water for
later examination.
Do not be alarmed if, upon reaching home, your colony
26 MICROSCOPIC TECHNIQUE
seems to have vanished. Place the plant in an aquarium
and await developments. It will presently appear and
afford many hours of study. These Infusoria can con-
tract with a suddenness that is most disconcerting. The
observer may be calmly examining a Vorticella under the
microscope when, for no apparent reason, it disappears
like a flash, and one feels that the slide has been moved.
Soon, however, it may be seen separating itself from its
support, the coiled stem growing longer and straighter,
until the whole animal is again in view. Sometimes it is
barely extended when it again leaps from sight. If the
student has an aquarium he will do well to introduce
several colonies of these interesting animals, for they are
always worth studying.
Several species of Stentor may be found, the most com-
mon being Stentor molymorphus, illustrated in the draw-
ing (Fig. 27) . In shape the bodies arc somewhat variable
at will, and vary slightly in the same species. The largest
are trumpet-shaped and are, as a rule, permanently at-
tached at the narrow end of the body to some fixed sup-
port. Some species (Stentor Barrett?) form a lorica into
which they retreat when disturbed, folding the frontal
border to form a covering over the lorica. A few are free-
swimming by means of cilia and vibratile hairs. In color
they may be green, red, blue or almost black.
Pararnecium, illustrated in the drawing, is sometimes
called the slipper animalcule because of its shape. It is
frequently found in ponds but may be easily raised in a
tumbler of water to which a few small pieces of hard-
cooked egg have been added. Pure cultures of enormous
numbers of Paramecia may be thus reared in a few days.
On one surface of the animal is an opening that leads to
the mouth. The entire body is covered with cilia, while
MICROSCOPIC OBJECTS FROM WATER 27
one species has a caudal tuft of setae (stiff hairs) at the
posterior extremity. It reproduces by asexual division.
Two contractile vesicles may be seen, one in each half
of the body.
Volvox globalor, another free-swimming form, is illus-
trated in the drawing. This beautiful little animal may
be found in profusion early in spring, and will multiply
freely in an aquarium. It is quite large, in fact large
enough to be seen with the naked eye, and swims about
freely in a drop of water. The illustration shows an indi-
vidual containing a number of sporific inclusions that
will presently leave the parent cell and develop into new
individuals.
With this short introduction to the many beautiful and
interesting objects to be found in water, we must leave
that subject and go back to the business of preparing the
material for examination.
In addition to the collecting net a few more accessories
will be required. These, like the net, can be made by
the student. The easiest and most efficient way to remove
drops of water from a gathering to the slide is with a clip-
ping tube. This is merely a length of glass tubing of small
bore drawn out to a point in the flame. Such tubing is
obtainable in various diameters at any chemical supply
house. Tubing of about three sixteenths to one quarter
of an inch is about right. Several dipping tubes should
be provided, one straight, just as it is received, two with
points of different diameters drawn out in the flame, and
two with curved drawn-out points. These tubes are used
by holding the moistened finger over one end and im-
mersing the other end in the water. When the finger is
removed from the free end the water will rush up into
the tube until it reaches the same level as the water on the
28 MICROSCOPIC TECHNIQUE
outside. In this way a drop or a larger quantity of water
may be transferred easily and quickly to the live cage for
examination.
The live cage is necessary in the study of aquatic or-
ganisms because by its use we can keep the objects alive
for a long time and study them in life. The usual prac-
tice of students is to drop a cover glass on the specimens
and thus restrain their movements and confine them to a
limited space. This method, while it works after a fash-
ion, requires constant additions of water to keep the ani-
mals alive. This is bothersome and can be avoided by
using a live cage or box.
HANGING DROP COVER GLASS
RECESS IN SLIDE
HANGING DROP, GLASS CELL
Fig. 29. Diagrammatic views of live boxes. Depression
slide and cell slide.
One form illustrated in Fig. 29 is made from a ring of
glass, celluloid, metal, wood or cardboard cemented to
the slide. The first three materials need no preparation
before use, but wood or cardboard rings should be given
a coat of shellac to make them waterproof.
Glass tubing of large diameter and thin wall, such as
a large test tube, makes an excellent cell. With a tri-
angular file or a glass cutter score two marks on the out-
side of a test tube all around it, about one quarter of an
inch apart. Heat a rod of metal and touch one of the
MICROSCOPIC OBJECTS FROM WATER 29
marks with it. The tube will break off neatly at the mark.
Repeat at the other mark and you will have a cylinder of
glass one quarter of an inch deep. Now take some
Canada balsam and apply a ring of it to a slide, making
the ring of the same diameter as the glass cylinder and
using a generous quantity. Heat the slide over the spirit
lamp until the balsam is nearly hard, then press the
warmed glass ring into it and set it aside to harden. When
hard take fine sand and water, or emery powder and water,
and working on a piece of glass, grind the uncemented rim
of the glass ring flat. If desired, the depth of the cell thus
formed may be reduced to one eighth of an inch or less
by continued grinding. When the desired depth is at-
tained wash the slide and cell in water, dry it and paint
a ring of balsam around the outside of the joint and allow
to dry thoroughly, when the cell will be ready for use.
Such a slide may be used for a hanging drop or by filling
the cell with the water to be examined. In either case
paint a ring of vaseline on the rim of the cell before ap-
plying the cover glass, to prevent evaporation. Rings
made of brass tubing may be made up on slides in this
same way, or in a pinch a washer may be used.
Illustrated in Fig. 29 is another type of slide that may
be used for holding a drop of water. This is known as
a depression slide, since it has a shallow depression ground
into it. These slides are available from supply houses.
Equipped with a collecting net, dipping tubes and a
live box the rnicroscopist has all the equipment needed to
afford him hours of entertaining study of pond life. All
of the forms mentioned may be found without any
trouble, as well as thousands of others that will delight
and fascinate. Furthermore, not all of these minute
specks of life have been found and described, so there is
30 MICROSCOPIC TECHNIQUE
ample field for research work in this branch of micros-
copy. It is just as easy for a student worker to discover
new forms as it is for the trained scientist, and every ob-
server should be on the alert for new species.
As mentioned earlier, many forms of aquatic life are
difficult to mount permanently on a slide. Others are
comparatively simple to mount and the student may want
to know how these may be preserved. The greatest diffi-
culty encountered in mounting Infusoria, Rotatoria,
Rhizopoda, Hydra, Obelia, etc., is that of killing them in
such a way that they remain expanded. Most killing
agents stimulate them to such an extent that they imme-
diately contract into a shapeless mass. To prevent this
we first apply a reagent that will anesthetize them in an
expanded state, then kill them with another reagent,
usually combined with a fixing agent to prevent the post-
mortem changes that take place almost at once.
Many forms may be successfully anesthetized with Ep-
som salt made up into a saturated solution and added
slowly to the water. Separate a small quantity of the
water containing the desired forms and slowly add a satu-
rated solution of Epsom salt until the desired effect is
secured. Keep the water as quiet as possible to prevent
contraction, and add the solution very cautiously. One
very good way is to place a piece of string in the end of a
dipping tube, drop some of the solution in the tube and
suspend this so that the string just dips into the water.
In this way the salt solution slowly diffuses into the water
without setting up any currents to disturb the animals.
Crystals of menthol or chloral hydrate may be placed in
a filter and floated on the water to achieve the same re-
sult. The author has had considerable success with Vor-
ticella, Stentor, Paramecium, etc., by subjecting them to
an atmosphere of formalin, which kills the forms quickly
MICROSCOPIC OBJECTS FROM WATER 31
in an expanded state. The method used is to place the
water in a cell-slide in a large dish and leave until all
motion in the water has subsided and the forms are fully
expanded. A few drops of formalin are then introduced
into the dish by means of a previously attached dropping
tube and the dish is covered with a plate of glass to con-
fine the vapors. The slide is allowed to remain a few
minutes and is then taken out, the water transferred to
albumenized slides and these treated as follows.
The slides to be used are first prepared with Mayer's
albumen fixative (See Chapter IV) . A drop of water
containing the desired organisms is then placed on each
slide and allowed to become nearly dry, assisted by gently
blowing on the drop of water. When nearly dry plunge
the slide in 70% alcohol to coagulate the albumen and
hold the objects to the slide. Now place the slide in one
of the fixing agents (See Chapter III) . Shaudinn's solu-
tion is the one usually recommended for Infusoria, but
Worcester's fluid works just as well. Leave until thor-
oughly fixed, then wash as instructed for the removal of
mercuric chloride fixatives. The washed slide is now
stained, using alum-haematoxylin and eosin, or picro-
carmine and methyl green according to the directions
given in Chapter VI. Following staining, the slide is de-
hydrated, cleared and mounted in balsam.
Taking advantage of the mildly anesthetic properties
of clove oil the writer has recently conducted a series of
experiments in which this reagent was used to anesthe-
tize strongly contractile aquatic organisms prior to fixing
them. The method was tested on Hydra, Rotatoria, Vor-
ticella and other difficult forms with results almost com-
parable with those resulting from the use of cocaine and
Novocaine. After several techniques were tested the fol-
lowing was adopted as a satisfactory procedure.
32 MICROSCOPIC TECHNIQUE
Bring the objects from the aquarium or collection into
a small watch glass that can be placed on the stage of the
microscope for observation. Make the transfer with a
dipping tube, including enough depth of water to permit
the organisms to expand fully. When a sufficient num-
ber of individuals has been transferred to the watch glass,
allow the water to become quiet and observe through the
microscope. When the forms are fully expanded place
a drop of clove oil on the surface of the water, using a
pipette. Bring the tip of the pipette close to the water
and ease the oil out very gently to avoid disturbing the
water and making the organisms contract. The oil film
will spread over the water and its anesthetizing action
will slowly take effect.
Have ready a pipette full of a suitable fixing fluid, such
as Bouin's or Worcester's fluid. Observe the progress of
the anesthesia carefully and as soon as all signs of motion
cease, add the fixing fluid. It is important that the fixing
fluid be added at the earliest possible moment after the
forms are quiet. General death of the individual follows
closely upon anesthesia and many organisms start to dis-
integrate immediately after death. It is the purpose of
the fixing agent to arrest this disintegration by killing and
fixing the organism in one operation. Regulate the pro-
portion of water in the watch glass so that about an equal
volume of fixing fluid may be added.
Allow this fluid to act about fifteen minutes, then drain
off most of it and add pure fixing fluid. After the objects
are thoroughly fixed, they are washed and treated as re-
quired by the fixing fluid used (See Chapter III) .
Temporary staining may be effected by drawing neu-
tral red stain under the cover glass in the live cage with
blotting paper in the manner described in Chapter IV.
CHAPTER III
Killing, Fixing & Preserving
Living material is of very little use in microscopical
study. Accordingly the animal or plant must first be
killed so that the elements desired for study may be dis-
sected out. Fresh tissue from recently killed subjects
should be selected in all cases. Killing usually refers to
the general death of the subject and does not apply strictly
to the life processes of individual cells, which continue
for some time after the general death of the specimen. It
is the purpose of the fixing agent to terminate this life
process, which it does effectually only if it is applied be-
fore total cessation of vital activity. Destructive post-
mortem changes follow almost immediately upon the
death of the cells and unless this is arrested by the action
of the fixing agent, serious alteration of the tissue takes
place. Fixation, then, is the process of terminating cell
life and hardening and preserving the tissues in a condi-
tion as nearly like that obtaining in life as is possible.
The process is effected through the chemical action of
certain materials which are accordingly called fixing
agents.
Preservation of structural elements to present a faith-
ful impression of their true character is a fundamental
requirement in the preparation of microscope material.
The subsequent operations of dissecting, staining, sec-
tioning and mounting serve only to make the examina-
tion more intelligible by clarifying the material and dif-
ferentiating structures that have been brought into
proper condition by fixing. Considerable thought and
33
34 MICROSCOPIC TECHNIQUE
effort must sometimes be expended upon the selection
and application of an appropriate fixing agent for the
material in hand. Its composition, reaction and applica-
tion must be such that it will penetrate and kill the tissues
quickly to preserve their active characteristics. It must
do this with a minimum of alteration in their structure,
and it must harden and preserve them enough to prevent
post-mortem changes, yet leave them amenable to the
subsequent processing by other reagents in the operations
of staining, sectioning and mounting.
No one chemical yet discovered possesses all these vir-
tues. A reagent that fixes one class of tissue may act as
a macerating agent on another class. Most of the reagents
that fix nuclei satisfactorily are known to alter or destroy
certain cytoplasmic inclusions. Hence we combine in
one mixture a group of reagents to secure a desired effect.
The more generally used fixing agents contain one or
more of the following chemicals in varying proportions,
alcohol, formalin, acetic acid, mercuric chloride, chromic
acid or its metallic salts, picric acid, nitric acid and osmic
acid. Many combinations of these, as well as a number of
other reagents, have been proposed, tried and discarded.
Relatively few of those to be found in microscopical writ-
ings are of value to the student. The few that are of value
have been selected and described below, together with a
short list of the materials for which they are satisfactory.
The alert student will be able to select from the formulae
given one or more fixing agents to be used on the material
with which he is working.
The first fixing agent to be considered is pure methyl
alcohol. It is usually used in strengths of 70% to 95%-
This is an acceptable fixative for crude work and for ro-
bust organisms like insects and Crustacea. It is entirely
KILLING, FIXING b PRESERVING 35
unsuited to soft, delicate tissues or those containing much
water, for it shrinks them badly. It causes little or no
chemical alteration, except in the solution of fats and oils.
Weak alcohol (30-35%) is useful for fixing tissue that is
to be dissociated by maceration. It dissolves intercellular
cement substance and fixes cell contents with little shrink-
ing or alteration.
Acetic acid is a weak fixative for some tissues and has
a decided tendency to make them swell. Taking advan-
tage of this property we combine it with alcohol in such
proportions that the shrinking propensity of the one re-
agent is balanced by the swelling action of the other, as in
the following formula:
CARNOY'S FLUID
Glacial acetic acid 10 ccm
Alcohol (85%) 85 ccm
Chloroform 30 ccm
Mix immediately before use.
This mixture penetrates and fixes with fair speed and is
a suitable medium to use on rough work with fairly ro-
bust material. When fixation is complete, transfer the
material to 80% or 90% alcohol.
Very impermeable material should be fixed in the fol-
lowing mixture, which will penetrate and kill even the
most refractory organisms:
CARNOY AND LE BRUN'S FLUID
Alcohol, absolute 50 ccm
Glacial acetic acid 50 ccm
Chloroform 50 ccm
Mix immediately before use.
Saturate the above mixture with mercuric chloride.
36 MICROSCOPIC TECHNIQUE
Use this fixative only for the most impermeable ob-
jects, such as nematodes, and heavy-shelled eggs. When
the material is fixed it must be treated with iodine as de-
scribed under the use of mercuric chloride fixing agents
listed further along in this chapter.
The following mixture is very good for insects, crusta-
cea and botanical specimens of many kinds:
FORMALIN- ACETIC-ALCOHOL
Alcohol (85%) 85 ccm
Formalin 10 ccm
Glacial acetic acid 5 ccm
Keep a stock solution of alcohol and formalin and
add the acid to measured quantities of this when
needed for use. Fixing is complete in from one to
twenty-four hours, when the material should be
transferred to 85%, alcohol for preservation.
Formalin is an important fixative with numerous de-
sirable properties. It does not dissolve carbohydrates or
fats, penetrates rapidly, preserves tissues indefinitely with-
out serious alteration, leaves them in good condition for
a large variety of stains and is convenient and inexpensive
to use. Alone, however, it does not harden tissues suffi-
ciently to withstand the shrinking effects of absolute al-
cohol as used in dehydration, or the action of clearing
reagents and heat, hence it cannot be used alone for ob-
jects that are to be imbedded in paraffin.
For rough work use a 10% solution of commercial for-
malin in water.
Tissues from vertebrate animals may be fixed in:
Formalin 10 ccm
Physiological salt solution 90 ccm
KILLING, FIXING & PRESERVING 37
Invertebrate animals and tissues therefrom may be
fixed in:
Formalin 10 can
Water 90 ccm
Glacial acetic acid i ccm
If the student proposes to do any work in plant cy-
tology, such as the preparation of material for study of
mitosis, the following fixative of Karpenchenko is excel-
lent. It penetrates rapidly, producing a delicate and
beautiful fixation of mi to tic figures in root tips, ovaries
and anthers.
KARPENCHENKO'S FLUID
Chromic acid 0.5 g
Water 54.5 ccm
Glacial acetic acid 5.0 ccm
Formalin 40.0 ccm
Keep a stock solution of i % chromic acid in water
and add to measured quantities of the other reagents
lor use.
Mercuric chloride is a powerful and rapid fixative for
a large number of tissues, including invertebrate forms
that are to be mounted entire, such as many aquatic or-
ganisms, some Infusoria, Cestoda and flat worms. One
of the most useful of the many mixtures that have been
proposed is:
GILSON'S FLUID
Mercuric chloride 5 g
Nitric acid (80% sol.) 4 ccm
Glacial acetic acid i ccm
Alcohol (70%) 25 ccm
Distilled water 220 ccm
38 MICROSCOPIC TECHNIQUE
Several precautions must be observed when working
with solutions of mercuric chloride.
1 . Never use anything but distilled water for solutions,
except when working with marine organisms, when sea
water is used.
2. Never bring metallic instruments into contact with
the solution. Use glass or wood rods for handling the
materials.
3. Materials should be left only as long as is required
for complete penetration of the fixing fluid.
4. Every trace of mercury must be removed from the
material before it is mounted, or small, needle-shaped
crystals will eventually form and ruin the mount. Ma-
terials fixed in aqueous solutions of mercuric chloride
should be washed in running water for six to twelve hours,
then passed up the ascending alcohol series to 80% alco-
hol. Materials fixed in Gilson's fluid or any other alco-
holic solution of the salt should be washed in several
changes of 50% 70% alcohol, then transferred to 80%
alcohol. The residual mercury must always be removed
by treating the material with iodine, which combines
chemically with the mercury. Add a concentrated solu-
tion of iodine to 90% alcohol by dropping until the
solution is a deep amber color. After a lapse of time, de-
pending upon the amount of mercury present, this color
will be discharged. More iodine must be added as this
takes place. When the reaction is nearly complete, as in-
dicated by a slowing down of the rate of decolorization,
the iodine must be added very cautiously to avoid an ex-
cess that would stain the material. If enough mercury
is present to necessitate the addition of iodine after the
third addition, change the entire solution to prevent pre-
cipitation of mercuric iodide on the material.
KILLING, FIXING 6- PRESERVING 39
For general histological work Zenker's fluid is one of
the very best. It preserves nuclei, cytoplasm and connec-
tive tissue in excellent condition.
ZENKER'S FLUID
Mercuric chloride 5 g
Potassium bichromate 2.5 g
Distilled water 100 ccm
Glacial acetic acid 5 ccm
Keep a stock solution containing the mercuric
chloride and the potassium bichromate and add the
acetic acid to measured quantities immediately be-
fore use.
WORCESTER'S FLUID
(Modification according to Galigher)
Mercuric chloride, sat. aqueous solution . 100 ccm
Formalin 5 ccm
Glacial acetic acid 5 ccm
Mix immediately before use.
The original formula as proposed by Worcester con-
tained more formalin and less acetic acid than the modi-
fication. It was recommended for fixing all forms of
Protozoa. It gives splendid results with amoebae and
many flagellates, such as Euglena. It does not produce
as fine a fixation on Paramecia and other Infusoria as
Kleinenberg's fluid, which should be used for all forms
with a relatively soft cuticle.
The fixing agent should be poured upon the expanded
material contained in a small amount of water. Allow
to remain in the fixative at least an hour. Decant the
fluid, wash in several changes of water and pass up the
alcohol series to 80% alcohol. Final removal of the mer-
curic chloride must be effected with iodine.
40 MICROSCOPIC TECHNIQUE
Picric acid is an excellent fixative for many types of
material, preserving structures with great fidelity. It
does not, however, render nuclei in the best condition for
staining, so a small part of acetic acid is added to the satu-
rated aqueous solution of picric acid to improve its nuclei-
fixing property. Another shortcoming is that it does not
harden tissues sufficiently to withstand the macerating
action of water or weak solutions of alcohol, to which end
formalin is added to the mixture. Picric acid leaves the
material in excellent condition for absorbing most stains,
if proper precautions are taken to remove the picric acid
by the methods given below.
Water and weak alcohol exert a strong macerating ac-
tion on picric fixed material, for which reason all materials
fixed in picric acid mixtures are transferred directly from
the fixative to 70% alcohol. Never use water or alcohol
of less than 50% strength for washing picric-fixed ma-
terial. Wash until every last trace of the yellow color
imparted by the acid has been removed. In practice,
pour off the excess fixing fluid, leaving just enough to
cover the material, and then add an equal amount of 70%
alcohol. After a few hours have elapsed this solution may
be poured off and pure 70% alcohol added. Change the
alcohol frequently and do not stop the washing until every
trace of acid is removed, as indicated by the absence of
any color in the alcohol. Warming the alcohol to 35C.
accelerates the removal of the picric acid, but the tem-
perature should go no higher.
The picric acid fixative given below is probably the
most generally useful medium to use, since it gives satis-
factory results with more materials than any other for-
mula now in use. Nearly all stains may be used with
entire success, and it is specially useful for haematoxylin
KILLING, FIXING if PRESERVING 41
and eosin staining. Carmines work very well with ma-
terials fixed in it. Its action is so delicate that it fixes
such structures as cilia and the achromatic elements of
mitotic figures faithfully, making it a valuable fixative
for the study of cytology. The majority of embryological,
histological, botanical and zoological materials to be
stained for general study are most safely handled in this
fixative.
Aquatic organisms to be mounted entire, such as amoe-
bae, Hydra and hyclroids, annelids, soft Mollusca, tissues
of Insecta and Crustacea, embryos of fishes, birds, mam-
mals and reptiles, and most adult vertebrate tissues may
be successfully fixed by this reagent. Due to the free acid
content it must be avoided where calcareous structures
are to be preserved, arid it is unsuited to the fixation of
flagellate and ciliate Protozoa, sponges, Medusa, fiat-
worms and insects or Crustacea that are to be mounted
whole.
BOUIN'S FLUID
Picric acid, saturated aqueous sol 75 ccm
Formalin 25 ccm
Glacial acetic acid 5 ccm
Materials are fixed quickly, but are unaffected by pro-
longed immersion, although delicate structures are best
removed as soon after complete fixation as possible. Con-
tractile forms of Protozoa are best killed first in a solution
of Bourn's diluted three to one with water. When they
have settled to the bottom, the dilute solution may be re-
placed with full strength reagent. Twenty-four hours
should be sufficient for thoroughly fixing most materials,
and many specimens are thoroughly hardened in much
less time. The material should be transferred directlv
42 MICROSCOPIC TECHNIQUE
from the fixing agent to 70% alcohol, never to water. In
this connection see the instructions given earlier for the
handling of picric materials.
When very impermeable objects such as insects and
Crustacea are to be sectioned, or similar refractory forms
are to be dealt with, a modification of the above formula
is used by substituting alcohol for the water as a solvent
of the picric acid, thus:
ALCOHOLIC BOUIN'S FLUID
Picric acid, saturated sol. in 70% alcohol . 75 can
Formalin 20 ccm
Glacial acetic acid 5 ccm
Material fixed in this solution should be handled
in the same way as detailed above.
The next mixture is valuable for the fixation of many
delicate structures which would be distorted by other re-
agents. It penetrates well and causes a minimum of
shrinking. It is very useful for Protozoa with a thin
cuticle, such as Paramecia, but is not recommended for
Rhizopoda or Flagellata, like Euglena. Protozoa are fixed
in a few minutes but may be left for several hours without
harm. Naturally the time for complete hardening in-
creases with the density and permeability of the material.
KLEINENBERG'S FLUID
Cone, sulphuric acid 2 ccm
Distilled water 100 ccm
Saturate this solution with picric acid.
Handle material fixed in Kleinenberg's in the
same way as all other picric acid fixed material, by
washing in alcohol, never in water.
CHAPTER IV
Dissociation
The critical examination of many forms of animal and
plant tissues under the microscope may be greatly facili-
tated if the structures are isolated. This permits ex-
amination from every angle without interference from
other structures and allows the use of selective stains on
individual cells and structures to delineate their various
features more clearly. The exact methods to be used in
each case will vary with the nature of the material, the
nature of the connective tissue and the structures. Three
general methods of dissociation are recognized, each one
of which performs a specific function. They are teas-
ing, maceration and corrosion. General details of each
method will be given here, with enough data for the stu-
dent to select the method or combination of methods
which will best dissociate the material in hand.
The simplest method of dissociating elements for mi-
croscopic study is teasing. By this method fibrous tissues
not firmly united by intercellular cement may be pulled
apart without previous treatment. Nerve and tendon
tissues are examples of this class of material.
The actual work is performed with dissecting needles
as described in Chapter XI. Two sets of needles are used,
one pair of rather heavy needles with blunt points, one
pair with extremely fine points to be used in the final
separation of individual cells or fibers. Examine the
needles carefully before starting work to be sure they are
perfectly clean and sharp. If they are not sharp grind
them to a fine point on a carborundum hone. When the
43
44 MICROSCOPIC TECHNIQUE
work is finished, or whenever work is discontinued for
any length of time, clean the needles thoroughly, then
they will always be in condition to resume work.
TEASING On the stage of the dissecting microscope
place a slide containing a small bit of tissue in a drop of
the medium from which it was taken. With one of the
stout needles in the left hand hold the material down
firmly, far enough from the edge to afford a secure hold.
With the other needle pull the material apart, starting at
the edge and working in toward the center as the mar-
ginal structures are separated. Be careful not to crush
or break the structures and avoid pressing it down on the
slide. The proper technique is a pulling apart and not a
crushing action. Properly done, the material is divided
into fine shreds. The stout needles are now laid aside and
work is continued with the fine ones. Separate the shreds
carefully to isolate the individual fibers or cells of which
the material is composed. When this seems to have been
accomplished, transfer the slide to the compound micro-
scope and examine under a medium power. If the indi-
vidual units are not sufficiently separated, continue the
work with fine needles under the compound microscope
until you are sure that further division is impossible.
Remove any large pieces of material that remain undi-
vided and transfer the dissociated material to the proper
reagent for mounting.
Temporary mounts of teased material may be pre-
pared for quick study by teasing the material in physio-
logical salt solution. It may then be studied unstained
or it may be stained by applying a cover glass to the prep-
aration and drawing the stain under the glass. The pro-
cedure is easy. Simply place a drop of stain at one edge
of the cover glass and apply a piece of filter paper to the
DISSOCIATION 45
opposite edge. The filter paper will absorb the fluid
under the glass and as this recedes toward the filter, capil-
lary attraction will draw the stain underneath. When
the entire cover glass area is fdled with stain remove the
paper and allow the material to absorb the stain. Then
repeat the same operation with distilled water and wash
away all superfluous stain. 70% alcohol may be used in
the same way to remove refractory stain residues. If
permanent stains are used the mount may be preserved
by drawing a drop of glycerine under the cover and seal-
ing with a ring of gold size. Liquid mounts of any kind
are rather unsatisfactory for a number of reasons and so
are not recommended. It is better to make permanent
mounts in Canada balsam as follows.
This practice is by far the best way of treating teased
material of any kind for permanent mounts. By selection
of appropriate stains the process may be adapted to ani-
mal as well as vegetable material, making it universal.
First fix the material in 10% formalin for at least
twelve hours. Delicate structures should be pinned to
thin pieces of wood, but must not be stretched.
Staining precedes teasing in this process, and is best
effected by using haematoxylin as a nuclear stain and
eosin as a counter stain. (For formulas covering stains
see Chapter VI.) First wash the formalin-fixed material
in several changes of distilled water, or for several hours
in running water. When completely free of formalin,
cut the material into coarse pieces and place in several
times its volume of dilute alum-haematoxylin stain.
After several hours' immersion pour off the stain and wash
in a few changes of distilled water. Tease apart a small
piece and examine it under the microscope. If the nuclei
have absorbed enough stain to give them a strong, sharply
46 MICROSCOPIC TECHNIQUE
defined color, with other structures stained only slightly
or not at all, wash the entire lot of material in running
water for a few hours. While overstating is not very
likely, it may occur, and the material will then need to be
destained by immersing it in a half-saturated solution of
ammonium-alum until examination shows that all parts
except the nuclei have been destained. Now wash in run-
ning water for several hours. Dehydrate by passing the
material through the ascending series of alcohols. Add
to the 80%, 95% and first absolute alcohol baths enough
eosin to color the solution a deep pink. Leave for an hour
or two in the first absolute alcohol, then pour this away
and replace with new absolute alcohol without eosin and
leave for an hour longer.
The material is now ready for clearing. Replace the
last absolute alcohol with 50-50 alcohol and creosote-
xylol as described under clearing insects and leave for one
hour. Pour off this solution and add creosote-xylol, re-
placing this after a few hours with fresh solution. Then
add balsam to the creosote-xylol containing the material,
adding balsam every few hours until the liquid assumes
the consistency of a medium syrup. The prepared ma-
terial is then teased apart in balsam on a slide. When
sufficiently isolated structures are secured remove any
large pieces of tissue that may remain, add a drop of thick
balsam and a cover glass. When setting the cover be sure
to lower it very slowly to exclude air bubbles and to pre-
vent the material from spreading and running to the edge
of the cover.
MACERATION Many tissues are firmly cemented to-
gether with an intercellular cement substance which
would defeat any success in simple teasing. Such ma-
terials must first be subjected to the action of a reagent
DISSOCIATION 47
which will dissolve or soften the cement and make dis-
sociation of cell units possible by teasing or shaking.
While a great many macerating agents, as such liquids
are called, have been proposed and recommended from
time to time, there are only a few that need be considered.
These give excellent results with most of the material the
student is likely to encounter. The first is pure water,
either cold or warm. Most substances, especially vege-
table substances, may be satisfactorily macerated in water.
Heating may be resorted to for quicker action, but the
temperature must not exceed 50 C. (150 F.) . Sub-
stances containing albumen should be macerated in cold
water only, for heat coagulates the albumen and renders
it hard.
Choice of a macerating agent is governed by the char-
acter of the material to be treated. A reagent must be
selected which will exert a minimum of alteration in the
tissues, yet it must perform its work thoroughly in order
to separate the units completely. This is admirably ac-
complished by Ranvier's One-third Alcohol solution:
Alcohol (95%) one part
Water two parts
This is especially recommended for epithelia, glands
and smooth muscle cells. Its slow action preserves tissues
with the least alteration, yet so thoroughly macerates it
that epithelial tissue may be shaken apart after 36 hours
immersion.
Another very excellent reagent for animal tissues is
Gage's Formalin Solution, made by mixing:
Formalin 2 ccm
Physiological salt solution 1000 ccm
48 MICROSCOPIC TECHNIQUE
Vegetable tissues of many kinds may be satisfactorily
macerated in pure water or water and glycerin in the
proportion of one part glycerin to six parts water. Ob-
jects must be left in the solution until thoroughly rotted,
when the elements may be teased apart. The constituent
cells of plant stems may be separated nicely by simple
water maceration. To procure annular vessels for ex-
amination take the stem of maize and cut into pieces
about one-half inch long. Then slice these longitudin-
ally. Macerate in water until rotten. Place under the
dissecting microscope and pick out the annular vessels.
Some difficulty may be experienced in procuring ma-
terial which is quite free of debris. In such cases use a
fine-pointed water color brush to pick out the desired ma-
terial. The grade of brush known as a No. oo red sable
is the best to use. The hairs in this brush are springy,
hold their shape well and can be turned to a fine point.
If the amount of desired material greatly exceeds the
amount of debris present it may be better to eliminate the
latter.
Sometimes the difference in weight between wanted
and unwanted material may be used to separate the two
groups. Repeated washings with periods of settling in
between will frequently clean the material very well. Or,
if a decided difference in specific gravity exists, one of the
two groups will remain suspended or sink more slowly
than the other group. In such cases allow most of the
heavy material to settle to the bottom and draw off the
supernatent liquid with a dipping tube. This operation
may need to be repeated several times before perfectly
clean cells are secured, but one is amply repaid for the
work involved by greater clarity and beauty of the com-
pleted mounts.
DISSOCIATION 49
Scalariform vessels and spiral vessels from vegetable
stems may be isolated in the same manner as was de-
scribed for annular vessels. The former may be procured
in abundance from the rhizome of the fern Pteris aqui-
linia, and the stem of rhubarb (Rheum officinale) affords
splendid examples of the latter.
To mount animal material which has been macerated
and teased in water, coat a perfectly clean slide with
Mayer's albumin fixative, made as follows:
Whip the whites of several eggs slightly, two or three
dozen strokes being sufficient. Allow to stand one hour,
skim off the foam and pour the remaining liquid into a
clean graduate. Add an equal volume of glycerin and
one gram of sodium salicylate for each 100 ccm. of liquid.
Shake thoroughly and filter through paper into a clean
bottle. Filtration is very slow. If a vacuum filter is avail-
able it should be used. Lacking this, the operation
may be accelerated by placing only a small portion of
solution in the filter at a time and replacing the filter
paper frequently. Keep a working stock in a small vial
fitted with a dropping rod, and the reserve stock in a
tightly closed bottle.
The slide is prepared by placing a drop of the fixative
on it and smearing it back and forth with the clean finger,
rubbing it quite vigorously to make sure it adheres to
every portion of the slide. Wipe off the surplus with the
moist finger until only a very thin film remains. For a
method of cleaning slides see Chapter V.
Place a drop of water containing the desired material
upon the albumenized slide and tilt it back and forth a
few times to spread the material. When almost dry, dip
the slide in 95% alcohol to coagulate the albumen and
hold the material fast to the slide. Pass the slide down
50 MICROSCOPIC TECHNIQUE
through the descending series of alcohols to water, then
to haematoxylin stain, if this is to be used. Leave until
the nuclei are stained deeply. Wash in running water for
fifteen or twenty minutes and then pass up the ascending
alcohol series to 95% alcohol. Counter stain for two
minutes in 0.5% eosin in 95% alcohol. The depth of the
eosin stain may be reduced in 95% alcohol if too deep.
Place the slide in crcosote-xylol for several minutes, or
until clear, transfer to pure xylol for a few minutes, then
add a drop of balsam and a cover glass.
If stains other than haematoxylin are to be used, the
technique should be altered to coincide with that re-
quired for the particular stain selected. Consult Chapter
VI for methods.
CORROSION The dissociation method known as cor-
rosion makes use of the power of certain chemicals to de-
stroy some structures without affecting others. This
process makes it possible to study such structures as fish
scales, sponge spicules, and chitinous insect skeletons.
Another application of corrosion consists in injecting the
internal cavities of organs, insects, shells, etc. with wax,
then corroding away the soft parts with a strong reagent,
leaving perfect casts of the interior surfaces.
Since calcium is soluble in acids, calcareous structures
such as some sponge spicules, Foraminifera, fish scales
and the like, must be freed of soft tissues by maceration in
strong alkali solutions. Concentrations of 30-35% of
sodium or potassium hydroxide in water are the usual
strength. At room temperature several days to as many
weeks may be required to completely dissolve the soft
tissue. The action of the reagents may be accelerated by
boiling. While this is harmless to calcareous structures
it must not be attempted with insects, for it would destroy
DISSOCIATION 51
them. When all unwanted parts have been dissolved,
wash the material thoroughly in water to cleanse it of any
alkali, dehydrate and mount in balsam.
Siliceous structures such as radiolarians, diatoms and
the spicules of siliceous sponges are cleaned with strong
nitric acid. The material is immersed in the cold acid
and left to dissociate, or it may be boiled for more rapid
results. Wash, dry and mount the cleaned specimens in
balsam.
Hard materials such as bone, teeth, horn, claws, coal
and minerals require different treatment, to be described
in a succeeding chapter.
CHAPTER V
Section Cutting
In order to complete the detailed microscopic study of
organisms and tissues with the various parts in their cor-
rect relative positions, it is necessary to cut sections thin
enough to transmit light. This must be accomplished
without distorting the specimen or crushing any of its
parts. Some vegetable materials such as soft stems, roots,
fleshy leaves, etc. may be sectioned free-hand after a little
practice, but animal tissues must be properly supported
in a plastic medium to hold the delicate parts in their
proper positions.
If work with a large variety of tissues is contemplated a
mechanical device called a microtome is necessary. This
instrument holds the material to be cut and passes it over
an extremely sharp knife which shaves off thin sections.
It is automatic in action and very accurate, advancing the
specimen at predetermined intervals between each cut, so
that any number of uniform sections may be made. This
instrument, however, is expensive and is beyond the reach
of the average student. To meet the demand for an in-
expensive device to serve the same purpose the hand
microtome or well microtome was contrived. This per-
mits the cutting of sections to fulfill every requirement
of the student. A home-made well microtome is illus-
trated in Chapter XI. Anyone with the necessary facili-
ties can build such a microtome, or have it made in a ma-
chine shop at small cost.
As each section is cut from the specimen, it is placed on
a piece of clean paper, keeping the sections in their cor-
52
SECTION CUTTING 53
rect consecutive order. In this way the entire morphol-
ogy of a specimen may be reconstructed. The sections
may be placed serially on the slide and a progressive study
of the entire organism effected.
Animal tissues must be supported in some medium
when they are to be sectioned. Three general methods
are in use, namely, paraffin imbedding, celloidin imbed-
ding and freezing. When materials are to be used for
diagnostic purposes or for immediate examination sec-
tions may be obtained by freezing. Celloidin (nitrocel-
Fig. 30. Bausch & Lomb Minot Rotary Automatic
Microtome for laboratory use in paraffin sectioning.
lulose) is used for sectioning a few materials, such as
whole brains or some watery tissues which would shrink
in hot paraffin. Aside from these exceptions, paraffin is
used for supporting practically every material the student
is likely to encounter, except calcareous tissues and min-
erals. It is the most valuable method of microtechnique
and is essential to practically every field of biological
science. A complete understanding of this operation as
well as considerable skill in its execution should form part
of the equipment of every student.
54 MICROSCOPIC TECHNIQUE
After the material has been properly fixed and washed,
it must then be dehydrated. This is accomplished by re-
placing the water with purified methyl alcohol. Alcohol
has the property of shrinking tissues and distorting the
cells when the material is placed directly into a strong so-
lution. For this reason the alcohol is diffused gradually
by using dilute solutions. Dehydration must be gradual
and it must be thorough. If the least trace of water re-
mains in the tissue it will prevent proper penetration of
the clearer and the paraffin, thereby rendering the whole
operation a failure.
The alcohol series usually starts with a 15% solution
f 95% methyl alcohol which need not be absolutely
precise. For practical purposes the solution may be pre-
pared by adding 15 ccm. of 95% alcohol to 85 ccm. of dis-
tilled water. All other percentages may be made in the
same way; 35% solution is 35 ccm. alcohol to 65 ccm.
water, 50% solution is 50 ccm. alcohol to 50 ccm. water,
70% solution is 70 ccm. alcohol to 30 ccm. water, etc.
Steps of 15%, 35%, 70%, 80%, 95% and 100% or abso-
lute alcohol are generally used for all but the most deli-
cate material.
Wide-mouthed bottles with well-fitting stoppers are the
proper containers. It must be possible to transfer the
material quickly from one jar to the next, so containers
with even moderately narrow necks should be avoided.
Mayonnaise jars in the eight-ounce size are excellent.
If the material has been washed in water it is placed
first in the lowest member of the alcohol series. If it has
been washed or preserved in alcohol, such as material
fixed in any of the picric acid mixtures, dehydration has
started and should be continued from that point by trans-
ferring the material to the grade of alcohol next higher to
SECTION CUTTING 55
that used for washing or preserving. Successive changes
to higher concentrations are then made until absolute
alcohol is reached. The volume of alcohol must at all
times be several times that of the material.
The student may experience some difficulty in han-
dling small objects when transferring them from one
reagent or solution to the next. Large objects are easily
handled by pouring off one grade of alcohol, and adding
the next. This is difficult with small, light specimens
such as mosquito larva, plant lice, aquatic organisms and
the like, so these are transferred on a filter.
Cut a three-inch disc of filter paper from a larger sheet,
and place it in a small funnel measuring one and one-half
inches across. Pour the material to be dehydrated, with
its containing liquid, into the filter. If the liquid is to
be retained allow it to filter into an appropriate container.
If of no value discard it. The next solution, say 15%
alcohol, is now poured into the filter and allowed to drain
into the container. Now remove the filter from the fun-
nel and place it in the container of solution, where it is
allowed to remain until the time comes to transfer it to
the next higher step, when the filter is drained, replaced
in the funnel and the appropriate solution allowed to
drain through it and again placed in the container. This
method, while somewhat wasteful of alcohol, permits the
handling of large groups of small objects without loss.
Another method of drawing off solution is by means of
a pipette. In this method test tubes or vials may be used
as containers, cutting down the amount of alcohol needed.
An ordinary straight glass tube or a regular pipette serves
the purpose. The disadvantage of this method is that
small objects are easily drawn into the pipette and thus
lost. To avoid this possibility the author ties a piece of
56 MICROSCOPIC TECHNIQUE
filter paper over the immersion end of the pipette. First
a small wad of cotton is stuck loosely into the pipette end,
leaving a small ball projecting from the tube. The filter
paper is then folded loosely over the end and secured with
thread. If cotton is not used the pipette end is apt to
puncture the paper, thus defeating its purpose. If diffu-
sion through the filter paper is slow it may be speeded up
by gentle suction applied to the free end of the pipette.
The period of immersion in the various alcohol steps
cannot be stated accurately. Small porous objects may be
completely permeated in less than an hour, while dense
tissues, such as liver, will require several hours for the
liquid to penetrate to the center. It is not necessary for
material to remain in each step until completely pene-
trated, since the weak alcohol in the outer layers will
penetrate to the interior while the stronger grade is en-
tering the outer layers. As a matter of fact no material
should remain longer than six hours in an alcohol solu-
tion of less than 50% as the macerating effect of a weak
solution will disintegrate the tissue. The time in alcohols
of 70%, 80% and 95% should be sufficient to insure com-
plete penetration, and may safely be extended to days.
Change the absolute alcohol at least once and prefer-
ably twice, and be sure to leave the objects until com-
pletely dehydrated. A good rule is to leave it until you
are sure every trace of water has been removed, then leave
it as long again.
The dehydrated material is now in condition to absorb
a reagent in which paraffin is soluble. This process is
called clearing because the oils or other reagents which
dissolve paraffin also tend to clear the objects and render
them somewhat transparent.
Many clearing agents have been tested, but experience
SECTION CUTTING 57
shows that toluol, a by-product of coal-tar, is the best. It
penetrates rapidly, does not harden tissues too much and
is so thin and volatile that it is quickly removed from the
paraffin. Many writers on microtechnique recommend
xylol for clearing, but this chemical frequently renders
tissues as hard as stone, making cutting difficult or impos-
sible. The student is well advised, therefore, if he uses
toluol for clearing in all paraffin work.
Mixtures of alcohol and toluol must be stored in tightly
covered containers as they quickly absorb water from the
air. For the same reason material must be handled as
rapidly as possible when changing from one solution to
the next.
The old practice of taking material out of a reagent of
one specific gravity and placing it immediately into one
of another gravity accounts for many poorly prepared mi-
croscope slides. The transfer from absolute alcohol to
toluol is a case in point. Clearing, like dehydration, must
be done gradually if the material is to remain in the best
condition. The steps need not be as numerous as those
recommended for dehydration, except in the case of ex-
tremely delicate material, three steps being sufficient for
student material.
After removing the material from the last absolute alco-
hol bath, place it in a mixture of one part toluol to three
parts absolute alcohol. Experience is the only guide to
the length of time material must remain in each bath.
As a general rule allow it to remain in each alcohol-toluol
mixture as long as it remained in each of the higher grades
of dehydrating alcohol. Next transfer the material to a
mixture of equal parts of alcohol and toluol, and, after
the necessary immersion time, transfer it to pure toluol.
Properly cleared material will present the same appear-
58 MICROSCOPIC TECHNIQUE
ance throughout. If dark spots are seen when it is held
against a light or if these spots appear white and opaque
when the specimen is held in a bright light against a dark
background, they are an indication that the clearing is not
complete and the material must be returned to pure
toluol, preferably a fresh batch, for further clearing. If
the spots still persist after prolonged immersion in the
clearer, the trouble is caused by incomplete dehydration.
This can be rectified only by replacement in absolute al-
cohol for a longer period and again clearing in three
stages.
Over-immersion in toluol does the material no harm,
so leave it until there is no question of incomplete pene-
tration. Then replace the used toluol with a fresh solu-
tion and leave it for another period of time to insure
complete removal of every trace of alcohol. Unless pene-
tration of the toluol to the very center of the material is
complete it cannot be infiltrated fully with paraffin.
Two grades of paraffin are used for sectioning. One,
soft paraffin, is suitable for imbedding materials to be cut
into rather thick sections ( 1 4 ju or over) . This has a melt-
ing point of 45 C. (115 F.) . Hard paraffin melts at
54 C. (130 F.) and is used when thin sections (12 ju
or less) are to be made.
Commercial paraffin varies considerably in quality.
Each lot must be tested for melting point and homogene-
ity before it is used. First test the melting point, which
will probably be low. Then fill a small pill box with
melted paraffin and cool it as quickly as possible in cold
water. Examine it critically and if it shows white, opaque
spots it is unfit for use.
When a satisfactory lot of paraffin is found it must be
filtered to remove foreign substances. A heated funnel
SECTION CUTTING 59
is the best way to filter paraffin. If a small quantity of
wax is heated well above its melting point it may be fil-
tered through a previously heated metal funnel before it
solidifies. The filtered paraffin may be collected in con-
tainers which have been smeared with a thin coat of
glycerin to prevent the wax adhering to them.
The grade of paraffin known as Parawax is available in
all grocery stores, and serves quite well as a soft paraffin.
The author has used it for hand-sectioning with the well
microtome with very gratifying results. Each lot must
be tested for melting point and filtered before use. This
product varies considerably in melting point so that by
testing several lots one may be found with a melting point
high enough to make it available as a hard paraffin.
The melting point of paraffin may be raised to produce
a hard paraffin by adding bleached beeswax to it. No
exact proportions can be given because of wide variations.
It is advisable to melt Parawax and then add beeswax a
little at a time. Take samples frequently and chill them
in cold water to solidify. When the samples are hard,
test for melting point. Samples of Parawax and beeswax
have been found which when combined produced a hard
paraffin melting at 60 C. (140 F.) , with good cutting
quality. When a suitable product has been secured guard
it carefully, for it is a rare treasure.
Another method of raising the melting point of paraf-
fin is given by Lee in his Vade-Mecum. " Paraffin of
about 50 C. melting point is taken and heated in a porce-
lain capsule (evaporating dish or crucible) by means of
a spirit lamp. After a time disagreeable white vapors are
given off and the mass shrinks a little. This result is ar-
rived at in from one to six hours, according to the quality
of the paraffin. The mass then becomes brownish-yellow,
6o MICROSCOPIC TECHNIQUE
and after cooling shows an unctuous or soapy surface on
being cut. The melting point will be found to have
risen several degrees."
Save all scraps of paraffin, such as unused portions of
blocks, shavings, chips and trimmings, for reheating tends
to improve the cutting quality. When a quantity has
been collected melt it, filter and determine its melting
point.
The material is now in pure toluol and ready to be in-
filtrated with paraffin. Gradually saturate the toluol with
paraffin at room temperature by adding finely shaved
paraffin to it. If the material is very fragile the paraffin
must be supported while passing into solution. This is
done by tying the paraffin up in a cheesecloth bag and sus-
pending it in the toluol by a string so that the paraffin dips
below the surface of the toluol but does not touch the
material. As the paraffin dissolves, acid more until the
liquid is saturated.
Material may be left in this mixture for days without
suffering any ill effects, and should be left for at least
twenty-four hours before going on to the next step. Now
place the container in a warm place where it will receive
very gentle heat. If a paraffin oven is available it may be
set on top of this, or it may be placed on a water bath or
sand bath that is held at a temperature lower than the
melting point of the paraffin. Add paraffin until no more
will dissolve. Allow to stand over night with the con-
tainer uncovered to permit evaporation of the toluol.
Now place the container in the paraffin oven and allow
the paraffin to melt completely, gently agitating the dish
occasionally to thoroughly mix the contents. The oven
should maintain a fairly constant temperature in the
neighborhood of 54-55 C. (130 F.) for either hard or
SECTION CUTTING 61
soft paraffin. (For description of a paraffin oven see
Chapter XL)
Penetration of the paraffin to the interior of the speci-
mens will depend upon their size and permeability.
Large pieces of fairly dense material may remain two
hours, small pieces or less dense tissues one hour, and
small, easily permeable material for twenty to thirty min-
utes. At the end of this period pour off half of the paraf-
fin-toluol mixture and make up to original volume with
pure melted paraffin, agitating the container to mix thor-
oughly. At the end of the respective periods pour off
all of this mixture and replace with pure melted par-
affin. Change the pure paraffin several times while the
material is in the oven to insure absolute elimination
of toluol.
The period of immersion in pure paraffin is again de-
pendent upon the material. Small, easily-permeable ob-
jects will require about two hours, large pieces of dense
material will require more time. Six to eight hours
should suffice to infiltrate even the most refractory sub-
stances, if the preliminary infiltration has been thorough.
Prolonged immersion extending several hours beyond the
actual time needed will do no harm if the oven is at the
proper temperature. High temperatures are injurious,
and material which has been cooked is worthless.
The infiltrated material is now ready to be imbedded
for sectioning. The objects are taken from the paraffin
bath, placed in a shallow container with enough melted
paraffin to cover them and arranged in the desired posi-
tions for cutting. Two requirements must be met in im-
bedding. First, the individual units of material must be so
arranged in the paraffin cake that they may be cut apart
and sectioned in the desired plane. Secondly, the paraf-
62 MICROSCOPIC TECHNIQUE
fin must be cooled as quickly as possible to prevent crys-
tallization.
Choice of an imbedding container may be made from
several available objects. For small objects there are
watch glasses, which may be had in a number of sizes from
laboratory supply houses. Paper trays made as shown in
Fig. 31 are inexpensive and very efficient. Select a
heavy bond paper or light cardboard and fold as indicated.
The tray has the advantage that any required data may be
written directly upon it, dispensing with separate labels.
The operation of imbedding should be done directly
in front of the paraffin oven, or close to it. A small spirit
Fig. 3 1 . Diagram of paper
imbedding tray. Fold
along lines a-a' and b-b'.
Without unfolding make
the folds A'-B', c-c', d-d'
and C'-D'. Indent and
fold along A'-A, B'-B,
C'-C and D r -D. Unfold
and refold so the area
A-B-D-C forms the bot-
tom of the tray. Fold the
ends down on the outside.
a fa
1
1
A'
i i
^r t^ ^~ i
\ |
~7
X ^
N X
,..,.. ^>V , . ,/ r
TA B 1
1
1
1
1
1
1
1
d
___ xl c___D,
X . N.
,' 1 X
c'
J
_^
1 1
1 1
1 1
a 1 b 7
lamp, two fine dissecting needles, fine-pointed curved for-
ceps, imbedding containers and a dish of cold water
should be at hand.
When all is ready smear the inside of the imbedding
container very lightly with glycerin. Only the thinnest
SECTION CUTTING 63
film is necessary and all surplus should be wiped away.
Pour into it enough paraffin to cover the material thinly.
Now, with warmed forceps quickly transfer the objects to
the dish and arrange them properly. If several objects
are being imbedded arrange individual pieces in an or-
derly progression, allowing several millimeters of paraffin
between each. This is to make it possible to separate one
unit from the others without destroying them. It is usual
to place objects so that the intended plane of section is
parallel with the bottom of the dish. This is obviously
impossible with some material, for long, thin specimens
cannot be stood on end. Handle objects in melted paraf-
fin with the greatest of care as they are very apt to crumble
or break.
Should the material consist of very small or fragile ob-
jects which cannot be individually handled, pour off most
of the melted paraffin, then quickly pour the remainder
containing the objects into a prepared dish. Arrange
them with warmed needles. Should the paraffin begin to
solidify before arrangement is complete it may be kept
fluid by warming the dish just a little by placing it on top
of the oven or on a warmed metal plate. Be extremely
careful not to get it too hot. If a film of semi-solid paraf-
fin forms on the surface warm a section lifter and pass it
lightly over the surface. The upper layers of paraffin
may be kept fluid for a few minutes in this way. Heating
of paraffin with imbedded objects is recommended only
as a last resort, for the objects sink to the bottom of the
dish, leaving their under sides unsupported, and the
heated dish retards cooling enough to permit some crys-
tallization of the paraffin.
Now take the dish in the fingers and move it carefully
to the dish of cold water where it is held almost sub-
64 MICROSCOPIC TECHNIQUE
merged. Blow on it gently to hasten cooling of the top
layers of paraffin. As soon as a thick film has formed on
the surface of the paraffin lower it slowly in the water and
hold it completely submerged until it solidifies. This
must not be done too rapidly or the rush of water over the
rim will cause the center to spurt upwards and ruin the
preparation. If a paper tray is used for imbedding it is
only necessary to float the tray on the surface of the cool-
ing water.
If the tap water is warm it is advisable to add ice to the
chilling water, for the object is to cool the paraffin as
quickly as possible to prevent crystalli/ation. When the
cake is completely cool it should float free of the imbed-
ding dish and rise to the surface of the water. If this does
not take place in half an hour run a thin blade around the
edge of the cake to loosen it. Never heat a dish to release
the block of paraffin.
Examine the paraffin blocks carefully. They should
present a homogeneous appearance, without white spots
or bubbles. If opaque areas occur around the imbedded
objects it is an indication that all the clearing agent was
not removed. The only remedy for this condition is to
put the blocks back into the oven, melt the paraffin and
keep it fluid for several hours to evaporate the clearer,
changing the paraffin once or twice, and then reimbed
the material. Objects thus imbedded may be sectioned
at once or kept in a cool place protected from dust.
When the time comes to cut sections the paraffin cake
must be separated so that each small block contains only
one object. If the objects are very small and close to-
gether several may be included in one block. To cut
objects apart make a scratch on the surface of the cake to
indicate the intended cut, then gradually deepen this un-
SECTION CUTTING 65
til the cake may be broken apart. Trim the edges square,
leaving several mm. of paraffin surrounding the object on
all sides. Be sure the intended plane of section is correct
and then true up the edges so that a true rectangle will
be presented to the knife.
If the sections are to be cut free-hand, that is, without
a microtome, it is advisable to fasten the paraffin block to
a support to afford an easy grip in the hand. Such a sup-
port is provided by a small block of wood, about three-
Fig. 32. Cutting sections free-hand with a razor.
quarters of an inch square by three inches long. Dip the
end of this into melted paraffin to coat it to a depth of
about one-eighth of an inch. This stratum of paraffin is
used to fasten the paraffin block to the support and should
be left permanently. If the student has access to a well
microtome or an automatic microtome the following in-
structions will apply with equal effect.
Place the paraffin block with the imbedded material on
the table, with the face to be cut on top. Warm a flat
66 MICROSCOPIC TECHNIQUE
metal instrument such as a scalpel handle and apply it to
the paraffin on the support block until this is slightly
melted. Remove the warm instrument and quickly press
the paraffin block into the melted paraffin. Now heat a
needle or the scalpel handle again and melt the paraffin
at the joint between the two blocks, welding them to-
gether solidly. Immerse the whole in water for several
minutes, when it will be ready for cutting. Trim the
object block to a perfect rectangle before beginning to cut
sections.
Where many sections are to be cut an automatic micro-
tome is almost necessary, but for the student who makes
only a few sections at infrequent intervals it is not essen-
tial. Section cutting free-hand is not difficult and after a
few trials very thin sections may be produced. Fig. 32
shows the method of holding the knife and the object for
free-hand sectioning. The well microtome, shown in use
in Fig. 33, is an improvement over free-hand cutting.
This instrument is excellent for student use, since it is
inexpensive and reliable. By fastening the object block
to the support block in the microtome the object is held
securely while the large table provides a solid, steady sur-
face on which the knife slides. Adjustment for section
thickness is secured by turning the screw at the bottom
of the well.
One item of great importance in successful sectioning
is the knife used. This must be stiff and very sharp.
A safety razor blade may be used if it is fastened to a
handle to stiffen the blade and hold it rigid. Blades
of the single-edge type backed with metal heavier than
the blade are stiff enough for sectioning, but the double-
edge blades are too flexible. Better than any safety
razor blade is the old-fashioned straight razor used
SECTION CUTTING
67
by barbers. This is heavy and stiff and if of good quality
will retain its edge for a long time. Give it the same care
you would your shaving razor, for the edge must be just
as keen for sectioning as it should be for a comfortable
shave, or even sharper. Keep the razor in perfect condi-
tion by honing on a good hone, and by frequent strop-
Fig. 33. Cutting sections by hand in
a well microtome. Sections imbedded
in paraffin.
ping. If any difficulty is experienced in keeping the razor
perfectly sharp, take it to a barber and have him put it
in condition. Once properly sharpened the razor may
be used for cutting many sections before sharpening will
again be necessary.
Several difficulties may be encountered in section cut"
68 MICROSCOPIC TECHNIQUE
ting, and it is well to know their cause and remedy. If
the material or sections are split or cracked in the direc-
tion of the cut, the knife may have a nick in it or there may
be hard particles in the material. Clean the knife with
xylol and try again. If the scratches disappear they were
caused by hard particles from the material or paraffin.
They may be corrected in the same way if they reappear.
If this expedient has no effect, try using another por-
tion of the knife and note results. If the cracks disappear
the knife was at fault and should be ground and honed
to clear it of nicks. If the material crumbles and tears
out of the paraffin this may be caused by insufficient infil-
tration. This is indicated by a soft, mushy consistency of
the paraffin around the object. It may be caused by in-
complete dehydration or insufficient time in the paraffin
oven. To correct it dissolve the paraffin with xylol, de-
hydrate and reimbed. If the material is hard and brittle,
the clearing agent may be at fault or the material may
have been subjected to too high a temperature in the
paraffin oven. To remedy this condition read again the
paragraphs covering these two subjects.
As the sections are cut they should be placed in a row
on a sheet of paper perfectly free from dust or lint. Ar-
range them in serial order so that a series of sections may
be placed on the slide in the order which they occupied
in the live object if desired. In this way the entire mor-
phology of the specimen may be reconstructed on the
slide.
Cut the sections with a straight motion of the knife.
Hold the microtome in the left hand, as shown in Fig. 33,
with the knife in the right hand. Pass the knife straight
through the material and paraffin with a quick, steady
motion. Do not see-saw the knife back and forth as this
SECTION CUTTING 69
is very likely to leave zig-zag lines on the section. Some
practice will be necessary, of course, before the correct
technique is mastered, but once attained, uniform results
may be secured with certainty.
PREPARING THE SLIDES The slides must be perfectly
clean and free from any trace of grease. Clean the slides
by boiling in the following mixture:
Potassium bichromate 20 g
Water 100 ccm
Cone, sulphuric acid (commercial) ... 75 ccm
Rinse the cleaned slides in water, wash with ammonia,
then distilled water and store in 90% alcohol until
needed. When they are removed from the alcohol for
use they will dry almost instantly and be perfectly clean.
Mayer's albumen fixative is needed. Prepare a slide
as directed in Chapter IV by smearing with Mayer's fixa-
tive. Place a drop of distilled water on the slide and lay
the section on it in the place it is to occupy permanently.
The section will be curled somewhat and must be flat-
tened by the application of gentle heat. Be extremely
careful not to overheat as high temperatures are sure to
distort the cells, even to the point of producing character-
istic cracks in which cell masses are pulled apart, often in
parallel lines. This distortion becomes more evident
after the paraffin is removed and the sections stained.
The process of flattening sections is called stretching,
and as a general guide it may be said that sections in soft
paraffin should be stretched at about 37C. (g8F.) ,
those in hard paraffin at 40 C. (lor/F.) . The slides may
be placed in the paraffin oven which must be adjusted to
maintain the above temperature, or a warming stage may
be made by filling a pan with sand and equipping it with
70 MICROSCOPIC TECHNIQUE
a thermometer. Bring the sand temperature up to the
required point, cover it with a sheet of glass and lay the
slides on the glass.
The length of time required to stretch sections depends
upon their thickness, smoothness and the character of the
material. Sections of cylindrical objects are especially
hard to flatten. Some sections flatten perfectly in ten min-
utes at the above temperatures while others will take an
hour or more. Particularly difficult subjects may require
heating high above an open flame, but be very careful not
to melt the paraffin. As a last resort, to be used only in
the most obstinate cases, the sections may be flattened
with a rolling motion of the little finger. Add distilled
water as required to make up that lost by evaporation.
Sections must be kept moist all the time.
When the sections are flattened, remove the slides from
the warming pan and carefully pour off any excess water,
leaving them just wet enough to prevent the sections from
sticking. If the sections have shifted their position take
a needle and carefully move them into place, touching
only the paraffin and not the material itself. Now place
the slides in the oven or on the warming pan for about
half an hour, when they may be placed in boxes for later
finishing. If they are to be finished at once they may be
dried in the oven at 37 C. (98 F.) or allowed to stand
twenty-four hours if not dried by artificial heat. Slides
thus prepared may be stored for years in tightly covered
boxes in a cool place and finished whenever needed.
Laboratory supply houses furnish special containers
for the reagents used in treating paraffin sections affixed
to slides. These are called Coplin jars, are made of glass
and are provided with vertical slots to hold the slides.
The student can get along quite well with substitutes in
SECTION CUTTING 71
the form of straight-side vials with bakelite screw caps.
These are procurable from drug stores in a size one and
one half inches in diameter by four inches high, just a
convenient size for slides. One or several slides may be
handled at the same time by making a little basket of
brass wire to hold them. Thus the slides need not be
touched and the work is reduced to a minimum.
Arrange the reagent jars as in the diagram (Fig. 34) .
In front of each row of jars place a strip of blotting paper
to be used in absorbing the excess liquid that drains from
the slides.
Fig. 34. Diagram showing arrangement of reagent jars for
handling and staining sections fixed to slides.
REMOVAL OF PARAFFIN Place the slides in the first
jar at the left, labeled, "Paraffin Xylol No. i." Leave
for two to three minutes, remove and allow to drain into
the jar, touch the ends to the blotting paper and quickly
place in the second jar labeled, "Paraffin Xylol No. 2."
They may remain in this jar for a long time without
harm, although several minutes is sufficient.
On removal of the slides from the second xylol place
them in 50-50 xylol-alcohol to begin removal of xylol.
Two or three minutes is sufficient. Transfer them
quickly to the absolute alcohol without draining or blot-
ting. At this stage and on transfer to thin celloidin, great
72 MICROSCOPIC TECHNIQUE
caution must be observed to work quickly, for the slides
must not at any time be allowed to dry. Both of these
reagents are highly volatile.
COATING WITH CELLOIDIN Transfer the slides
quickly from absolute alcohol to a thin solution (about
1%) of celloidin in 40% ether-6o% alcohol. After sev-
eral minutes lift the slides and allow them to drain thor-
oughly. Watch them closely and when the celloiclin ad-
hering to them forms a soft film, plunge them into the jar
of 70% alcohol. Lower them to the bottom of the jar
with a single motion as any hesitancy will cause a ridge
to form in the celloidin. Allow the slides to remain in
70% alcohol about three minutes.
If these directions have been followed carefully the sec-
tions should now be firmly fixed to the slides. If they
fall off or loosen and become wrinkled it is probably be-
cause the slides were not perfectly free from grease, the
sections were not perfectly flattened, or they were made
too hot during the stretching operation.
TRANSFERRING TO STAIN The sections are now ready
to be stained. If an alcoholic stain is to be used they are
transferred directly from 70% alcohol to the stain. If
a water stain is used for nuclei the slides are passed down
the series of alcohols to water as instructed in Chapter VI,
which should be consulted for staining and counterstain-
ing techniques. The student will be well advised to use
alum-haematoxylin for a nuclear stain with eosin as a
counterstain in his early efforts, and should master these
before proceeding with alcoholic stains.
CLEARING Properly stained, counterstainecl and de-
hydrated, the slides are now cleared in xylol to make the
tissues transparent. Xylol exerts a strong shrinking ac-
tion on tissues, so the sections are protected against this by
SECTION CUTTING 73
recoating with celloiclin. Transfer the slides from ab-
solute alcohol to i % celloidin in ether-alcohol and treat
exactly as described for the first celloidin coating, except
that instead of hardening the celloidin in alcohol we now
plunge the slides into a jar of chloroform where they may
remain for a minute or two, but no longer.
Transfer the slides from chloroform directly to 5050
creosote-xylol for final clearing. They should remain
until every trace of cloudiness has disappeared. When
cleared, pass them to a jar of pure xylol and leave for three
to five minutes. Longer immersion is not advisable be-
cause of the shrinking and hardening effect of the xylol.
MOUNTING Canada balsam is used almost exclu-
sively for mounting paraffin sections. Have at hand a
supply of clean cover glasses, a bottle of balsam, line-
pointed forceps, and a small spirit lamp.
Take a slide from the jar of xylol, drain thoroughly
and with a soft cloth wipe the back and front of the slide
clean, up to the section. Hold the slide in the left hand,
place a drop of balsam on the section and with the forceps
in the right hand pick up a clean cover glass. Warm this
over the flame and lower it quickly into place. Properly
done there should be no air bubbles, but if there are small
ones they will probably work out as the slide dries. Large
ones, however, must be treated by dissolving off the cover
with xylol and setting a new cover.
SECTION CUTTING BY FREEZING When sections are
required in a hurry and time does not permit paraffin
imbedding, satisfactory sections may be cut from pieces of
fro/en tissue. While not generally suited to the cutting
of sections intended for critical study, the method is still
of considerable value for diagnostic, identification and
demonstration purposes. Fresh tissue may be frozen and
74 MICROSCOPIC TECHNIQUE
sectioned without any previovis treatment although this
is not the best practice. Whenever time is available the
material should first be fixed in a 10% solution of for-
malin in water or physiological salt solution. If the pieces
are cut rather thin, fixation will be complete in ten to
twelve hours with most materials. Most of the routine
staining techniques may be used with entire success on
formalin-fixed material. If the sections have been cut
from unfixed material the best practice is to place them
in 10% formalin at room temperature for ten to fifteen
minutes before staining. When freezing slices of material
which have been fixed in formalin, wash them for several
hours in running water before cutting sections. If sec-
tions are required from material preserved in alcohol,
run the material down the alcohol series to water before
freezing.
Fixed and washed material may be frozen and sec-
tioned "as is/' but better sections will usually result if
it is infiltrated with a gum and syrup mass. This sup-
ports the various elements and freezes without the forma-
tion of ice crystals that might otherwise injure delicate
tissues.
To make the infiltrating mass take:
Gum acacia (gum arable) 40 g
Water 60 ccm
Carbolic acid crystals 0.5 g
Dissolve the gum in the water and add the car-
bolic acid. To this solution add 40 ccm of syrup
made by saturating water at room temperature with
cane sugar. Do not heat the mixture.
Transfer the thin slices from water to this solution and
leave until thoroughly infiltrated. This may take twenty-
four hours, or the material may be left for days without
SECTION CUTTING 75
harm. The prepared material is placed on the object
carrier of the microtome with a few drops of water and
frozen.
The usual method employed in laboratories is to
freeze the material in special apparatus using liquid car-
bonic acid gas. The average student will not have such
equipment at his disposal, so he must resort to other
methods. Two alternatives are available, one in which
solid carbon dioxide or dry ice is used, the other by the
use of ethyl chloride. The worker will have to select the
method best suited to his conditions.
Dry ice may be purchased in small quantities from
large dairies or ice cream manufacturers. A cube meas-
uring one inch square is ample for freezing several pieces
of tissue. Place the dry ice in a box made of corrugated
cardboard and lay the tissue directly on the ice. Then
cover the box with another piece of board and leave until
frozen. Cut it free from the ice and place it on the ob-
ject carrier of the microtome, which has been prepared
by placing a few drops of water on it. Now lay the dry
ice on top of the tissue and leave for a few minutes until
thoroughly frozen. Remove the ice and cut sections with
a chilled knife.
CAUTION: When handling dry ice be very careful not
to allow it to come into contact with the skin. Al-
ways handle it with forceps as the extremely low
temperature can and does cause painful burns.
The ethyl chloride method is perhaps slightly more
convenient to use, since the freezing agent is more readily
available. Ethyl chloride may be procured from any
druggist in 50 g and 100 g tubes, costing about two dol-
lars for the larger size. It comes in liquid form in glass
tubes fitted with a release valve which liberates the liquid.
76 MICROSCOPIC TECHNIQUE
The chemical works on the principle of lowering the
temperature by rapid evaporation. It is so highly volatile
and evaporates at such a rate that by this evaporation it
extracts heat so rapidly that it freezes the tissue with
which it comes in contact.
To use ethyl chloride for freezing, place a few drops of
water on the object carrier of the microtome and freeze
this by directing a stream of ethyl chloride against it from
the nozzle on the tube. Quickly place the tissue on the
object carrier and freeze in the same manner. When
frozen cut sections in the usual way as rapidly as possible.
When sectioning any frozen material, work rapidly and
keep the knife chilled with ice. Dip the knife in ice water
before each cut, for unless the knife is kept cold the sec-
tions will stick to the blade. If the work is being done
on a rotary or a sliding microtome, a piece of ice may be
placed on the back of the knife, where by melting it will
soon shape itself to the blade and remain in place. This
is obviously impossible when using a well microtome, so
the knife must be kept cold by dipping it in ice water.
If the tissue has been frozen too hard the sections will
crack or roll. In this case wait a short time and try again.
When the consistency reaches the point where perfect sec-
tions may be cut, work rapidly and cut as many sections as
are needed. If the material becomes too soft before the
required number of sections are cut, freeze it again and
continue the work.
HANDLING LOOSE SECTIONS Sections cut from frozen
material, or free-hand sections of hard substances, are
called loose sections, as distinguished from paraffin sec-
tions affixed to slides for processing. These are some-
times handled through the operations of staining and
mounting simply by transferring them from one reagent
SECTION CUTTING 77
to the next until xylol is reached, when they are mounted
in balsam. Better results are secured by fastening the
sections to slides by Wright's method, which permits more
convenient handling and prevents loss of loose portions.
WRIGHT'S METHOD Change the water on the sec-
tions several times to wash out all gum. Select a good
section and pass a slide under it in the containing water.
Lift the slide carefully so the section is carried with it,
holding it in place with a small brush if necessary. See
that the section lies perfectly flat on the slide. Drain off
superfluous water, being careful that the section does not
move, and blot off the surplus water up to the section
with filter paper. With a pipette flood the section and
the slide immediately around the section with absolute
alcohol, but do this gently to avoid moving the section.
If it should move be sure that it lies perfectly flat and is
free from folds before continuing to the next step. Allow
the absolute alcohol to remain a few seconds, then drain
the slide and immediately place a few drops of 1% cel-
loidin on the section. Tilt the slide several times to dis-
tribute the celloidin, then raise to a vertical position and
blot off the excess solution with filter paper, leaving a thin
film on the section and slide. During this manipulation
be sure the section has not slipped or folded. As soon as
the celloidin film begins to set, plunge the slide in 70%
alcohol to harden the celloidin.
The sections are now firmly attached to the slide and
may be treated as instructed for paraffin sections. All
operations are identical with that schedule, except for de-
hydration in absolute alcohol. This reagent is a solvent
of celloidin, hence would dissolve away the film that
holds the sections to the slides. In place of alcohol we
make use of the dehydrating property of creosote-xylol
78 MICROSCOPIC TECHNIQUE
by transferring the slides from 95% alcohol (in which
they should remain no longer than five minutes) to equal
parts of creosote and xylol. Change this solution after
every trace of cloudiness disappears from the slide, then
allow it to remain in a second change of creosote-xylol
twice as long as it remained in the first solution. When
clearing and dehydration are complete mount in balsam.
CHAPTER VI
Staining
The primary purpose of preparing materials for the
microscope is to study their various elements. The
greater proportion of microscopic elements are colorless
or nearly so, and, in order to increase their visibility or
to distinguish certain elements from others, the material
is subjected to various dyes or color-bearing chemicals
which impart color to parts or all of the specimen. Cer-
tain cellular and intercellular substances absorb these
stains in varying degrees. Some parts will be strongly
colored while others retain little or no stain, thereby af-
fording an opportunity to differentiate sharply between
various elements. Some stains are designated as nuclear
stains because they possess an affinity for the nuclei of
cells, coloring them deeply, while the cytoplasm is colored
lightly or not at all. Other stains fall into the group
known as general or plasma stains. These impart a color
to the cell plasm, affording us an opportunity to stain the
nucleus with one color and the plasm with a contrasting
color, thus presenting details with maximum contrast.
The most widely used stain for most sections and for
some whole material is haematoxylin, a dye prepared
from logwood. The active principle is haematin, a com-
plex compound of carbon, hydrogen and oxygen. When
combined with salts of aluminum, iron and some other
metals that act as mordants, this dye produces lakes of a
deep blue or black color in the nuclei of cells, imparting
to them a strong, precise, permanent color.
79
8o MICROSCOPIC TECHNIQUE
Haematoxylin alone has almost no staining power but
when dissolved in water it oxidizes to form haematin, the
staining agent. This oxidation process is referred to as
ripening. A solution of haematoxylin in which oxida-
tion has progressed to the stage where it produces only a
weak stain is called unripe; one in which oxidation has
progressed to the point where it produces a sharp, clearly
defined stain, ripe, and an old solution that produces a
diffused muddy stain because of over-oxidation, over-
ripe. It will be seen that the oxidation process is pro-
gressive, resulting finally in a worthless solution. A well-
ripened but not over-ripe solution is essential to the
production of the well-defined differential stain neces-
sary for critical microscopic study and photography.
If the solution is kept in a loosely covered container
natural ripening takes place in from three to six weeks,
depending upon the temperature. Oxidation is slow and
the solution remains in good condition for from six weeks
to three months if it contains only haematoxylin, or for
many months if it is made up with alum according to the
formula given here.
Haematoxylin may be ripened artificially by adding an
oxidizing agent such as hydrogen peroxide. Several in-
termediate compounds seem to result from this addition,
however, rendering the solution unstable so that it be-
comes over-ripe in a short time. Generally, natural
ripening is to be preferred.
The next group of stains contains carmine. This is
a deep red dye made from cochineal insects. It, like
haematoxylin, forms colored lakes when combined with
suitable mordants. These stain the nuclei a brilliant red
or purple-red. Structures thus stained are brilliantly
differentiated, yet quite transparent. For this reason car-
STAINING 81
mine is the favorite stain for objects to be stained and
mounted whole, such as aquatic forms, small insects and
their larvae, flukes, tapeworms, etc.
The third group of stains consists of the aniline dyes
in a large number of brilliant colors, of which compara-
tively few are of any use in microscopy. Safranin, for
example, is a brilliant and powerful stain for nuclei, while
eosin is the most precise and delicate stain yet discovered
for cytoplasm, connective tissue and cell membrane.
Staining is a large field embracing many combinations
of methods which it is impossible to cover completely in
a work of this size. For this reason only general staining
methods will be considered, with outlines complete and
fully applicable to a large part of the material encountered
by the student. For more detailed and amplified instruc-
tions one of the treatises mentioned in the bibliography
should be consulted.
When working with histological material a nuclear
stain is nearly always used first. Alum-haematoxylin is
usually used, although some of the anilines give excellent
stains for some material. Iroii-haematoxylin is the most
valuable stain for cytological preparations. Objects to be
mounted entire are usually stained with carmine, with in-
dulin as a counter-stain.
Since certain stains exhibit a selective affinity for some
classes of tissue, it is possible, by a proper selection of
stains, to color each class of tissue differently. Thus a
chromatin stain may be selected for the nucleus, a plasma
stain for the cytoplasm. Thus treated, the material will
reveal the other tissues of which it is composed and ap-
propriate stains may then be selected to differentiate the
particular tissues to be studied.
The material to be studied, either sections or whole
82 MICROSCOPIC TECHNIQUE
objects, is gradually brought from the preserving fluid
into the solvent used for the nuclear stain. If a water
solution is used and the material has been preserved in
70% alcohol it is passed down through the descending
series of alcohols until water is reached. If an alcoholic
stain is used it is passed up or
down the series as far as neces-
sary, or until that concentration
of alcohol is reached which
equals the concentration of the
stain.
The material is now placed in
the nuclear stain, filtered before
use, where it remains until ex-
amination shows the nuclei to be
deeply colored. Two methods
are in use, one known as pro-
gressive staining, in which the
material is stained in a dilute
solution of the dye, the other
called regressive staining, in
which the material is first stained
toxylin, with indulin deeply in a strong solution and
as a counter stain. then destained in another re-
X 5 agent which extracts the excess
stain. A more precise color differentiation usually re-
sults from the regressive method.
When staining has progressed far enough to insure
complete impregnation, the object is transferred to a
washing medium to remove adhering excess stain. This
will be water or alcohol, depending upon the stain used;
water for aqueous stains, alcohol for alcoholic stains.
The objects are now counter-stained by bringing them
Fig. 35. Female Cy-
clops. An aquatic ani-
mal that can be reared
in an aquarium. Fixed
in Bouin's, stained in
standard alum-haema-
STAINING 83
up or down the alcohol series to match the concentration
of the solvent of the counter-stain. They remain in this
for a few minutes and are then rinsed a moment in water
or alcohol as required by the counter-stain.
Dehydration of the material is accomplished by bring-
ing it into absolute alcohol by graduated stages. If the
counter-stain is one which washes out easily, dehydration
must be effected as quickly as possible to retain the stain.
The dehydrated material is now ready for clearing
and mounting. For details, see Chapter VIII.
The following formula for alum-haematoxylin is taken
from Galigher. It is called by him " Standard Alum-
Haernatoxylin " and is a modification of Harris's formula.
Galigher says of it, " it is a thoroughly satisfactory nuclear
stain for general histological purposes."
STANDARD ALUM-HAEMATOXYLIN
Haematoxylin, white crystals 0.5 g
Aluminum ammonium sulphate 0.3 g
Alcohol (50%) 100 ccm
Mercuric oxide (red) 0.6 g
Dissolve the haematoxylin and alum with the aid
of heat. When solution begins to boil add mercu-
ric oxide and boil for twenty minutes. Then add
enough 50% alcohol to make up the original vol-
ume. Allow to cool and stand over night. Then
filter through two thicknesses of filter paper and
stopper tightly.
This stain is ripened by the addition of mercuric oxide
and is ready for use at once. Its staining power will in-
crease slowly for a month or more. If properly stored, it
will keep six months to a year.
When using this stain for sections the progressive proc-
ess is preferable. The time and effort involved are re-
MICROSCOPIC TECHNIQUE
Fig. 36. Mitosis in onion root tip. Alum-haematoxylin
stain to show chromatic figures and nucleus. No counter
stain. Xgoo
duced to a minimum, the depth of this stain is easy to
control and the results usually equal those of regressive
staining. Small and easily permeable whole organisms
may be treated in the same way. The following direc-
tions apply to either class of material.
The solution used in progressive staining is made by
diluting 12 ccm of standard alum-haematoxylin with
88 ccm of saturated solution of ammonia alum in distilled
water. Allow the mixture to stand three or four weeks
before using it. After this period of oxidation the stain-
ing power remains nearly constant for six to eight months.
i. Pass the material down the descending series of al-
STAINING 85
cohols to water. Wash in water two or three minutes. If
the material has been fixed to slides, such as paraffin sec-
tions, aquatic organisms or minute insects, set the slides
in a pan through which water is running. Loose sections
or free organisms may be handled by simply changing the
water several times.
2. Transfer to staining solution and allow to remain
about twenty minutes. Remove a piece of the material,
rinse in water and examine under the microscope. If the
nuclei are strongly stained, with the plasm colored slightly
or not at all, remove the remainder of the material and
wash for fifteen minutes in running water as in step i.
If the stain is weak, replace the test piece in the stain and
at the expiration of ten to fifteen minutes examine again.
If a satisfactory stain is not achieved in forty-five min-
utes the solution is too weak or the material too imper-
meable. The stain may be tested for strength by trying
another piece of material. If this test proves positive,
stain the material by the regressive method; if negative
add a few drops of standard alum-haematoxylin and im-
merse the material again.
The final washing in tap water serves a double purpose.
First, it removes the last traces of alum which if left in
the material would soon cause the stain to fade; secondly,
the slight alkalinity of the water turns the stain blue.
3. Dehydrate as described earlier up to 80% alcohol.
To the 95% alcohol arid the first wash of absolute alcohol
add enough eosin to color the liquid a deep pink. Leave
until thoroughly counter-stained, wash quickly in abso-
lute alcohol and place in the clearer.
To prepare standard alum-haematoxylin for regressive
staining add one drop of concentrated hydrochloric acid
to each 100 can of stain. Filter the solution before use.
86
MICROSCOPIC TECHNIQUE
1. Pass down through alcohols to water as in step i.
above.
2. Place in concentrated stain for twenty minutes.
3. Rinse material in distilled water and place in 0.5%
Fig- 37- Cross section through eye of rat.
Stained with borax-carmine, whole. Photo-
graphed for contrast with the background.
X 5 o
solution of hydrochloric acid to remove excess stain.
Watch material carefully and examine under the micro-
scope frequently to ascertain the exact amount of stain
remaining. Loose objects or sections may be placed in a
watch glass on the microscope stage and observed through-
out the process. When sufficiently destaincd transfer the
material to tap water and wash thoroughly.
4. Dehydrate, clear and mount in balsam.
The carmine stains may be used after a large variety of
fixing agents. Material fixed in Bouin's fluid or any of
STAINING 87
the picric acid fixing agents stains beautifully with car-
mine, as does material fixed in alcohol and formalin.
Grenadier's alcoholic borax-carmine yields beautiful
stains when used according to Lynch's method. For
whole objects such as small Crustacea, Daphnia, some
Polyzoa, Hydra, tunicate larvae, small Insecta, small
flukes and the like it gives excellent results.
GRENACHER'S ALCOHOLIC BORAX-CARMINE
Carmine 3 g
Borax 4 g
Distilled water 100 ccm
Boil until carmine is dissolved, or better still,
allow to stand until this takes place. Then add:
Alcohol (70%) 100 ccm
Allow mixture to stand several days and filter.
The procedure is:
1. Transfer material from 50% alcohol to stain and
allow it to stand over night.
2. Cautiously add concentrated hydrochloric acid drop
by drop, gently agitating the container, until all carmine is
precipitated as a heavy, brick-red substance. After stand-
ing a short time the supernatant liquid must be a trans-
parent red containing no trace of the deep translucent
solution originally used. Allow to stand over night.
3. Add an equal volume of 3% hydrochloric acid in
70% alcohol to the solution in the container and agitate
to mix the contents thoroughly. Allow material to settle
and then decant or pipette off the carmine. Most of the
precipitated carmine will remain in suspension and may
be easily withdrawn. Examine the withdrawn liquid
carefully for organisms that might have been drawn out
88 MICROSCOPIC TECHNIQUE
with it. If any amount of desired material is present
place the liquid in a shallow dish, from which the wanted
material may later be reclaimed. If no material is present
discard the liquid. Refill the container with acid alcohol
and allow it to settle, after which it must be drawn off
again. Repeat the washing with acid alcohol until all
precipitated carmine has been removed from the material.
4. Place the material in a watch glass with sufficient
acid alcohol to cover it well, and observe under the micro-
scope. The acid will slowly extract the stain and must
be replaced with fresh solution as often as it becomes
deeply colored. The destaining operation should extract
most of the stain from cytoplasm and leave the eggs, gland
cells, digestive tract, trachea, etc. deeply colored. Do not
overdo the destaining or the residual tint will be too weak.
When destaining seems to have progressed far enough,
pick out several organisms, place them in absolute alcohol
for a few minutes, clear in creosote and examine carefully
under a cover glass. In this way the staining of the bulk
of material may be carefully controlled to give exactly
the stain desired.
5. When properly differentiated, draw off the acid al-
cohol and replace with 80% neutral alcohol. Change
this several times, depending upon the size and quantity
of the specimens, in order to remove all traces of acid.
Allow material to remain in last neutral 80% alcohol at
least one hour.
6. Dehydrate, clear, infiltrate with balsam and mount.
When a solution composed of carmine, picric acid and
ammonia is evaporated to dryness there results a reddish
brown substance called picro-carmine, which is somewhat
soluble in water. This solution gives a beautiful trans-
parent stain to chromatin and is excellent for large flukes,
STAINING 89
proglottids of tapeworms, and other forms in which the
spaces between the organs consist of a dense parenchyma.
It is equally well adapted to staining many aquatic forms,
such as Paramecia, Volvox, Rotifera, Daphnia, Cyclops,
etc., differentiating the organs with great clearness and
precision.
RANVIER'S PICRO-CARMINE To a saturated solu-
tion of picric acid in water add a strong solution of
carmine in ammonia until a precipitate begins to
form. Add a small crystal of phenol to prevent de-
velopment of fungi and let the solution stand in an
open jar until evaporated to one fourth its original
volume. Filter to remove precipitate and allow
filtrate to evaporate to dryness. To use for stain-
ing make a saturated solution of the residue in dis-
tilled water and add a trace of phenol or salicylic
acid.
To stain with picro-cannine pass the material down the
alcohols to water, then transfer to the stain, which should
be several times the volume of the material. Small
aquatic forms should remain several days, larger forms
a correspondingly longer period. When examination
shows a satisfactory stain, wash the material in several
changes of water, then up-grade to 70% alcohol. Change
this several times at one-day intervals to remove the picric
acid. Destain in 70% acid alcohol (0.5% to 1.0%) and
examine frequently as directed under carmine staining.
Destaining should be carried on until most of the color
has been extracted from all structures except nuclei and
the denser varieties of cytoplasm.
Dehydrate, counter-stain, clear and mount in balsam.
As has been mentioned, many of the aniline dyes are
used both as nuclear and general stains, but their use in
go MICROSCOPIC TECHNIQUE
the hands of the student is not likely to produce as satisfac-
tory results as haematoxylin or carmine. Many of these
dyes depend upon a delicate hydrogen ion concentration
which is difficult or impossible to secure without a well-
equipped laboratory. Hence only a few of the anilines
will be considered. All of those mentioned have been
used by the writer and worked out satisfactorily.
EOSIN This is one of a class of acid aniline dyes that
is used quite generally as a counter-stain with haema-
Fig. 38. Embryonic seeds in ovary ot
hyacinth. Alum-haematoxylin for nu-
cleae, with eosin for plasm. X25
toxylin. It imparts a strong transparent color to cyto-
plasm, muscle fibers and intercellular structures, giving
a valuable contrast with blue nuclear stain. When
mounted in neutral balsam eosin is quite permanent.
The variety of chemical known as Eosin Y is a very
good stain, easily soluble in water or alcohol. It is ad-
visable to use an alcoholic solution because the aqueous
STAINING 91
solution stains slowly, gives a rather diffuse stain and
washes out easily. It is somewhat dependent upon the
pH concentration for best results. A 0.5% solution in
90% alcohol is adjusted to a hydrogen ion concentration
of 5.4-5.6 by adding 3.2 ccm of .1 N hydrochloric acid
per hundred ccm of dye solution. This should result in
the solution which will give the best results.
Eosin may be used following formalin or Bouin's fluid
for fixing, the latter giving better preservation and ex-
cellent staining.
INDULIN A blue dye which provides a beautiful
counter-stain for whole objects stained with carmine.
Fig. 39. Spiral vessels from stem oi : rhubarb. Mal-
achite green stain. Mounted in glycerine jelly.
Xioo
The product marketed by Griibler under the name
"Indulin Griinlich, nach Rawitz " is the only really de-
pendable one. American-made indulins have proved un-
trustworthy. It is a valuable stain for bringing out clearly
such appendages as cilia, bristles, spines, flagella, etc.
Once in the tissues, this dye is difficult to remove, hence
92 MICROSCOPIC TECHNIQUE
it is used as a progressive stain in very dilute solution.
Prepare for stock a 0.16% solution of the dye in 90%
alcohol. When ready to use it, add enough stock solution
to the 90% dehydrating alcohol to color it lightly. Im-
merse the material and examine it frequently, as the stain
acts quickly. When the desired depth of color is at-
tained, transfer the material immediately to clear 90%
alcohol. Be sure the alcohol used is neutral or slightly
acid, never alkaline, as the slightest trace of alkali will
prevent staining or produce a degraded color.
MALACHITE GREEN A deep green aniline dye, solu-
ble in water or alcohol. It is useful for staining vegetable
sections or teased material, aquatic forms, insects and
larvae.
To prepare the stock solution take:
Malachite green 2 g
Distilled water 90 ccm
Glycerin 30 ccm
Mix water and glycerin and dissolve the dye.
To use add enough stock solution to distilled water to
color it strongly, and filter. Immerse the material until
stained. Destain in 50% alcohol acidulated with 0.5%
acetic acid.
Soluble aniline blue in a 2% aqueous solution, filtered,
may be used for leaf hairs, epidermis, vegetable sections,
etc., in the same way as malachite green.
CHAPTER VII
Preparing and Mounting Hard Objects
When hard animal structures such as bone, teeth, horn,
nails, etc. are to be studied, methods different from any
thus far discussed must be used to secure thin sections.
Minerals, too, require a different technique, as will be
described later.
Since bone is easily procurable and a very interesting
object for the microscope, especially the polarizer, let us
first examine the methods of sectioning it for study.
Two methods of preparation are in use, one a grinding
process in which the natural bone is reduced to a thin
section in the dry state, the other a decalcifying process
in which a reagent is used to soften the bone to the point
where sections may be cut with a knife. Both methods
have their adherents, so it will be well to describe both.
GRINDING SECTIONS OF BONE Use only fresh bone
that has been placed in water as soon as the surrounding
soft parts have been removed. Bone that has been per-
mitted to dry naturally does not give a true view of its
character because of infiltration of fat from the medullary
canal which takes place as fast as the water evaporates.
The same is true of bones that have been boiled. There-
fore, procure perfectly fresh material and immerse it in
water as soon as possible. Cut the bone while wet into
pieces as thin as possible, using a hack saw and the finest
toothed blade procurable. Cut both transverse and longi-
tudinal sections and immerse them in water, where they
remain until all soft tissue has unquestionably dissolved
out or corroded away. No other reagent need be used,
93
94 MICROSCOPIC TECHNIQUE
but plenty of time, several months, is required to secure
proper cleaning. Change the water from time to time,
shake the contents occasionally and, when all soft parts
have been destroyed, wash the bone thoroughly with clear
water and allow it to dry.
Take the section of dried bone, place it on a fine-cut
file and rub it back and forth, first on one side, then on
the other, until it is quite thin. Change to very fine
emery or garnet paper and continue grinding until the
section seems to be so thin that it will transmit light.
Examine it under the microscope and you will be sur-
prised to find that it does not transmit sufficient light to
reveal details, but is merely translucent. Now transfer
the section to a stone hone and continue grinding until
the section is extremely thin and transparent when ex-
amined with the microscope. Do not press hard while
grinding for the pressure cannot be distributed equally
and some spots will be ground thinner than others. Fur-
thermore, it is very easy to break the thin sections. Try
to keep the surface level by uniform grinding. Bone
grinding is a slow, tedious process, but careful work is
certainly justified by the beauty of the specimen when
well prepared.
When the section is thin enough, put it into a test tube
or shallow vessel and wash with a gentle stream of water
from a pipette to remove bits of ground bone. When
clean, place it in absolute alcohol for a few minutes, then
lay it between two slides and let it dry thoroughly.
Take a small quantity of regular balsam and drive off
a good part of the solvent with gentle heat, until a drop
of it solidifies as soon as it cools.
Put a small piece of this hard balsam on a slide and
another piece on a cover glass. Warm both of these until
PREPARING AND MOUNTING HARD OBJECTS 95
the balsam melts and spreads slightly, then allow to cool
until solidification takes place. Place the ground bone
section on the balsam on the slide, lay the cover glass on
the section, balsam side down, and warm the slide very
carefully until the balsam begins to melt. Quickly press
the cover clown on the section with some soft, blunt in-
strument, to spread the balsam to the edges of the cover.
If properly done the balsam will infiltrate the bone ma-
trix and render it transparent, while the Haversian canals,
canaliculi, and lacunae will be left filled with air. Be-
cause of the difference in refractive index these will seem
black against the white background of the bone, thus
making greater contrast. If the balsam is too warm it will
enter the cavities and make them transparent, thereby de-
stroying the structure of the bone.
Decalcified osseous tissue may be cut in the microtome
like paraffin sections, and if properly done serves the same
purpose as ground sections without entailing the tedious
grinding of the dry process.
Hydrochloric acid is the most widely used decalcifying
agent. It is not the best to use because it makes tissues
swell badly. Nitric acid does not do this. However,
nitric acid in strong solutions exerts a powerful gelatin-
izing action on bones, which may fortunately be con-
trolled by the addition of alcohol. Busch (Lee's Vade-
Mecum) recommends the use of one part of concentrated
nitric acid in ten parts of water. Fresh bones are placed in
95% alcohol for several clays and then transferred to
the nitric acid, which is changed every day for eight or
ten days. The specimens must be removed as soon as they
are decalcified or they will turn yellow.
Another very excellent decalcifying reagent is 3%
nitric acid in 70% alcohol. Bones should be softened in
MICROSCOPIC TECHNIQUE
Fig. 40. Forming bone in the foot of a rat.
Alum-haematoxylin and eosin. Decalcified
for cutting sections. Approximately Xioo
this mixture for from several days to a week or more, de-
pending upon their size. The liquid must be changed
every other day. As soon as a needle can be thrust into
the bone it is soft enough to cut. Remove from the acid
alcohol, wash in 95% alcohol and place in 95% alcohol
containing an excess of precipitated chalk to free the ma-
terial from all traces of acid. Wash until the alcohol fails
to give an acid reaction to litmus paper. Place in abso-
lute alcohol to dehydrate, clear in toluol and infiltrate
with hard paraffin. Sections may be cut in the same man-
ner as paraffin sections, and may be stained in the same
way.
Transverse and longitudinal sections of teeth make
very interesting slides, but the difficulties in preparing
PREPARING AND MOUNTING HARD OBJECTS 97
them are great. The student should know how it is done
even though he does not make actual sections. Thin
slices are cut from the whole tooth with an emery saw.
This consists of a thin piece of flat sheet metal, such as
a strip of clock spring, or even a piece of tin. The cut-
ting is done by flooding the work with water in which is
suspended powdered emery. First make a scratch on the
tooth's surface with a metal-cutting saw to afford a starting
place for the cut, then start the emery saw, using it ex-
actly as you would any saw. Using plenty of water and
emery, work the metal strip back and forth and in a short
time the cut will be made. The particles of emery take
the place of saw teeth and cut rapidly. New emery must
be added as rapidly as it is used up. If only one section
is to be made from a tooth, considerable time may be
saved by grinding the tooth down on an emery wheel
until the place is reached where the section is to be made.
Then only one cut with the emery saw is required.
The section is now reduced to the requisite thinness by
rubbing between two pieces of plate glass. If the sawed
section is thick and requires considerable grinding to re-
duce it, emery powder and water are used between the
plates. If the sawed section is thin, use finer powder.
When reduced to comparative thinness, wash the plates
and the section with water, substitute pumice powder for
the emery and continue grinding. When reduced to al-
most the proper thinness, again wash plates and section
in water, then continue the grinding with rotten stone.
Examine the section from time to time and when the nu-
merous minute scratches have disappeared, wash again
and replace the rotten stone with precipitated chalk and
water. When examination shows that the section is thin
enough to reveal all details and all scratches have been
98 MICROSCOPIC TECHNIQUE
polished out, remove to 95% alcohol, then to absolute
alcohol to dehydrate, when it is ready for mounting.
Sections of teeth seem to suffer some loss of fine detail
when mounted in balsam, so they are usually mounted
dry, with gum acacia. Make a solution of the gum in
water by dissolving 5 g of the dry powder in 100 ccm of
distilled water. To this add 6 drops of glycerin and 25
ccm. of 95% alcohol.
Fig. 41. Turntable for ringing slides and turn-
ing cells.
Spin a ring of gold size on the slide in the turntable.
When this is dry, paint a thin film of gum water on the
slide inside the ring and allow this to dry thoroughly in
a place free of dust. A desiccator is a very good place to
dry slides. When perfectly dry, take the section of tooth
out of the 95% alcohol, place in absolute alcohol for
several minutes, and permit the alcohol to evaporate.
Breathe lightly on the gum film on the slide, quickly set
the section in place in the center of the ring, and press
into contact. The condensed moisture from the breath
will soften the gum enough to make it adhesive and the
section of tooth will adhere to the slide. Set in the des-
iccator until dry, spin another ring of gold size over the
first one and quickly set the dry cover glass.
PREPARING AND MOUNTING HARD OBJECTS 99
Fig. 42. Section through coal. Ground section,
showing original cell laminae. X5o
Coal, being of vegetable origin, affords opportunity
for the preparation of very instructive slides. Not all
samples of coal will reveal the woody character, but some
pieces may be found to display a most interesting struc-
ture. Generally the softer grades of anthracite or the
harder bituminous coals afford the best material. Sec-
tions may be procured either by grinding or by cutting.
GRINDING SECTIONS OF COAL To grind sections of
coal first cut thin slices with a metal-cutting saw or the
emery saw. Then make one side of the section smooth
by rubbing first on a file, then emery paper, then crocus
paper laid on a piece of plate glass. Use the finest grade
of paper available in the last stage of paper grinding.
Now transfer the section to a hone, using plenty of water,
and grind down on this until perfectly smooth and free
from scratches when examined with the microscope.
Scratches that are invisible to the unaided eye will show
ioo MICROSCOPIC TECHNIQUE
up like deep valleys under the microscope. When the
section is perfectly smooth, it must be polished by rub-
bing on a dry piece of fine linen stretched taut on a piece
of plate glass, using jeweler's polishing rouge as the agent.
The polished section is placed on a slide, polished side
down, by putting a drop of hard balsam on the slide,
warming until the balsam melts and spreads, and pressing
the section into contact. When cold, the section must be
firmly attached to the slide at every point or it will break
out at the free places when it is reduced to the required
thinness.
Because of its color, coal must be ground thinner than
light-colored objects, to reveal its structure. If the sec-
tion attached to the slide is not very thin, it may be re-
duced quickly by filing. If already thin, proceed at once
to the fine emery paper and crocus paper, grinding the
free side of the coal. As the section becomes thinner ex-
amine it frequently with the microscope until the details
begin to appear. Then transfer the slide to the hone and
work it smooth, using plenty of water. Keep honing
until the section is reduced to such a thickness that the
details are clearly discerned. Now transfer to the polish-
ing cloth and polish the specimen. Work slowly to avoid
heating the slide enough to soften the balsam and dislodge
the section. When polished, transfer to clear water and
wash thoroughly to remove all traces of abrasive. Dry
the slide and section thoroughly in the desiccator or oven.
When perfectly dry, acid a drop of xylol-balsam and a
cover glass to protect the specimen.
The foregoing method of grinding hard specimens ap-
plies to all minerals and such animal subjects as Echino-
dermata, shells of Mollusca, corals, fossil wood, etc.
CUTTING SECTIONS OF COAL " The Micrograph ic
PREPARING AND MOUNTING HARD OBJECTS 101
Dictionary " recommends a process by which coal is ren-
dered sufficiently soft to permit section cutting with a
razor. The author has had no experience with this
method, which is somewhat vague in that the strength of
the macerating fluid is not given. If the student wishes,
however, he may start experimenting with a 25% solu-
tion. If this is too energetic, or too weak, alterations in
strength will be indicated and the solution may then be
adjusted either way to give the desired results.
The method given advises macerating the coal for a
week to ten days in a solution of potassium carbonate. At
the end of that time it should be possible to cut moder-
ately thin sections with a razor. These are then placed in
strong nitric acid, covered and heated gently. They soon
turn brownish, then yellow, when the reaction must be
stopped immediately by throwing the whole into a large
volume of clear water, or the coal will be dissolved. The
sections should be dark amber and very transparent, and
should exhibit the structure clearly. Sections thus pre-
pared are best mounted in glycerin jelly, as balsam seerns
to make them more or less opaque.
This opacity resulting from balsam mounting is prob-
ably due to water of inclusion. The remedy may be
found in complete dehydration in absolute alcohol, fol-
lowed by clearing in toluol and mounting in balsam. As
previously noted, balsam is the best mounting medium
for most objects and every effort should be made to bring
them into condition where it can be used. Absolute dry-
ness is essential, for the least trace of water will cause the
balsam to become cloudy and the specimen more or less
opaque.
Friable objects such as calcareous shells should be in-
filtrated with balsam before grinding. Saw thin sections
102 MICROSCOPIC TECHNIQUE
of the material and immerse them in xylol for a week to
insure complete penetration of the solvent. Then place
in thin balsam and allow this to evaporate until it is quite
thick. Place on a slide and warm gently to drive off the
remaining xylol and harden the balsam. When hard,
grind and polish the exposed face of the section. Wash
thoroughly, dry and warm the slide just enough to allow
removal of the section. Place the polished face in contact
with the slide and fasten with balsam. Now grind to the
required thinness, wash, dry and mount in balsam.
CHAPTER VIII
Preparation of Animal Material
We have already examined some of the microscopic
members of the animal kingdom in our study of aquatic
organisms. Now let us turn our attention to another pro-
lific source of material for the microscope, as represented
by the larger forms of life with which we are more
familiar. This class includes all animal tissues, such as
nerves, muscles, teeth, horn, bones, feathers, hair, skin,
etc., and that group which provides such a wealth of
microscopic material, the insects.
The group of animals scientifically called Arthropoda
is the largest biological group in existence. It contains
more families, genera and species than any other group.
It is the most widely distributed geographically, and ex-
hibits more diversity of form, habitat and life history than
any other group. Since this group will provide the stu-
dent with the greater part of his microscopic material, let
us digress for a moment and examine these creatures
more carefully.
The Arthropoda are distinguished from all other ani-
mals by the fact that their bodies seem to consist of a series
of rings or segments, apparently joined together, and
bearing certain hard jointed appendages. Examine a
caterpillar under a hand lens. You will see that it consists
of a long cylindrical sack which is folded in upon itself at
regular intervals, giving it an articulated appearance.
Some of the arthropods, such as the true insects and the
Crustacea (crabs, lobsters, crawfish, etc.) have what we
call an exoskeleton, that is, the skeleton is on the outside
103
104 MICROSCOPIC TECHNIQUE
of the body instead of on the inside as it is in humans,
cattle, dogs, etc. This exoskeleton is composed of a hard,
horny material called chitin, which, when the animal dies
and dries, breaks readily into perfect rings that clearly re-
veal the articulated structure. It is by the grouping and
arrangement of these rings, as well as by the appendages
(legs, antennae and wings) , that the various groups of
Fig. 43. Fresh water crustacean.
X7o. Slide and photograph by Ir-
ving L. Shaw.
Arthropoda are classified. The true insects, for example,
are called Hexapoda because they are provided with six
feet, terminating as many legs, all of which issue from one
part of the body called the thorax. An animal belong-
ing to the group loosely referred to as bugs is not a hexa-
pod if it has more or less than six legs. Nor is the speci-
men necessarily a bug, for the true bugs are a separate
group of insects, known correctly as Hemiptera. Spiders
are considered insects and are included in the Arthropoda,
their specific classification being Arachnida.
This division of body segments for the performance of
specific duties may be observed in the larval stage of all
true insects. For example, any small, elongated, tubular-
PREPARATION OF ANIMAL MATERIAL 105
Fig. 44. Foot of beetle. Fig. 45. Wing of house fly.
Xi8 ' Xis
Photographs by Irving L. Shaw
bodied crawling animal is to many people a worm. Sci-
entific classification is not so loose. The true worms have
been classified into one order, the Vermes, distinguished
by specific relationships. While the second or larval
stage of insects may resemble worms in general, there are
very pronounced differences apparent to even the casual
observer. While the bodies of both are articulated, in the
insect larva certain segments have certain duties to per-
form. Let us examine the caterpillar (larva) of any moth
or butterfly, preferably one without hairs and a large one
in which the segments may be easily seen. Such specimens
may be found feeding on cabbages, potato vines, sassafras
leaves, tomato stalks and any number of similar places.
Almost every plant has an insect larva that feeds upon it in
preference to any other, so it is not difficult to find suit-
able specimens.
The first segment of the body is always the head, dis-
io6 MICROSCOPIC TECHNIQUE
tinguishable by the presence of large eyes and a horny
texture different from that found anywhere else on the
body. The second, third and fourth segments will each
bear a pair of legs bilaterally placed, the true walking legs
of the creature. These are hard and terminate in hooked
feet visible under low magnifications. Further along to-
ward the posterior end of the body of some larvae are
found the pseudopoda (false feet) or claspers. There are
ten of these, two each on the seventh, eighth, ninth and
tenth segments, with the fifth pair on the last or thirteenth
segment. This pair of claspers may be modified to bear
but slight resemblance to the other eight, yet its purpose
is exactly the same. The pseudopoda are distinguishable
from the true feet in that they are a great deal larger in
diameter, soft and pulpy in texture and flat on the ends.
They are used not as feet, to propel the animal, but as a
means of taking hold of a twig or branch while the head
end of the body swings about in the air in search of a new
feeding place. This phenomenon may frequently be seen
among larvae such as the so-called measuring worm, a
green caterpillar that humps itself up in the middle as it
travels along, then stops and rears itself on its pseudo-
podac, holding the forward part of the body nearly erect
while the head waves about hunting a feeding place.
When the insect undergoes the third stage of its life
cycle, called the pupal stage, in which it remains dormant
for a period, the three anterior leg-bearing segments are,
in a manner of speaking, fused into one. This, the mid-
dle portion of the body of an adult insect, is the thorax,
from which issue the six legs that distinguish the order.
This distinguishing characteristic may be found in every
true insect, including the Diptera (flies) , the Lepidoptera
(butterflies and moths) , Hymenoptera (bees, wasps, hor-
PREPARATION OF ANIMAL MATERIAL 107
Fig. 46. Sylvanus surinamensis. Balsam mount. Photo-
graphed to show detail. Note female reproductive or-
gans at anal end.
nets, etc.) , Orthoptera (grasshoppers) and Neuroptera
(dragon flies) . In the order Coleoptera, which includes
the beetles, the thorax is extended to form the prothorax,
which carries two pairs of legs, while the thorax carries
the anterior pair. This arrangement of legs is well il-
lustrated in the photomicrograph of the beetle Sylvanus
surinamensis^ Fig. 46.
Any one of the insect orders will provide the student
with abundant material for study. The order Lepidop-
tera, for example, is rich in a diversity of material. All
moths and butterflies lay eggs which may be found by dili-
io8 MICROSCOPIC TECHNIQUE
gent search among the leaves of the specific food plant of
the species. Each insect prefers one particular plant on
which to feed, and since it is the larva that does the feed-
ing, the adult always lays its eggs on that plant so that
suitable food is immediately available when the young
larvae emerge from the eggs. Of course the beetles and
bugs, as well as the butterflies, lay eggs which may
be found attached to the surface of food-plant leaves or
stems.
These eggs exhibit a variety of forms, one of which is
shown in Fig. 76. These are the eggs of Podisus spi-
nosus, found on the leaf of a wild cherry tree. Most in-
sect eggs have some form of ornament such as the spines
in the illustration. In one species they resemble tiny
mushrooms, while in another the entire surface is studded
with tiny bumps. Some eggs are transparent. If the stu-
dent is fortunate enough to secure some of these he can
place them in a cell for protection and watch the develop-
ment of the embryo within the shell from clay to day.
The next stage of insect development is the larva.
This is the feeding stage, in which most insects do their
destructive work. Many adults feed voraciously, espe-
cially some of the beetles, such as the Japanese beetle
(Popillio Japonicd) , and the rose chafer (Macrodactylus
subspinosus) and the potato beetle (Doryphora decern-
lineatd) , to mention only a few commonly-known species.
Others, particularly the moths and butterflies, feed little
or not at all during the adult stage, and then only on nec-
tar which they obtain from flowers, for their mouth parts
consist only of sucking tubes. These are curled up into a
close spiral resting between two extensions on the head
called the maxillary palpi. The adult exists only to prop-
agate the species. This the male does by fertilizing the
PREPARATION OF ANIMAL MATERIAL 109
eggs which the female then deposits on the appropriate
food plant.
The larval stage of insect development is of interest to
the microscopist because of the opportunity it affords to
study the anatomy of the caterpillar. Many of the larvae
are transparent, or at least translucent. If one is placed
on the microscope stage many of the body functions may
be observed in life. Others are colored green by the chlo-
rophyl of the leaves they eat. Usually this green color can
be extracted with alcohol, when the alimentary canal will
become visible through the skin of the larva.
An interesting experiment may be performed on some
caterpillars to show the course of the alimentary canal, the
circulatory system and the respiratory system. It is deli-
cate and will require considerable care, but successfully
accomplished is well worth the effort. It consists of in-
jecting into the body solutions of stains to color each
system differently so that they may be easily discerned.
The best subject is a large caterpillar with a smooth
skin, such as the larva of the tomato hawk moth (Macro-
sila quinque-maculatd) . This may be found feeding on
tomato and potato vines. Examine it carefully with a
hand lens to find the mouth opening, then examine the
sides of the larva to locate the spiracles or openings to the
breathing tubes.
Three stains will be needed, as well as injection appara-
tus of some sort. The subject is small so will not retain
much fluid, ten ccm of each being quite sufficient for sev-
eral specimens.
For the alimentary canal use:
Acid aniline green 0.5 g
Distilled water 100 ccm
Glycerin 35 ccm
no MICROSCOPIC TECHNIQUE
Add glycerin to water and dissolve acid aniline
green in this to make a stock solution. For use take
5 ccm of stock solution to 25 ccm of distilled water.
For the circulatory system use:
Carmine 3 g
Borax 4 g
Distilled water 100 ccm
Dissolve borax in water and add carmine. Heat
gently to dissolve, then add 100 ccm 70% alcohol,
filter and keep well stoppered.
F"or the respiratory system use:
Methyl violet 0.5 g
Distilled water 100 ccm
The best form of injection apparatus is a hypodermic
needle. Lacking this a satisfactory substitute may be
made by drawing glass tubing out to a very fine point in
the flame and equipping the pipette thus made with a
small rubber bulb such as is used on medicine droppers.
In fact, medicine droppers may be used for injections if
the points are drawn out to a fine needle shape. A sepa-
rate pipette should be provided for each stain so that one
color is riot contaminated by another. Fill each pipette
and lay them ready to hand so that they may be used in
quick succession.
Have everything in readiness. Grasp the caterpillar
firmly in the left hand but do not crush it. Insert the tip
of the injector containing green stain into the mouth of
the larva and compress the bulb. Proceed very gently
and continue the injection until the stain is forced out of
the anal end of the specimen. Now that the alimentary
canal is stained, proceed to inject the circulatory system
with red stain by puncturing the back of the larva until
PREPARATION OF ANIMAL MATERIAL in
the tip of the injector just passes through the blood tube.
This is the most delicate part of the operation and great
care must be taken not to puncture both walls of the tube
or the stain will enter the body cavity and fill it, thereby
destroying the effect. The blood tube, which in insects
takes the place of the heart in humans, lies directly under
the skin on the dorsal side, running directly through the
middle of the back from end to end. If the blood tract
alone is stained it will appear as a thin red line running
along the back. Sometimes, although by no means al-
ways, the alcohol in this second staining kills the cater-
pillar. In either case proceed at once to stain the respira-
tory system by injecting violet stain into the trachea or
spiracles, as the breathing pores along the sides are called.
Inject one spiracle and observe the result. Sometimes it
is necessary to make a separate injection into each opening
because of blocking of the tubes, and each side must be in-
jected separately since there are no interconnecting tubes
between the two sides. This injection will kill the speci-
men if not already dead because it shuts off its air supply.
When all three systems have been injected place the sub-
ject in 50% alcohol until the natural green color is gone,
when the injected systems should be clearly discernable.
If the subject is not entirely clear and transparent it must
be dehydrated slowly by passing it up through a series of
graded alcohols until absolute alcohol is reached. To do
this remove it from the 50% alcohol and place it in 70%
alcohol for about two hours, then in 80% alcohol for the
same length of time, then in 95% and finally in absolute
alcohol, where it must remain until it is completely free
from water. Any trace of water will make it impossible
to clear properly. Leave in absolute alcohol one hour if
a medium specimen, two to three hours for large ones.
112 MICROSCOPIC TECHNIQUE
It might be well to explain the reason for the graded
series of alcohols. Alcohol has the power to extract water
from material placed in it. Since somewhere in the neigh-
borhood of 98% of all animal matter is water this means
that the supporting cells of animal matter retain their
shape largely because of their water content. If this is
removed too rapidly the material will shrink unequally,
resulting in a wrinkled appearance which bears no re-
semblance to the subject in life. On the other hand, if
the water is extracted slowly, the tissues shrink somewhat,
but the progress is more uniform and the relative posi-
tions are retained in a more naturalistic arrangement,
being fixed by the alcohol as it extracts the water.
When the specimen is completely dehydrated it must
be cleared by replacing the alcohol with a chemical to
render the tissues transparent. For this we use a mixture
of equal parts of xylol and beechwood creosote. This has
the property of penetrating the tissues and making them
transparent, while at the same time it is soluble in Canada
balsam so that the specimen may be infiltrated with bal-
sam for preservation if desirable. Pour off the absolute
alcohol and quickly replace it with creosote-xylol in which
the specimen must remain until it is perfectly clear and
transparent. If there are any whitish or opaque spots the
subject was not completely dehydrated and must be re*
turned to absolute alcohol for another period and again
cleared. When it appears to be perfectly cleared pour
away the used creosote-xylol and replace it with fresh so-
lution, in which the specimen may remain for several
hours, when it will be ready for infiltration with Canada
balsam or paraffin. For either of these steps consult the
chapters in which they are described.
Should the specimen be entirely satisfactory after it has
PREPARATION OF ANIMAL MATERIAL 113
been bleached in alcohol to remove the chlorophyl, im-
merse it in distilled water over night to remove the alco-
hol, when it may be preserved indefinitely by imbedding
in glycerin jelly. To make this take:
Gelatin (U. S. P. grade) 8 g
Distilled water 52 can
Glycerin 50 ccm
White of egg 5 ccm
Carbolic acid crystals o.i g
Soak gelatin in water for one hour, then dissolve
with gentle heat. Add glycerin and white of egg,
stir until thoroughly mixed and heat to 75 C. for
30 minutes. Filter through flannel while hot and
add carbolic acid to the filtrate. Store in well stop-
pered bottles. This must be warmed for use to
make it fluid.
To imbed a specimen in glycerin jelly soak the subject
in water until every trace of alcohol is removed. Warm
the jelly just enough to make it fluid. Place the specimen
in a shell vial and fill to the top with warmed jelly. Stop-
per well and dip the neck in melted paraffin to seal. Thus
prepared the specimen will keep indefinitely.
Insect larvae may be prepared for sectioning by hard-
ening the fresh subject in alcoholic Bouin's fluid, the for-
mula for which may be found in Chapter III. This will
harden in about three days and will render the material
in good condition to cut paraffin sections. If more rapid
hardening is necessary for quick sections on routine exam-
ination work, harden in formalin 100 ccm, water Goo ccm.
This will harden in two days, but the cutting quality of
the specimen is not as good as that of Bouin's fixed mate-
rial. After fixing, imbed in paraffin and cut sections as ex-
plained in Chapter V.
ii4 MICROSCOPIC TECHNIQUE
Dissection is an important operation in the preparation
of microscopic material and the study of insects gives the
microscopist an excellent opportunity to become familiar
with it. Insects are abundant, so no fear need be enter-
tained of ruining a specimen through clumsiness in the
first dissection. Some specimens will necessarily be sacri-
ficed in the process of acquiring skill in the use of dissect-
ing needles. Working with fine needles under the micro-
scope is quite different from performing the same work
using both eyes. First, the three dimensional effect is
lacking because of the monocular vision. Then, only a
very small part of the specimen is seen at one time and it
is somewhat difficult to direct the movements of the
needles in the small field covered by the lens. If a lens of
the triple aplanat or simple magnifying type is used the
image will be right side up, as seen by the unaided eye.
This is also true of the binocular microscope of the Green-
ough type. But if the dissection is carried out under the
low powers of the compound microscope the work is ren-
dered more difficult because of the inverted image pre-
sented by this instrument. Right becomes left and up
becomes down, movements that are difficult to coordinate
at first.
A dissecting microscope of some sort will be necessary
for dissection of small parts of insects, vegetable fibers,
animal fibers, etc. Such an instrument need not be elabo-
rate or expensive, as long as the optical system is good.
The lens may be either simple or compound, depending
upon the magnification desired. Powers of ten to sixteen
are ample for general student work. The lens should
have a flat field, the largest field that the desired magni-
fying power will permit, and the longest working distance
commensurate with its power. All of these requirements
PREPARATION OF ANIMAL MATERIAL 115
are fulfilled by the type of lens known as a triple aplanat.
This is somewhat expensive compared with simple lenses,
but its corrections are so good that it is the ideal monocu-
lar lens to use for dissecting work. The only improve-
Fig. 47. Bausch & Lomb Grcenough
type Binocular Microscope.
merit over this type of lens is the Greenough type binocu-
lar microscope, an expensive instrument carrying two
objectives and two eyepieces, which is the most comfort-
able microscope to use for dissecting and examination of
whole objects in general. The image is presented to the
eye erected and, because of the binocular arrangement in
which the subject is viewed with both eyes, the effect of
perspective is excellent.
A support of some sort must be provided for both the
lens and the subject. In the absence of a proper dissect-
ing microscope these may be improvised, the only require-
ment being that the lens be held in a fixed relation to the
subject so that it will stay in focus. Some provision should
be made for illumination of the subject from below, a val-
uable aid in many cases of dissection. Some material may
n6 MICROSCOPIC TECHNIQUE
be satisfactorily manipulated in reflected light. In either
case, there should be enough light to avoid working in
semi-darkness, but not enough to be glaring. An excel-
lent way to provide a pleasant working light is to set up
a screen of tracing cloth or paper between the light source
and the work. In this way the light will be diffused
evenly, avoiding hard confusing shadows. It is also an ex-
cellent plan to wear an opaque eyeshield on the forehead,
to intercept the light rays and prevent their falling on the
upper surface of the dissecting lens and introducing dis-
concerting reflections. Occasions will arise when a con-
centrated light is necessary. This may be provided by
introducing a condensing lens into the light beam and
focusing it upon the work. This purpose is served very
nicely by an ordinary Florence flask such as is used in
chemical laboratories. The flask may be filled with water
colored to give any type of light desired. Alternatively,
a plano-convex lens may be used.
Many dissections may be successfully carried out under
the lens of a tripod magnifier such as is sold for linen test-
ing. This lens has a magnifying power of seven to seven
and one half times. It is uncorrected for color arid flat-
ness of field, but it has a long focal length which permits
free working with the needles under it. The self-con-
tained legs permit its use in trays or directly on the body
of large specimens. One serious disadvantage is the pres-
ence of the three legs, which sometimes interfere with
free manipulation of the needles, but extreme portability
and low cost help to compensate for this. In Chapter XI
several improvised dissecting microscopes are described,
any one of which may be procured at low cost.
The tools for dissection are simple to make and easy to
procure. First a set of needles will be needed. In their
PREPARATION OF ANIMAL MATERIAL 117
simplest form these consist of fine and coarse needles
thrust into wooden handles, as described in Chapter XI.
We will also require two pairs of forceps, one with fine
curved points and one with strong blunt points. Fine-
pointed scissors that cut at the very extreme tip of the
blades are needed, also a scalpel or lancet. Numerous
improvisations of the last item may be made from safety
razor blades, but none is quite as satisfactory as a surgical
instrument. Considering the low cost of a really good
scalpel and the pleasant working it affords, it is false econ-
omy to attempt to work with substitutes. The Bard-
Parker company make a line of low priced surgical knives
which serve the purpose splendidly. The instruments
are arranged so that the blades may be removed from the
handles in a manner similar to safety razors. The handles
are bought separately and the blades are available in sev-
eral styles. The number 1 1 blade, made to fit the num-
ber 3 handle, is good for general use, while the collection
may be completed by the addition of numbers 10 and 12,
the former a curved blade, the latter scalpel shaped. The
number 4 handle is much larger and takes larger blades,
and will seldom be needed.
The last piece of equipment needed is a shallow tray or
dish in which to perform the dissection. Most dissections
should be carried out in a fluid of some sort, physiological
salt solution being generally preferred. A goo'cl selection
for this is the type of glassware known as a Petri dish, a
shallow cylinder with a flat bottom. These are purchased
in pairs, one of which acts as a cover. Very large speci-
mens may be handled nicely in glass photographic trays.
The requirements are few; the receptacle must be water-
tight, large enough to contain the specimen easily, and
preferably transparent for substage illumination.
n8 MICROSCOPIC TECHNIQUE
In some dissections portions of the specimen must be
held rigidly while work is being done on another part.
Since both hands are needed for manipulation of the dis-
secting needles, the subject is transfixed with needles
thrust into the bottom of the dissecting tray. This means
that the bottom of the tray must have a soft lining. Such
a lining may be added to any glass tray by fastening strips
of thin cork to the bottom with shellac. Cut strips of
gasket cork (procurable from auto accessory stores) one
sixteenth of an inch wide, using a sharp razor blade and a
straight-edge. Paint one side with thick shellac and fasten
to the bottom of the tray, allowing about one quarter of
an inch between them. Next cut short strips of cork to
lengths that will just fill the spaces between the long
strips and shellac these in place, forming a lattice on the
tray bottom, which will permit fastening the subject to
the tray with needles. It may be illuminated by the sub-
stage mirror, and both hands are free for work. Large
dissections that need not be performed under the lens
may be carried out in a tray lined with cork shellacked to
the entire bottom. In this case the light must be arranged
to cast a minimum of shadows, and plenty of light must
be provided.
The physiological salt solution referred to for use in
dissections is made by dissolving table salt (not the iodized
variety) in water. Six-tenths of a gram in one hundred
ccm of water is the correct concentration. If a larger
quantity of solution is required it may be made by dis-
solving six grams of salt in one liter of water.
Most insects, in fact all but butterflies and moths of
which the wing scales are to be mounted, may be collected
in alcohol when wanted for dissection. Use the ordinary
70% rubbing alcohol and drop the specimens into it as
PREPARATION OF ANIMAL MATERIAL 119
they are collected. If they are being collected for sec-
tioning they should be killed in cyanide gas. Potassium
cyanide is a deadly poison and must be kept out of reach
of children. Place a small quantity of the dry salt in
the bottom of a wide-mouthed bottle or jar fitted with
a tight cover. A layer one-eighth of an inch deep is ample.
Over this place four layers of blotting paper cut into discs
that fit closely into the jar. Force these into place with a
stick and close the jar. It is ready for use at once. Drop
the insects into the jar and close the cover. The gas aris-
ing from the cyanide (hydrocyanic acid gas) will kill in-
sects in a few seconds, leaving them little time to beat
about and destroy their wings. As soon as the specimens
are dead they must be removed from the jar and spread
if they are to be preserved as whole objects, or placed in
Bouin's fixing solution if they are to be sectioned. Speci-
mens intended for dissection may be preserved in alcohol,
or they may be preserved for a few days without hardening
by immersing them in glycerin one part, water three parts.
This keeps the material soft and pliable, and also acts as
a macerating agent, rendering the tissues very soft so that
they may be teased apart or even shaken apart with little
difficulty. Hence the material must not be allowed to
remain too long in the solution or it will be worthless.
The preparation of insects for microscopic study takes
several forms. Small species may be mounted whole, in
the round, or flat. They may be sectioned to show the
internal arrangement of organs, or they may be dissected
to show individual organs. The student will gain valu-
able experience in mounting by preparing a set of slides
of various small species, mounting them flat as described
below.
To mount an insect whole, flat, select some of the small
120 MICROSCOPIC TECHNIQUE
flat forms such as gnats, small ants, small spiders or small
dorsiventrally flattened species of beetles, such as the
Cucujidae genus, a good example of which is shown in
Fig. 46. This is Sylvanus surinamensis, a small brown
beetle frequently found in corn meal, flour and other pre-
pared cereals. As already stated, the exoskeleton of adult
insects is composed of chitin. In many species this is
strongly pigmented and must be bleached before mount-
ing, or it will not transmit the light needed to observe de-
tails. The black ant is a case in point. To bleach out the
color, a weak solution of calcium hypochlorite in alcohol
is used. This chemical is readily available in the com-
mercial product Chlorox. To 10 ccm of 70% alcohol
add three to four drops of Chlorox and immerse the speci-
mens. Bleaching will require from a few days to as many
weeks depending upon the si/e, permeability and depth
of coloration of the insect. If bleaching is not complete
at the end of two days pour away the solution and replace
it with fresh. Repeat at two day intervals until bleached.
Do not use a stronger bleaching solution than that recom-
mended in an effort to hasten the process, for the liga-
ments that hold the various parts of the body together
may be destroyed and the specimen will fall apart.
When bleaching is complete, pour away the solution
and wash the material several times with alcohol to free
it of any remaining chlorine. Next place the specimen
under the dissecting microscope and spread the legs and
antennae. This may be somewhat difficult, for the alcohol
sometimes renders the subject too hard to spread. In this
case do not attempt to force the parts into place, for this
will result only in ruining the specimen. If any difficulty
is experienced, if the parts do not remain as they are
placed but snap back into their former position, place
PREPARATION OF ANIMAL MATERIAL 121
the specimen in warm water (40 C.) , for a short time,
when the appendages should be sufficiently softened to be
handled easily. This measure is to be adopted only in
the most refractory cases, for the water immersion will
destroy part of the work that has already been done. The
soaking in alcohol has started the process of dehydration;
some of the water has been extracted, and if the specimen
is placed in water to soften it, this water will have to be
again extracted before the subject may be mounted.
When the legs, antennae and wings have been spread to
your satisfaction, lay a narrow strip of paper on each side
of the specimen and press another slide down on it, hold-
ing the two slides together with a wrapping of thread, or
clipping them together with a wooden spring-type clothes-
pin. If the specimen was taken from 70% alcohol it must
now be placed in 95% alcohol by immersing the two
slides in the liquid. Leave for at least twenty-four hours.
If the specimen was softened for spreading by soaking in
water it must be placed in 70% alcohol for twenty-four
hours, followed by 95% alcohol for the same length of
time. The steps in the process are then the same for ma-
terial prepared in either way. From 95% alcohol transfer
the two slides with the specimen between them to abso-
lute alcohol for a few hours, separating the slides slightly
by releasing the thread that holds them together. Then
separate the slides, remove the specimen with a camels-
hair brush and allow it to remain in absolute alcohol for
several hours longer. Prolonged immersion in absolute
alcohol will not do the slightest harm, but if the specimen
does not remain in it long enough to become completely
dehydrated it will be impossible to clear it.
When you are satisfied that the material is perfectly
free from water it must be rendered transparent. This is
122 MICROSCOPIC TECHNIQUE
accomplished by using a chemical that is miscible with
both alcohol and Canada balsam. Xylol fulfils these con-
ditions but has the disadvantage of hardening too much,
thus rendering the tissues brittle. Furthermore, it will
not clear specimens that contain the least trace of water.
To overcome these disadvantages a mixture of xylol and
beechwood creosote, equal parts of each, is used. This
mixture clears the material perfectly, absorbs small traces
of water and does not make the structures too brittle.
To use the creosote-xylol clearer, pour off the last wash
of absolute alcohol and quickly add a mixture of equal
parts creosote-xylol and absolute alcohol. Leave the ma-
terial in this overnight, keeping the vessel well covered.
Now replace the creosote-xylol-alcohol with creosote-xylol
and leave the material until perfectly clear. When it
seems to be cleared, replace the old solution with new and
leave for several hours more, or until needed. If whitish
opaque spots appear in the material it was not completely
dehydrated and will have to be returned to absolute
alcohol and again cleared until the spots disappear. In
place of creosote-xylol, synthetic wintergreen oil (methyl
salicylate) may be used, although it presents no ad-
vantages.
When the material has been cleared it is ready to be
mounted on the microscope slide in Canada balsam, gen-
erally referred to simply as balsam. Hard, robust types
such as ants, beetles and the legs of chitinous species may
be transferred directly to thick balsam without any inter-
mediate treatment. Soft-bodied specimens such as aphids,
small larvae and the like must be slowly infiltrated with
a gradually strengthened solution of balsam to prevent
destructive osmotic currents from being set up by the
difference in density of the clearer and the balsam.
PREPARATION OF ANIMAL MATERIAL 123
The gradual introduction of balsam can best be ef-
fected by placing the material in a circular dish and cover-
ing it with creosote-xylol to a depth of about a quarter of
an inch. Now fold a disc of coarse filter paper into the
usual cone shape, adjusting the last fold so that a shallow
cone is formed, the edges of which rest on the rim of the
dish and the apex dips only slightly below the surface of
the liquid but does not touch the material. Into this
cone are placed a few drops of thick balsam which will be
slowly dissolved by the xylol. More balsam is added from
time to time until the liquid in the dish assumes the con-
sistency of thin syrup. The filter paper is then removed
and the mixture allowed to concentrate by evaporation
until it is almost as thick as the regular mounting balsam,
the vessel being meanwhile covered loosely with a glass
plate to prevent the entrance of dust.
Thus prepared, the specimens are ready to be placed
upon the slide. If the material has been carefully carried
through the above stages it should be in excellent condi-
tion for critical study under the microscope. Consider-
able time and material have been used in bringing the
work to this stage, and having arrived, it deserves proper
mounting to display every characteristic and preserve it
for future study. Work slowly, think out each step of
the process and be sure you know what to do and how to
do it before proceeding. Attention to a few details will
insure perfect mounts which will retain their beauty in-
definitely. Be careful to place the specimens in orderly
arrangement if they are large enough to be handled indi-
vidually and if more than one is included under one cover
glass. If only one is used see that the appendages are ar-
ranged in life-like positions. This can be done best under
the dissecting microscope with the aid of fine needles,
124 MICROSCOPIC TECHNIQUE
being careful not to pull off any of the appendages. Try
to estimate the amount of balsam necessary to just fill the
area of the cover glass and run out to the rim in a neat
beveled edge. This will not be easy at first, but after
some practice will not be difficult. Be sure to support
the cover glass over delicate specimens so they are not
crushed. This may be done in any one of several ways.
Thin glass rods made by drawing tubing out in the flame
make excellent supports for thick objects such as the
larger Crustacea, spiders, ants, flies, small beetles, etc.
Thinner specimens may be safely mounted by laying a
piece of horse hair on either side, or cells may be made
with shellac. Very small or thin objects require no cover
glass support. They are placed directly on the slide, a
small quantity of balsam dropped on them and the cover
glass lowered carefully to prevent inclusion of air bubbles,
which are very difficult to remove once they have formed.
A very useful accessory for mounting is made by ruling
a rectangle the size of the slide on a piece of paper and
drawing diagonal lines from each corner to locate the
center. If a number of small specimens such as Paramecia
are to be mounted they may be placed in a small heap in
the center of the slide. A drop of balsam is then placed
on them and the cover lowered into place. As many indi-
viduals of small species should be mounted as the supply
of material will permit, for in this way a possible diversity
of form will be presented. If deliberate arrangement is
possible some individuals should be placed dorsal side up
and others ventral side up, with a few showing the lateral
aspect. This arrangement will permit critical study of
every detail. Make every effort to prevent clumping of
the material, for overlapping specimens will not reveal
their characteristics. Extremely minute objects are best
PREPARATION OF ANIMAL MATERIAL 125
mounted by placing a very small drop of balsam on a slide.
Pick up the material with a fine pipette or a thin wire
bent into a tiny loop and place in the drop of balsam. Al-
low this to harden overnight in a place free from dust.
Next day more balsam, the supports and cover glass may
be added without disturbing the mount.
Choice of a cover glass will depend upon the specimens.
Small objects are best mounted under a circular cover,
while larger ones or a large number of small individuals
require a square glass. Select a size that will cover the
specimen and allow 2 mm to 3 mm overlap at each side.
Very small or thin objects to be examined under high
powers or with immersion objectives should be mounted
under No. i covers. Larger specimens for examination
with medium and low powers may be satisfactorily
mounted under the thicker No. 2 covers, which are less
likely to be broken in handling the finished slides.
Placing the cover glass so as to avoid air bubbles is some-
times a bit difficult. A few general hints may be of assist-
ance. Use a sufficient quantity of balsam to completely
fill the cover glass area and extend beyond the edge in a
rim from 0.5 to i mm wide all around. Balsam shrinks
somewhat in drying and if the excess is not sufficient it
may shrink back and expose the edge of the cover and even
draw in air bubbles that might mar the preparation.
When a sufficient quantity of balsam has been placed on
the slide pick up the cleaned cover glass in a pair of fine-
pointed forceps, warm it gently and quickly move it over
the preparation on the slide. Lower the edge opposite to
that held by the forceps until it touches the balsam and
rests on the slide or supports, then slowly let down the
held edge so that the balsam runs along the cover, but
without disturbing the material. Release the glass and
126 MICROSCOPIC TECHNIQUE
allow its weight to press down upon the balsam. Now
gently warm the slide on a warming stage or over a small
flame until the balsam spreads slightly beyond the edge of
the cover. Press lightly on the center of the cover with
the blunt end of some instrument, such as the handle of
a dissecting needle. If the quantity of balsam is insuffi-
cient to completely fill the cover glass a small amount may
be added with a needle applied to the rim. If there is an
excess beyond that required for a neat rim it must be care-
fully scraped away without disturbing the cover.
Fig. 48. Completed slide.
The slide is now ready to be labeled. The label is a
small sheet of gummed paper attached to the slide. On
this is recorded all pertinent information, including the
name of the object, the date it was received and mounted,
the fixing solution and the stain used, and the number of
the slide. This number should be recorded in a note book
together with the data contained on the label, and any
other data that may be of interest. Such data might con-
sist of the exposure time, the light and magnifying power
used, the filter, if any, and the type of film if the subject
was photographed. The methodical recording of such
data is of inestimable interest and value as a working basis
for subsequent photographs.
CHAPTER IX
Preparing Vegetable Specimens
Rich as the vegetable kingdom is in material for the
microscopist, the student will find excellent exercise in
the examination of plant stems for his first adventure into
vegetable microscopy. These important structures of
plant life are interesting subjects and will afford endless
hours of instructive study if pursued with the assistance
of a good book on botany. While a detailed discussion of
stem features is impossible here, we can at least make a
few introductory remarks to enable the student to ap-
preciate some of the things he sees when he places a stem
section under the microscope.
The stem of a plant, and this includes all stems from
the short stubby stems of turnip to the enormous trunks
of our giant redwoods, serves two primary purposes. It
supports the foliage, holding the leaves up into the light
and air, and serves as a food canal, carrying the moisture
and food that is absorbed by the roots up into the aerial
parts of the plant to nourish them. A secondary function
of the stem is as a means of the sort of reproduction is
effected by branching and budding.
The botanist recognizes two well defined groups of
stems, known as endogenous, or inside growing, and ex-
ogenous, or outside growing. These terms refer to the
arrangement of the structural elements. Dicotyledonous
stems or dicots are examples of the exogenous type, while
monocotyledonous stems or rnonocots are of the endoge-
nous type. The steins in both systems are composed of
hard, woody bundles, called fibre-vascular bundles.
127
128
MICROSCOPIC TECHNIQUE
Fig. 49. Cross-section ot tansy Fig. 50. Cross-section of oak leaf
stem. X2o at midrib. X$2
Slides and photographs by Irving L. Shaw
These are surrounded by a mass of more or less soft ma-
terial, over which is the bark or protective coating. The
arrangement of the bundles in the two systems is quite
different. In the exogenous type the bundles are nearly
always arranged in a circle near the outside of the stem,
and the strength of the stem depends upon their compact-
ness. Such stems will increase in diameter as long as the
plant lives. This increase is the result of growth in the
layer of cells lying just under the epidermis or outside
layer. These growing cells form the cambium layer,
and by their growth each year give rise to the annual
rings that may readily be seen in a cross-section of a tree
trunk.
Endogenous stems are again divided into two groups,
herbaceous and woody. Herbaceous stems consist of a
mass of soft, pithy cellulose surrounded by a thin hard
layer of tissue containing the cambium and the epider-
PREPARING VEGETABLE SPECIMENS 129
mis. Scattered irregularly through the soft core are the
fibro-vascular bundles, or simply bundles as they are com-
monly called. All of the grass stems are endogenous, as
are those of rushes, iris and numerous other plants. Corn,
which is a coarse grass, is a good example of an endoge-
nous herbaceous monocot. There is another type of en-
dogenous stem that is much firmer in texture, although
of the same general character. This is in the form of a
cylinder in which the bundles have been compressed into
a thin ring, making the stem tubular and of great strength.
The various grain stems and most common grasses are
examples of this type. Soft juicy stems in which the bun-
dles are small as compared with the surrounding material
are called herbaceous, while those in which the bundles
constitute the greater part of the stem substance are called
woody. Most of the strictly woody stems are exogenous
and show annual rings.
Some plants have underground stems that are fre-
quently mistaken for roots. The May apple (Podophyl-
lurn peltatum) is a good example. The underground
stern may be distinguished from the roots by the more or
less regular division of the stem into nodes and inter-
nodes. Branching from these underground stems always
occurs at the nodes, and is therefore regular. Roots are
without these nodal divisions, hence the branching is very
irregular. The common white potato is a good example
of an underground stem of the tuber type. Leaf scars
are present in this type of stern in the indentations we call
eyes. Each of these eyes is a bud and a potential potato
stalk.
Rhizomes, or as they are more commonly called, bulbs,
are another form of underground stem. Cut a median
longitudinal section of a tulip bulb and the laminated
130 MICROSCOPIC TECHNIQUE
structure will at once be evident. These laminae are in
reality modified leaves that surround the embryo. When
the plant grows and before it has developed to the point
where it can manufacture its own food, it draws its nour-
ishment from these modified leaves. That is why many
tulips received from greenhouses as full grown plants
die in a short time. Growth has been so rapidly forced
in soil of low fertility that almost the entire food supply
has been consumed in growing leaves and flowers. When
the small remaining amount of available food has been
used the plant dies and the bulb becomes a shriveled
mass.
This matter of food storage for the young plant is quite
important. When the seed germinates it sends out the
root hypocotyl first in order that the process of supplying
nourishment and water may begin at once. Then the
plumule or young leaves start to grow. This growth is
very rapid, so rapid that the young roots are inadequate
to supply the food needed and the young plant would
starve, had nature not provided a source of food to keep
the plant alive until the roots begin to function. This
food supply is contained in the seed itself, and con-
sists largely of starch and a small amount of minerals.
Through the actions of enzymes produced within the seed
cells this food supply is made soluble, or otherwise modi-
fied so that it is available for the young sprout until its
own food manufacturing process begins.
Now let us return to the preparation of vegetable ma-
terial. In general, preparation for microscopical exami-
nation does not differ from the schedules already outlined
for animal material. Some authors recommend the use
of celloidin for imbedding because of the high water con-
PREPARING VEGETABLE SPECIMENS 131
tent of all vegetable tissue. Chamberlain, however, uses
paraffin with complete success in his work in plant his-
tology. There is therefore no reason why the student can-
not secure satisfactory results with the same imbedding
medium. The writer has been successful with subjects
such as ovaries of flowers, in which cut portions of embryo
seeds left unattached in the section were mounted on the
slide in the same relative positions they occupied in the
growing capsule.
The primary requisite for success in preparing vege-
table specimens lies in strict adherence to proper practice
in every detail of the various operations. Perhaps a sum-
mation of the principal steps will not be amiss.
1. Fix and harden all tissues thoroughly. Bouin's
fluids are as satisfactory as any fixative. The alcoholic
solution penetrates rapidly, fixing rose ovaries in one
hour if sections are required quickly. The aqueous solu-
tion is slower but just as thorough if given enough time
to act. Be sure to cut off one end of the ovary to insure
complete penetration of the fixing fluid. Bouin's leaves
the material in excellent condition for staining with any
stain the student is likely to use. Being a picric acid mix-
ture, Bouin's must be washed out of the fixed material
with 70% alcohol, and never with water.
2. Dehydrate by passing the material from 70% alco-
hol to 80%, then 95%, and finally complete the process
in absolute alcohol. Allow plenty of time for complete
extraction of the last traces of water, and change the abso-
lute alcohol at least once, allowing an equal period of im-
mersion in each change.
3. The transfer to xylol must be gradual. From abso-
lute alcohol transfer the material to:
132 MICROSCOPIC TECHNIQUE
A absolute alcohol, 75%, xylol 25%, then
B absolute alcohol, 50%, xylol 50%, then
C absolute alcohol, 25%, xylol 75%, then
D pure xylol
When the material appears clear and transparent
throughout, pour off the xylol and add pure toluol,
allowing it to remain in this for several hours.
4. Now pour off the toluol, add a quantity of fresh
solution and begin infiltrating with hard paraffin. Add
the paraffin gradually until the toluol is saturated at room
temperature. This is especially important with vegetable
materials since these contain much included air in minute
bubbles that must be displaced with paraffin, else the ma-
terial will break down in sectioning.
When the toluol is saturated, transfer the container to
the paraffin oven at a temperature of 35 to 37 C. and
allow the slushy mixture of paraffin and toluol to clear by
complete solution of the paraffin. Then add more paraf-
fin until the solvent is saturated at the oven temperature.
Let it stand in the warm oven overnight, then pour off
half the mixture and make up to its former volume with
finely shaved paraffin. Raise the oven temperature to a
point just above the melting point of the paraffin, as de-
termined by test. Keep at this temperature for twenty-
four hours. Do not allow the temperature to rise. At the
end of this time pour off all the toluol-paraffin mixture
and add fresh melted paraffin. Leave it in the oven until
all the residual toluol has evaporated.
5. Remove the container from the oven, place the ma-
terial in imbedding trays and add melted paraffin. Solid-
ify the paraffin in cold water.
All of the steps included in the above summary have
been described in detail in Chapter V. Several modifica-
PREPARING VEGETABLE SPECIMENS 133
tions have been introduced to adapt the process to vege-
table materials, but in the main the operations are identi-
cal. For instructions covering the cutting of sections see
Chapter V.
6. Stretch the sections on albumenized slides.
7. Remove the paraffin.
8. Coat with celloidin.
9. Downgrade to water.
10. Stain.
1 1 . Upgrade to absolute alcohol.
12. Counterstain.
13. Recoat with celloidin.
14. Clear and mount in balsam.
CONIFEROUS LEAVES The leaves or needles of conif-
erous trees are interesting botanical subjects for study.
To prepare sections take a bundle of needles, tie them
together with a strand of silk, imbed the bundle in paraf-
fin and cut transverse and longitudinal sections.
POLLENS The study of flower pollens embraces such
a vast amount of material that the student could devote
years of study to this branch alone. Pollen may be pre-
pared either as opaque or transparent material. Usually
both methods are used, frequently on the same slide.
Opaque mounts are made by spinning a shellac cell on
the slide, of slightly greater depth than the thickness of
the pollen grains. A disc of dead black, matte-surface
paper is then pasted to the inside of the cell with thin
shellac or celloidin. When this is perfectly dry, coat the
upper surface of the paper with gum water and allow it
to dry. Breathe on the paper and quickly dust the dried
pollen grains on it. The breath will moisten the gum
enough to make the grains adhere. Set the slide in the
desiccator to dry thoroughly, then spin a ring of shellac
134 MICROSCOPIC TECHNIQUE
on the cell and imbed the cover glass, finishing with
another ring of shellac.
Transparent mounts of pollen are somewhat difficult
to make because of the tenacity with which the grains re-
tain air bubbles. Many species of pollen are covered with
fine hairs, the interstices between them holding the air
imprisoned. Place the dried pollen in rectified spirits
of turpentine for several days, then transfer it to 70%
alcohol to remove the turpentine. Replace the 70% al-
cohol with 95% alcohol and allow to remain several days
longer. Transfer to absolute alcohol until completely
dehydrated, clear in toluol and mount in balsam, sup-
porting the cover glass with glass rods.
To mount pollen in balsam a slight variation must be
made in the usual technique. Place a drop of thin balsam
on the slide and spread it with a needle until it covers an
area equal to that of the cover glass, being sure to break
any bubbles that form. Drop the cleared pollen grains
into the balsam and set in the desiccator until dry. Now
add another drop of balsam and the glass rods, warm a
cover glass over the spirit lamp and lower it on the slide
carefully to avoid bubbles.
Deeply pigmented pollens like that from the tulip must
be bleached in chlorinated alcohol as described in Chap-
ter VIII. Colorless varieties may be stained to increase
visibility. The best stains for this purpose are those
anilines soluble in alcohol, such as eosin, f uchsin, safranin,
methyl violet, malachite green, methyl green, etc. Use a
rather strong solution, say 2-3% in 95% alcohol and stain
until the desired depth of color is attained. Povir off the
stain, rinse in absolute alcohol, clear in beechwood creo-
sote and mount in balsam. If the staining has been too
deep it may be reduced by immersion in alcohol.
PREPARING VEGETABLE SPECIMENS 135
GERMINATING SEEDS The dried seeds of cereals
(wheat, oats, barley, buckwheat, etc.) are soaked in water
for a few hours until they resume their natural shape as
nearly as possible. Then place a disc of blotting paper
in a Petri dish or saucer, moisten it and spread a layer of
swelled seeds on the paper. Cover this with another disc
of moist blotting paper and place in the warm oven
(about 25 C.) until the embryo begins to germinate and
sprout. Germination can be arrested at any desired point
by throwing the seeds in alcoholic Bourn's fluid. Allow
to fix thoroughly, then dehydrate, infiltrate and imbed in
paraffin and cut longitudinal sections.
Many vegetable materials, especially stems, may be cut
in the well microtome without imbedding. Soak the stem
in water until soft and then cut sections 20 to 30 /z
thick. As the sections are cut place them in 70% alcohol
for about ten minutes, then transfer them to 95% alcohol
and allow them to remain twenty to thirty minutes. Now
pour off the alcohol and replace with a 1% solution of
safranin in 50% alcohol, allowing this to act about twenty-
four hours. Pour off the stain (which may be used re-
peatedly) and replace with 50% alcohol to wash out the
excess color. Lignified structures retain the stain more
tenaceously than cellulose walls, which affords an oppor-
tunity to differentiate between the two. Destain until
most but not all the color has been extracted from the
cellulose walls and the lignified walls still show a strong,
brilliant color.
If the destaining is very slow it may be accelerated by
adding a small drop of HC1 to the washing alcohol.
Proper differentiation without acid should be complete
in from five to fifteen minutes. A longer period indicates
acid treatment.
136 MICROSCOPIC TECHNIQUE
Pour off the washing alcohol and wash the sections in
tap water, being especially thorough if acid has been used
for differentiating. The least trace of acid left in the
sections will cause the stain to fade in a short time.
Now stain in Delafield's haematoxylin for ten to fifteen
minutes. This will stain the cellulose walls but will have
little or no effect on the lignified structures. Wash the
sections in tap water for several minutes or until the origi-
nal red color is replaced by a rich purple. If the cellulose
walls show only a faint color replace in the stain for a
longer period. When the color is a deep purple or nearly
black, destain in acidulated water, using only the smallest
amount of HC1 and mixing it well with the water before
allowing it to touch the sections. As soon as a reddish
color appears in the sections, which may take place in
from four to five seconds, pour off the acid water and re-
place with tap water. Wash well to free from acid. This
acid treatment differentiates the stain by washing it out
of the lignified walls more readily than from the cellulose
walls, at the same time dissolving the precipitates that fre-
quently follow staining with Delafield's. The remaining
safranin is also washed out in this treatment, which is the
reason for allowing part of it to remain from the first
washing. If this were not done the stain would be weak
in the lignified walls.
Run the stained sections up through the ascending
series to absolute alcohol, clear in toluol or clove oil and
mount in balsam.
If sections are to be mounted in glycerin jelly they
may be mounted directly from the last wash water, since
they are not dehydrated for glycerin mounting.
Clove oil may work better for clearing than toluol or
xylol if the dehydrating absolute alcohol is not quite
PREPARING VEGETABLE SPECIMENS 137
100%. Clove oil will clear from alcohol of 99.5% or even
99%, so that if the strength of the alcohol is the least bit
questionable it is better to be safe and use clove oil. The
sections should then be placed in xylol for a short time to
facilitate hardening, since material cleared in clove oil
hardens very slowly in balsam.
The following schedule of the foregoing steps is given
as a tentative guide for staining,
i. Sections in 95% alcohol.
s. Safranin, 12 to 24 hours.
3. 50% alcohol until color is right, generally 2 to 10
minutes. Acid alcohol may be used if needed.
4. Water, running for several minutes, or 5 minutes
in water that is changed frequently.
Delafield's haematoxylin, 3 to 30 minutes.
6. Water, running, 5 to 10 minutes.
7. Water, slightly acidulated, 5 to 10 seconds.
8. Water, 20 to 30 minutes to wash out acid.
9. 50% alcohol, i minute.
10. 95% alcohol, i minute.
1 1 . Absolute alcohol, 5 minutes.
12. Toluol, i to 5 minutes.
13. Balsam.
If clove oil is used for clearing, finish as follows:
12. Clove oil, 2 to 5 minutes.
13. Xylol, i to 5 minutes.
The following schedule makes use of malachite green
and Congo red. Material is treated with 95% alcohol,
then passed down to water and stained as follows:
1. 3% aqueous solution of malachite green, 6 hours
or more.
2 . Wash in water.
3. Congo red, 1% aqueous solution, 15 minutes.
138 MICROSCOPIC TECHNIQUE
4. Wash in water.
5. Rinse in 80% alcohol. As soon as the green color
appears through the red, transfer quickly to,
6. Absolute alcohol.
7. Toluol.
8. Balsam.
Other stain combinations that arc easy to use are iodine
green and acid fuchsin; methyl green and acid fuchsin;
safranin and gentian violet. The schedules given above
may be followed with any of these combinations, with
slight modifications as indicated by the material in
process.
CHAPTER X
The Polarizing Microscope
No discussion of general microscopy would be com-
plete without some reference to the polarizing micro-
scope. Aside from the gorgeous colors produced by many
materials in polarized light, the polariscope is of immense
value in the study of mineralogy. No petrologist would
ever think of studying a new mineral or crystal without
using the polarizing microscope. Many minerals, be-
cause of their atomic structure, display the phenomenon
known as optical activity. This means that the ma-
terial takes on a different aspect in polarized light from
that presented in unpolarized light. The number of op-
tically active materials is legion. The characteristics of
many of these in polarized light are known and afford an
excellent and infallible means of identification, even
when mixed with other materials. Others are still wait-
ing for someone with enough time to investigate them,
affording the student another opportunity for original
research.
The polarizing microscope, or polarizer, as it is gener-
ally called, is almost like any other microscope. The dif-
ferences lie in the addition of two prisms of calcite (de-
scribed in Chapter XI) known as Nicol prisms, and a
rotating stage. For student use the rotating stage is not
absolutely necessary, but the nicols are. There are two
of these, one called the polarizer, the other called the ana-
lyzer. The polarizer is located under the stage so that the
light is acted upon before it reaches the slide. The ana-
lyzer is located between the slide and the eye of the ob-
'39
140
MICROSCOPIC TECHNIQUE
Fig. 5 1 . Crystals of potassium chlorate pho-
tographed in ordinary light. Crystals
grown in gelatine. Mounted in balsam.
server, usually in the draw tube of the microscope. One
(or both) of the prisms must be so mounted that it can be
rotated on its principal axis. This rotation makes possi-
ble alteration in the plane of rotation of the light so that
this plane may be made parallel with or at right angles
to the plane of vibration of the second prism. Thus,
when the plane of vibration of the two nicols is in the
same direction we say the nicols are parallel; when the
plane of vibration of the one nicol is at right angles to
the plane of vibration of the other we say the nicols are
crossed. As the planes of vibration of the two nicols pro-
gress by rotation from parallel to crossed the field gradu-
ally darkens until, when the nicols are completely crossed,
or exactly at right angles to one another, the field is quite
dark. This is called the point of extinction.
THE POLARIZING MICROSCOPE 141
Fig. 52. The same crystals of potassium
chlorate photographed in polarized light,
crossed nicols.
While we cannot enter into a detailed discussion of the
theory of polarized light, it may help the student if we
explain just a little of that theory. Polarized light, it
must be remembered, is just ordinary light. The differ-
ence lies entirely in the manner of vibration. In ordi-
nary light, either natural or artificial, vibration of the
ether particles that effect the transmission of light waves
is in every conceivable direction across the light path.
Now, when the nicol prism is introduced into this light
path it acts as an optical grating altering that heterogene-
ous vibration and making the light vibrate in one single
plane. Practically all of the incident light is transmitted
but its many planes of vibration have been transformed
into one constant plane. If another nicol is now allowed
142 MICROSCOPIC TECHNIQUE
to intercept the path of light from the first nicol and this
second nicol is arranged so that the direction in which it
permits ether vibrations to go is at right angles to the
plane of vibration of the incident light, it stops the inci-
dent light entirely, thus acting as an analyzer.
If the student is interested in a more detailed account
of the theory of polarized light he is referred to one of the
texts mentioned in the bibliography. For our purpose
the above brief description must suffice.
Most organic and many inorganic substances are op-
tically active and will respond to polarized light, showing
colors or interference figures. Aided by the home-made
polarizer described in Chapter XI the student can enjoy
the study of materials in polarized light. The following
short list of polarizer objects and their method of prepa-
ration is included in the hope that they may afford a start-
ing point for further investigation in this fascinating field.
Crystals of chemical compounds are great favorites.
These may be prepared in several ways and will vary with
each mode of preparation.
Method No. i . The usual method of preparing crys-
tals is to make a strong solution of the salt in distilled
water. Filter the solution and place a drop on a slide.
Several slides may be prepared, drying some naturally,
protected from dust, evaporating others over the spirit
lamp, and drying the rest in the desiccator. When thor-
oughly dry add a drop of balsam, except as noted later, and
a cover glass supported to prevent crushing the crystals.
Method No. 2. Make a saturated solution of the salt in
distilled water. Add a small quantity of gum acacia or
gelatine, enough to make about 35% solution, and filter.
Place a drop on the slide and set in the desiccator to dry.
Mount in balsam, except as noted.
THE POLARIZING MICROSCOPE 143
Method No. 3. Place a small piece of the dry salt on a
slide and lay a cover glass over it. Warm gently over the
spirit lamp until the substance melts, press the glass down
on the slide with a blunt instrument and allow to cool.
Method No. 4. Crystals of many salts can be produced
instantly by pouring some of the saturated aqueous solu-
tion into alcohol, when the chemical is not soluble in
alcohol. Use 95% alcohol and change it once. Then
pour off the 95% solution and replace with absolute al-
cohol. Change this once to completely dehydrate the
crystals. Mount in balsam except as noted.
Note. Some crystals, especially those of organic com-
pounds, are soluble in the solvent used to dissolve bal-
sam. These must be mounted in castor oil and given a
protecting ring of shellac.
To mount in castor oil the preparation is made on a
cover glass instead of on the slide. Dehydrate the crystal
either in alcohol or in the desiccator. Spin a ring of shel-
lac or gold size on the slide and allow to dry. When dry
add another ring, building up the depth of the cell thus
formed until it slightly exceeds the height of the cover
glass preparation. When the cell is nearly dry fill it with
castor oil, take the preparation in the fine forceps and
bring it, crystal down, over the cell. Lower the cover
glass in place gently to exclude air bubbles, then press the
cover on to imbed its edge in the shellac. Set aside to
dry. Clean off excess oil with xylol and spin a finishing
ring of shellac on the edge of the cover.
Crystals may be mounted dry in the same sort of cell.
All operations are the same except that no oil is used in
the cell. Cells for deep mounts may be made as described
for mounting insects in Chapter VIII.
Slides and cover glasses that are used for crystal growth
144 MICROSCOPIC TECHNIQUE
must be absolutely clean, free from any trace of grease or
dust. Any foreign matter on the slide or cover glass will
interrupt the formation of the crystals, preventing nor-
mal development.
An interesting experiment in crystal formation may be
conducted with a strong, but not saturated, solution of a
salt that is placed on the slide and allowed to dry in the
air of a room in which people are moving about. The
reason for using a strong rather than a saturated solution
is that crystallization might begin too soon. Place a drop
of the solution on a clean slide and set it where clust par-
ticles will fall into it from the air. Presently, as the water
evaporates and the liquid reaches the saturation point,
crystals will form about each dust particle'as a nucleus.
CRYSTALS OF METALLIC SILVER While on the subject
of crystals let us examine a method of making a very beau-
tiful slide of metallic silver crystals. These crystals do not
polarize, but are mounted in an opaque cell on the slide.
Place a perfectly clean slide in the turn table and spin a
shellac cell, making it of fair depth. While the cell is
drying make a 1% solution of silver nitrate in distilled
water. When the cell is perfectly dry place a drop of the
silver solution in the center and drop a few fragments of
copper, made by filing a copper wire, into the solution.
Set away in a place free from dust and in a short time
beautiful dendritic crystals of metallic silver will form
around the bits of copper dust. Leave until perfectly
dry, spin a ring of shellac on the cell and imbed the cover
glass. Paint the back of the slide with India ink or
opaque black show-card color to make a black back-
ground.
STARCHES All starches polarize beautifully, either
with or without the mica plate. Without the plate they
THE POLARIZING MICROSCOPE 145
show the characteristic cross in black, while with the plate
the grains are beautifully colored, the cross still remain-
ing. The shape and size of grains of starch varies with
each vegetable substance from which it was taken. It is in-
soluble in cold water, hence is easy to procure in a free,
unadulterated state from grains and vegetables. To ob-
tain starch grains from tuberous-rooted vegetables svich
as potatoes, sweet potatoes, yams, etc. simply scrape the
cut surface with a knife and place the scrapings in a test
tube of distilled water. When a sufficient quantity has
been collected shake it well and strain through linen fine
enough to retain the large particles of cellular tissue but
allow the starch to pass. Allow the starch to settle, which
will take place in a few minutes, then decant the water
and replace with clean water. Repeat the washing sev-
eral times until the starch is perfectly clean. Preserve in
distilled water.
Starch grains for the polarizer should be mounted in
balsam for best results. When the starch is clean, pre-
pare a slide with Mayer's albumen fixative (Chapter V) .
When nearly dry add a drop of water containing the
starch. Spread evenly with a needle and allow to stand
in a dust-free place until nearly dry. The film should be
just moist, not wet enough to run when the slide is tilted.
Plunge the slide in this condition into 70% alcohol to
coagulate the albumen, then transfer to 95% alcohol and
dehydrate in absolute alcohol. Clear in toluol until trans-
parent and mount in balsam.
Dried specimens such as cereal grains must be soaked
in cold water until soft enough to scrape, then treated as
above. Small seeds should be placed in a mortar with
water and broken into small bits. Prolonged maceration
of the seeds in cold water will eventually break up the
146 MICROSCOPIC TECHNIQUE
cells and liberate the starch grains, when they are treated
as above.
STARCH IN SITU To show the arrangement of starch
grains in their natural positions in the cells the material
is first cut into small pieces and dehydrated by passing up
the ascending series of alcohols to absolute alcohol. Since
the character of starch is altered by heat, starch-bearing
materials cannot be imbedded in paraffin for sectioning.
To cut sections make a cylinder of carrot to fit the well
microtome. Split this through the center longitudinally
and hollow out each half so that when they are placed
together the object to be cut will be gripped tightly.
Force into the microtome and cut fairly thick sections.
As the sections are cut place them in absolute alcohol.
Add to this alcohol enough of a 2% solution of methyl
violet in absolute alcohol to color it strongly. Prepare
the slide beforehand by applying a very thin film of di-
luted balsam, and allow to dry. While the balsam is dry-
ing the stained section should be clearing in toluol.
When it is perfectly clear place it on the prepared slide
with a section lifter. The toluol remaining in the section
will soften the balsam enough to make the section adhere
tightly. Add a drop of balsam and a cover glass, sup-
ported on glass rods.
Sections of watery materials may be mounted in gly-
cerin jelly if the student does not wish to go through the
more involved processes required for balsam mounts, al-
though glycerin preparations are not as clear as balsam
preparations. Pass the sections down the alcohol series
to water, stain in aqueous methyl violet or malachite
green, wash away excess stain with water and mount in
glycerin jelly. (Chapter VIII.)
RAPHIDES These crystals of calcium oxalate are pres-
THE POLARIZING MICROSCOPE 147
ent in a large number of vegetables and plants. Cacti
are very prolific, as are the cuticles of onions, garlic, hya-
cinth and lily. Rhubarb is a good source of raphides,
which may be procured by squeezing the juice from a cut
end of the stem onto the slide. The name is from the
Latin raphis, meaning a needle, and is suggested by the
form many of them present. Some are long and slender,
others are set in groups of rays like a rosette, while still
others are short and stout. They vary in size from ex-
tremely small ones to some as large as 1/25 of an inch.
They give brilliant colors with the polarizer.
Raphides are insoluble in water, hence can be pro-
cured by maceration in water until the tissue falls apart.
The larger pieces are picked out and the material washed
repeatedly in water. Between each washing the raphides
are allowed to settle. Pipette off the supernatent liquid
until no plant tissue remains. Dehydrate in absolute al-
cohol, clear in toluol and mount in balsam, supporting
the cover.
SILICIOUS SKELETONS Many of the grasses, canes and
cereal grain stems have a skeleton or cuticle of silicate
which makes a splendid polarizer object. The material
is first dried, either by heat or alcohol, and then immersed
in strong nitric acid and boiled. An effervescence will
take place as the tissue is destroyed, and when this stops
more acid must be added. Boiling and the addition of
fresh acid must continue as long as there is any efferves-
cence. Then replace the last acid with fresh and allow to
macerate until all tissue is dissolved. Wash in distilled
water until free of acid, using litmus paper as an indi-
cator, dehydrate in alcohol, clear in toluol and mount in
balsam.
LEAF CUTICLES The thin outer coatings of leaves are
148 MICROSCOPIC TECHNIQUE
very interesting subjects when separated from the leaf and
examined by polarized light. Many leaves are amenable
to simple maceration in water until rotten, when the cuti-
cle can be stripped off in fairly large pieces. Other leaves
of hard texture like the rhododendron, azalea, laurel, etc.
should be cut into pieces about a quarter of an inch
square, placed in a test tube with strong nitric acid and
heated until a separation of the cuticle begins as indicated
by the formation of blisters on the leaves. Throw the
contents of the test tube into a large volume of clear water
as soon as this occurs, to arrest the action of the acid, be-
fore the cuticle dissolves.
Separate the cuticles with dissecting needles, wash them
well in distilled water to free from acid, pass through
50%, 95% and absolute alcohol, clear in toluol and
mount in balsam. The cuticles may be stained in mala-
chite green by adding a few drops of an alcoholic solution
to the absolute alcohol.
LEAF SCALES AND LEAF HAIRS Leaf hairs exhibit an
enormous variety of forms and polarize beautifully. Sec-
tions of leaves containing hairs may be made as instructed
in Chapter V, if it is desired to show them attached to the
leaf. While this is instructive, the true arrangement of
the hairs is not always revealed because the paraffin im-
bedding flattens them. They may be procured as isolated
objects by hardening the leaf in alcohol and then cutting
off the hairs close to the leaf with a sharp scalpel or razor.
Collect the hairs on a filter and handle them through de-
hydrating and clearing as described in Chapter V. The
best results are secured when hairs are arranged in order
on the slide. It is tedious work but well worth the effort.
Prepare the slide as described for mounting starches.
Then, using the fine-pointed red sable brush to handle it,
THE POLARIZING MICROSCOPE 149
place each hair separately under the dissecting micro-
scope. They may be arranged radially, as the spokes of a
wheel, in rows, or in crosses. The latter arrangement is
especially interesting since it produces another color
where the two hairs intersect.
Leaf scales are procured by scraping the leaf surface.
These objects are very difficult to free of air, requiring
prolonged immersion in the clearing fluid. Dehydrate in
absolute alcohol, clear in toluol and mount in balsam.
TEXTILE FIBERS The fibers of cotton, flax, silk and
wool are all optically active. If the raw fibers are avail-
able they should be mounted individually as type speci-
mens for comparison with test subjects. Dehydrate in
absolute alcohol, clear in toluol or creosote-xylol and
mount in balsam.
Samples of woven fabrics may be prepared in the same
way and these afford especially beautiful polarizer objects.
RADULA OF SNAILS Tongues of snails are excellent
objects for dissection. If the specimen is large enough so
that the tongue may be easily dissected out, do so, being
sure the coiled up portion found inside the snail is re-
moved with it. Place in 10% potassium hydroxide solu-
tion for a few days to destroy the soft parts. Wash well
with distilled water and spread the tongue on a slide. Lay
a strip of paper on either side of the object to prevent
crushing it and cover with another slide. Tie the two
slides together, dehydrate in absolute alcohol, then clear
in creosote-xylol. When the object is cleared cut the
string binding the slides and separate them. The tongue
will probably adhere to one of the slides. Leave in creo-
sote-xylol for a while longer, then transfer to a clean slide
and mount in balsam.
HAIRS The hairs of all animals provide splendid
150 MICROSCOPIC TECHNIQUE
polarizer objects, both in transverse and longitudinal sec-
tions. The usual method of procuring sections of hairs is
that of shaving closely without the use of soap. While
this works after a fashion, the sections are usually diago-
nal-transverse, since hairs do not grow perpendicular to
the skin. A better method, in which exactly transverse
and longitudinal sections can be had is as follows.
Take a bundle of hairs and tie them together with
another hair. Make up a strong solution of gelatin by
solution in water, liquifying it on the water bath. Im-
merse the bundle of hairs in the gelatin and leave until
entirely impregnated. Remove on the point of a needle
and expose to the air until the gelatin cools and becomes
semi-solid. Push the mass off the needle into 70% alcohol
and allow it to harden. Dehydrate in absolute alcohol,
imbed in carrot and cut sections in the well microtome.
The sections are then dehydrated, cleared in creosote-
xylol and mounted in balsam.
The only objection to this method is the fact that the
imbedding gelatin is included in the mount. By mak-
ing paraffin sections this objection is overcome and the
isolated sections of the hairs only are on the slide.
To make paraffin sections the bundle of hairs is pre-
pared as above, dehydrated, cleared and infiltrated with
paraffin exactly as described in Chapter V. All operations
are carried out as described there, up to the point of de-
hydration in absolute alcohol. Since these sections are
not being stained they are not passed down the graded
alcohol series but are cleared in toluol directly from the
absolute alcohol and are then mounted in balsam.
HORNY TISSUES Structures that are composed of com-
pressed cells, such as quills, horns, hoofs, claws and nails
must be macerated in 30% solution of potassium hydrox-
THE POLARIZING MICROSCOPE 151
ide until soft. The period of immersion will depend
upon the degree of hardness of the material and the size
of the pieces. When soft enough to cut, wash the material
well in water to remove all trace of alkali and preserve in
alcohol until needed. Hollow objects, like quills, small
horns and small claws should be then dehydrated, infil-
trated with and imbedded in hard paraffin for sectioning.
Hoofs should be cut in carrot and the sections handled
as loose sections, or they may be flattened on albumenized
slides and handled like paraffin sections. Both transverse
and longitudinal sections should be made to demonstrate
the cell arrangement.
CHAPTER XI
Accessories
In the introduction that prefaced these chapters a state-
ment was made that microscopy is not expensive. Refer-
ences to chemicals and equipment may raise doubts as to
the veracity of that statement. Such doubts would be
quite justified if the student based his conclusions on the
cost of accessories as listed in the supply catalogs. Fortu-
nately there is another course open to him, for nearly
every piece of accessory equipment can be made at home,
or substitutes can be provided at low cost. There are
some items, such as dissecting knives, which, for the sake
of comfort in working, should be purchased. As for
chemicals, there are really only a few chemicals needed.
Most of them are comparatively inexpensive and only
small quantities of each are used. Therefore, the state-
ment that microscopy is not expensive is true, as it is the
purpose of this chapter to prove by discussing the equip-
ment needed, the ways of making it at home and the sub-
stitutes for commercially manufactured items.
DISSECTING MICROSCOPE A dissecting microscope is
quite essential for separating objects to secure parts to be
studied. Fig. 53 opposite shows an inexpensive form of
manufactured instrument. While such an instrument
would be pleasant to work with, a satisfactory substitute
can be made at a cost less than the cheapest manufactured
product by supporting a lens in a holder made of stiff
wire. The lens used has a magnifying power of 7.5 and
costs seventy-five cents. The student who has facilities
for doing the work can make a dissecting microscope like
152
ACCESSORIES 153
the cheaper one illustrated, with very little trouble, using
the lens mentioned above.
Fig. 53. Bausch 8c Lomb inex-
pensive Dissecting Microscope
for student use. X7-5
DISSECTING NEEDLES Dissecting needles were men-
tioned in Chapter VIII. The catalogs list these items in
several styles, with bone, metal or wood handles and
equipped with little chucks to hold the needles. These
instruments look very professional and cost in propor-
tion. Two are shown in the lower right corner of the
illustration, next to the large forceps.
Satisfactory substitutes for commercial dissecting
needles can be made at home for a few cents. After all,
the handle is not important. All it is for is to hold the
needle, and for this purpose wood is as good as any other
material. Hammer shanks from pianos, which are smooth,
round sticks of maple about three-sixteenths of an inch
in diameter, are excellent for this purpose and may be
procured from any piano repairman simply for the ask-
ing. Cut the ends square and bind one end to form a
ferrule by wrapping it tightly with fine copper wire for
about a quarter of an inch. The ends may be soldered
to hold them in place. Now take the needles you pro-
pose to use, break or grind off the eye and grind the end
to a point on a sharpening stone. Hold the needle in a
154 MICROSCOPIC TECHNIQUE
vise and drive the handle on it with light taps to a depth
of about a quarter of an inch. Thin needles are not likely
to split the wood if the handles are ferruled.
Handles for heavy needles such as are required for some
dissections must be prepared for the needle by drilling or
burning a small hole before the needle is driven home.
These, too, should be ferruled since they are sometimes
used under considerable pressure and might split. Lack-
ing a small drill, burn the holes with a needle smaller
than the one to be used.
The number and shape of dissecting needles is entirely
up to the student. They are so inexpensive that a suffi-
cient number should be provided to insure clean ones
always being at hand ready for use in emergency. Most
work may be done with a selection of four or five sets of
two each. One set should have extremely fine points for
the most delicate work, such as teasing apart single cells.
For these use the finest sewing needles, or the finest grade
of entomological pins. These, with the heads removed,
make excellent needles. One set of two needles should
have the points bent at right angles to the shank, while
two should be straight. One pair of curved and one pair
of straight needles in a heavier weight will be needed for
coarser dissections that would destroy the delicate points
of the fine needles. A third set of two straight and two
curved heavy needles with blunt points should be pro-
vided for very coarse work such as tearing apart large
pieces of tissue to be later teased with the finer needles.
Always keep the points clean and sharp by grinding on a
carborundum stone.
DISSECTING KNIVES In the upper right corner of the
illustration (Fig. 54) are four of the Bard-Parker knives
mentioned earlier. Directly under these is another style
ACCESSORIES 155
Fig. 54. Dissecting instruments.
made by the same manufacturer. These are the most
useful dissecting knives to purchase, for reasons given in
an earlier chapter. At the upper center is a group of four
lancets and a bone saw. While these delicate instruments
are very useful at times they are not absolutely necessary
if the student has a Bard-Parker handle with the No. 1 1
and 12 blades.
FORCEPS Three types of forceps are illustrated, one
with fine curved tips for setting cover-glasses, picking up
minute objects and for use in dissections. They may be
procured in most drug stores at very small cost. The
large forceps in the lower right corner is useful for taking
specimens out of bottles and holding slides in the flame
for warming, as well as in dissecting. The third forceps
in the upper center is known as a suture forceps. It is
used by surgeons to hold the suture needle when sewing
wounds, but it finds many uses in dissection work. For
example, when slitting a large piece of material or an
entire animal to get at some internal organ, this instru-
156 MICROSCOPIC TECHNIQUE
ment is used to hold one side of the subject and keep it
out of the way. The corrugated jaws afford a firm grip
on slippery material, while the handles are equipped with
a simple lock that keeps the jaws closed until deliberately
opened.
SHEARS Poor shears are worse than none at all. A pair
of good, small shears may cost a dollar or more, but are
worth every cent of it. The ordinary scissors sold by de-
partment stores for embroidery work are usually poor
specimens. Go to a cutlery store and select a good pair of
shears about four inches long. Be sure they have fine
points that meet perfectly and cut cleanly to the extreme
tip. This is important, as in dissections it is very fre-
quently necessary to make short cuts that can be accom-
plished only with the very tips of the shears. Keep them
sharp and clean and do not strain them by cutting hard
or tough materials beyond their ability, for this distorts
the points and destroys their fine adjustment. Manicure
shears with fine, curved points are sometimes useful but
not essential, for the lancet or scalpel may be pressed into
service if it is necessary to reach a difficult place.
SECTION LIFTER Loose sections cut from bulk objects
and small pieces of tissue can be handled nicely with the
instrument shown in the lower left corner, which takes the
place of a proper section lifter and costs much less. It is
a steel manicure instrument used for pushing back cuticle
and cleaning the nails. One end is flat and pointed, the
other broad and flat.
LAMPS Artificial illumination for visual examina-
tion and photomicrography of subjects may be provided
in several ways, some of them good, others poor. Visual
examination of transparent specimens may be made with
ordinary room lighting, although for the higher powers
ACCESSORIES
Fig. 55. Small
lamp for visual ex-
amination of mi-
croscopical sub-
jects.
Fig. 56. High power
lamp for photomi-
crography of micro-
scopical subjects.
this is inadequate. General practice in laboratories is to
provide individual lamps for each microscope. This af-
fords maximum control of the light and does not interfere
with other workers.
A desk light in a reflector, shown in Fig. 63, is one way
to provide light for both viewing and photographing
specimens. If the reflector is turned so that the light does
not enter the eyes of the person using the microscope, or
the camera lens, this type of lighting is quite satisfactory
for less critical work. Photomicrography, however, fre-
quently requires transmission of monocromatic light by
the introduction of suitable filters, when a proper lamp
housing with built-in provision for niters is superior.
Such a lamp is illustrated in Fig. 55. The housing for
this unit is a tin can in which sodium bicarbonate was
packed for sale. The can was procured at a drug store at
no cost. Fastened to the inside bottom of the can with
158 MICROSCOPIC TECHNIQUE
two machine bolts is a ten cent porcelain receptacle. Six
feet of fixture wire connects the receptacle to the supply
outlet. The reflector behind the bulb is a z\ inch watch
glass. The convex surface was sprayed with aluminum
paint, which provides a splendid reflecting surface. It is
held to the can by a tin spider of three radial arms 120
apart. They are a quarter of an inch wide and meet at a
three quarter inch disc in the center. Through this disc
passes a small machine bolt that holds the entire unit to
the can. The free ends of the spider are bent over to
secure the reflector. The lid of the can was slit with a
fine jeweler's saw. Five slits were made across the top.
Each slit terminates in a short slit at right angles, reaching
almost to the middle of the turned-up strips. These strips
were left attached to the lid by bits of metal about one
thirty-second of an inch wide. These hold the strips,
which were then turned up as shown, to provide ventila-
tion.
A fifty-watt daylight bulb was screwed into the recep-
tacle and the center of the filament measured from the
base. A hole one and one half inches in diameter was
then cut in the side of the can with a dull jack knife with
its center exactly in the center of the filament. A frame
to hold filters or ground glass was made of thin cardboard
and secured to the housing with small machine bolts, and
the lamp housing was finished with a coat of black lac-
quer. Entire cost was about forty-five cents.
The lamp house shown in Fig. 56 is larger and slightly
more elaborate, because it was made for photomicrog-
raphy entirely. In order to minimize the possibility of
vibration, the exposure was reduced by increasing the
power of the light to 250 watts. A concentrated filament
bulb was selected, with a standard screw base. The con-
ACCESSORIES 159
struction is the same as that of the smaller lamp house,
the only difference being the shape and size of the can,
which in this case is round. The exterior is provided with
a small aluminum shelf and a spring clip to hold a Flor-
ence flask, which serves three purposes. First, it acts as
a condenser to concentrate the light in a small spot. Sec-
ondly, being filled with water, it absorbs part of the heat
from the lamp. Finally, by coloring the water with suit-
able dyes or chemicals, a monochromatic light may be
produced. The segment of arc at the bottom, held by a
wing nut, permits adjustment to any angle. The entire
unit was assembled in three hours from scrap material,
and cost about fifty cents, excluding bulb and flask.
SLIDES These are inexpensive items to buy ready-
made, but when making a large number of mounts the
cost becomes a factor. These too may be made at home
at small cost. Any photographic supply house can
supply lantern slide cover glasses at about twenty cents a
dozen or a dollar and seventy-five cents a gross. They
measure 3^" by 4" and are cut from thin, clear glass,
usually free from bubbles. To make your own slides,
rule a piece of paper with a rectangle 3^" by 4". Draw a
line along one long side a quarter of an inch from the
edge. Now divide the remaining 3" by 4" rectangle into
four equal parts, the long sides parallel with the three-
inch side. Lay a lantern slide cover glass on the large
rectangle and with a glass cutter scratch a line at the three-
inch mark. Make similar scratches on each of the one-
inch marks and break the glass. If the cutter is sharp the
breaks will be true and fairly smooth. Grind the edges
on a carborundum stone or fine emery paper to make
them smooth. The cost per slide is thus very low and for
student work the material is quite satisfactory.
i6o MICROSCOPIC TECHNIQUE
SPIRIT LAMP Several operations in preparing micro-
scope slides require moderate heat. Bunsen burners, un-
less of the micro type, are too hot, so a spirit lamp or al-
cohol lamp is used. These may be purchased for from
fifty cents to a dollar, but one can be made for fifteen
cents. Most auto accessory stores and five-and-ten cent
stores sell small copper oil cans for ten cents. Buy one of
these and cut off the spout so that about one inch remains
in the screw cap. Five cents worth of dry asbestos pack-
ing, also obtainable at the accessory store, provides the
wick. A small empty cartridge-case or glass vial may be
used to cap the wick and reduce evaporation.
BALSAM BOTTLE A proper balsam container costs in
the neighborhood of fifty cents. As far as results are con-
cerned it is worth the price because it has an outside-
ground stopper, a very important feature on a balsam
bottle. Balsam has the nasty trick of cementing material
together, so that a ground-in stopper of the reagent-bottle
type would become cemented into the neck. Corks break
and the bits get into the balsam and cause trouble. So we
use an outside-ground stopper or cap on the balsam bottle.
The cap is ground on the inside and the bottle neck on the
outside. This keeps the balsam away from the two con-
tact areas, yet makes an air-tight seal.
A most efficient substitute for a purchased balsam bottle
may be had for the asking from a dentist. Many dentists
make what are called inlay fillings. Instead of filling the
cavity with silver-mercury amalgam they take an impres-
sion of the cavity, cast a solid metal filling from the im-
pression and cement this in place. The cement used for
this purpose is a dry powder that is mixed with metaphos-
phoric acid. The acid is sold in small (about one-half
ounce) bottles with outside-ground caps. Ask a dentist
DIAMETER-BAKELITE
OBJEC T DISC -BRA SS
SOLDER
\
2-56 SCREW
"BRASS PLATE
^-10-32 THREAD
14-40 THREAD
*-t"BRASS TUBE
BRASS PLUG
"SOLDERED IN
KNURLED KNOB
Fig. 57. Well microtome lor cutting sections.
i6 2 MICROSCOPIC TECHNIQUE
for one of these empty bottles, wash it thoroughly, dry it
perfectly and you have an excellent balsam bottle.
It may be improved for this purpose by providing the
cap with a dispensing rod for placing balsam on the slide.
Cut a piece of small-diameter glass rod slightly longer
than the distance from the inside of the cap to the inside
bottom of the bottle. Fire-polish one end in the flame of
the spirit lamp. Flatten the other end into a small flange
by holding it in the flame until soft, then pressing it
against a piece of heated metal. Coat the inside of the cap
and the flange on the rod with water glass (sodium sili-
cate) . Let this dry a little and press the two together,
holding them in position until the cement sets. The rod
will be permanently attached to the cap, which, used as a
handle, will keep the fingers free from balsam.
MICROTOME The microtome was mentioned in
Chapter V. This instrument is illustrated in use in Fig.
33, and as a unit in Fig. 57. Its purpose is to hold im-
bedded objects while thin sections are cut for mounting on
a slide. It consists primarily of a tube, closed at the lower
end, which is fitted with a fine-threaded screw by which
the specimen is advanced between each section cut. The
upper or open end is provided with a large flat plate that
affords a bearing surface for the knife.
All dimensions are given in the drawing, from which
the student can make or have made at small cost a simi-
lar microtome that will serve his purpose nicely. The
method of using the instrument is fully described in
Chapter V.
PARAFFIN OVEN Chapter V included numerous refer-
ences to a paraffin oven, used for imbedding objects to be
sectioned. Catalogs list these at prices ranging from
ten dollars for the simplest oven to several hundred dol-
ACCESSORIES 163
Fig. 58. Paraffin oven for paraffin imbedding.
lars for the most elaborate. This important item of
equipment can be built by any tinsmith in the form shown
in Fig. 58, or a substitute may be provided by the student
himself. This substitute is known as a Brown University
oven, because it was developed and is used extensively by
Brown University students' laboratories, both as a paraf-
fin oven and an incubator for micro-organisms.
The galvanized sheet iron oven shown in Fig. 58 was
made for the author by a local tinsmith at a cost of five
dollars. The inside dimensions are twelve inches wide,
eight inches high and eight inches deep. It is a double-
walled type of oven with a one-inch space between the
walls on five sides, and has a hinged door insulated on the
inside with celotex. Double walls were provided so that
the space between them may be filled with water, the oven
then being placed on an electric hot plate for heating,
making possible its use either as a paraffin oven or an in-
cubator for bacteria. At the rear left corner a one-inch
tube is soldered into the top. This communicates with
164 MICROSCOPIC TECHNIQUE
the interior of the oven so that a thermometer may be
inserted to measure the temperature. A similar tube is
provided at the rear right corner for filling the space be-
tween the walls with water. The oven may be heated by
using either the water jacket or an electric light bulb.
For some purposes, such as incubating bacteria cul-
tures, the oven is used with the water jacket filled. When
using it as a paraffin oven the electric bulb is used. The
space between the walls is then empty, the dead-air space
affording fair insulation to reduce heat-loss by radiation.
The thermometer is placed in one hole of a two-hole rub-
ber stopper, and the supply wire for the bulb goes through
the other hole. The heat from a fifty-watt bulb is just
enough to maintain a temperature of 56 58 C. By
using a smaller bulb a temperature of 37- 39 C. may be
easily maintained for flattening paraffin sections. Thus
a very useful accessory was provided at small cost.
The Brown University oven can be built even more
cheaply. It consists of a white enamel sauce pan without
a handle, a cover of sheet metal and an electric light bulb
to provide heat. The cover has a hole cut in the center
just large enough to permit inserting the lower part of a
bulb socket. A short length of metal tubing should be
soldered on some part of the cover to allow the insertion
of a thermometer. The material to be imbedded may be
set directly on the floor of the oven or suspended from its
sides in little wire baskets hooked over the rim of the
sauce pan. Changing the size of the bulb gives the re-
quired control of temperature. Since the factors of heat
input and radiation are constant, the only variation in
temperature will be that caused by variation in the am-
bient temperature. This, too, may be controlled within
rather narrow limits by immersing the oven in a large pan
ACCESSORIES 165
of water. Such an oven is very efficient and should cost
less than a dollar to construct.
Even this low cost might deter some students from do-
ing paraffin imbedding, so the suggestion illustrated in
Fig. 59. Test tube and Erlen-
meyer flask used tor imbedding
small objects in paraffin.
Fig. 59 is made. This is simply an Erlenmeyer flask con-
taining water, and a test tube for the paraffin and the ma-
terial to be imbedded. Heat is supplied by the spirit
lamp which is moved toward or away from the flask as re-
quired by the temperature to be maintained. Tempera-
ture control is not very accurate, but if the paraffin is kept
just above the melting point the material will not get too
hot and no fear need be entertained.
TURN TABLE This device is used for spinning rings
of gold size, shellac or lacquer for shallow cells, and for
i66 MICROSCOPIC TECHNIQUE
applying a finishing coat of black lacquer to the edge
of the cover glass. The practice of painting a finishing
ring half on the cover glass and half on the slide is rapidly
losing favor except for glycerin jelly mounts. Balsam
mounts are perfectly tight if made with a slight excess of
balsam, as already described, and gain nothing by ringing.
Glycerin jelly mounts may be made more secure by ring-
ing with shellac or gold size.
Neat shallow cells, however, do require the use of the
turn table. In its simplest form this is a block of wood,
on one end of which is a circular plate free to rotate about
its central axis. Provision must be made to hold the slide,
or centrifugal force will throw it off the table.
To make a turn table, take a block of wood six inches
long, four inches wide and seven-eighths of an inch thick.
In one end, near the edge, locate a center and drill with a
3/16" drill. Then take another piece of wood, quarter-
inch plywood is excellent, and cut a disc four inches in
diameter. Through the center of this disc run a 3/1 6"
flat-head stove bolt one inch long, with the nut on the
under side. Counter-sink the top so that the screw head
is flush with or slightly below the surface of the disc. The
slides may be held with spring clips like those used on the
microscope stage. Place the leg of the bolt in the hole in
the large block. On the face of the disc rule a rectangle
one by three inches in the exact center, to act as a guide
in placing the slide.
To use the turn table, dip a small camels-hair brush in
the gold size or shellac, start the table spinning with a
quick flip and lower the tip of the brush to the slide. The
result will be a circle of diameter depending upon the
distance of the brush from the center of the turn table.
POLARIZER Of the many accessories for the micro-
ACCESSORIES 167
scope, none can compare with the polarizer for sheer
beauty of results. The theory of polarized light has pre-
viously been discussed briefly, so let us examine the meth-
ods of securing polarized light at small cost.
Commercial polarizers and analyzers consist of natural
crystals of Iceland spar (calcite) , sawed in two longitudi-
nally, and the two halves cemented together with Canada
balsam. These crystals are called Nicol prisms, after their
inventor. One of these nicols, the polarizer, is placed be-
low the microscope stage; the other, the analyzer, is placed
above the stage, between the specimen and the eye of the
observer. Two types of polarizers are adaptable to stu-
dent construction, one the nicol type, the other a reflect-
ing type. The former is much more efficient, though
slightly more difficult to construct, while the second type
sacrifices efficiency for simplicity of construction.
Since the nicol type is the more efficient, and the diffi-
culties of construction not insurmountable, that is the
one to build. The first requisite is a pair of Iceland spar
crystals, which may be procured from any house that sells
mineralogical specimens to schools and collectors. When
ordering them by mail be sure to specify that good, clean,
clear crystals are desired. These are slightly higher in
cost than less desirable specimens, but are well worth the
difference. The one for the polarizer should be a rhomb
measuring about one half inch square. It will be some-
what rectangular for the four side faces are never equal.
The length is not very important, one half to five-eighths
of an inch being ample. The analyzer crystal need be
only about a quarter of an inch square and the same in
length.
Having procured suitable crystals they must be sawed
in half through the obtuse angles as shown in the diagram,
i68 MICROSCOPIC TECHNIQUE
Fig. 60. Iceland spar is a compound of calcium, and is
comparatively soft, so sawing will not be difficult. The
best way to hold the crystal while sawing it is to grip it in
a small vise between two pieces of thick cork, thus mini-
mizing the danger of breaking it. This danger is very
real, for while the mineral is soft it has very well defined
cleavage planes along which it breaks with great ease. Be
Fig. Go. Nicol prism,
showing plane of sec-
tion for polarizer and
analyzer.
very careful while sawing to keep the saw moving in a
straight line, without twisting, or the crystal will cleave
and be ruined.
Sawing is done with the emery saw as described in
Chapter VII. First make a deep scratch along the in-
tended line of division with a sharp-pointed instrument,
such as a heavy needle, in order to start the saw. Then
use the emery saw with plenty of water. After the cut has
extended to the center of the crystal it is advisable to take
it out of the vise and place it upon some hard support,
holding it with the fingers to complete the cut. When
about a sixteenth of an inch remains to be cut proceed
ACCESSORIES 169
very slowly, using very light pressure on the saw, for at
that point the crystal is very apt to break with a ragged
edge. Be warned again, to prevent cleaving keep the saw
going in a straight line and do not twist or turn it. When
one crystal has been sawed proceed in the same manner
with the other one.
The divided crystals are now polished to a glass-like
surface at each end and on the cut faces. The student
may do this himself or have it done by a manufacturing
optometrist. If it is done at home the faces are first
ground flat on a piece of plate glass, using very fine emery
powder as the abrasive, and plenty of water as the vehicle.
When the faces arc flat, wash plate and crystals with water
to remove every bit of emery. Then continue the grind-
ing with pulverized rotten stone. Commercial rotten
stone frequently contains large particles that might
scratch. To prevent this tie the powder in a piece of fine
silk or bolting cloth and shake it over the glass, thus sift-
ing out only the finest material. This grinding will pro-
duce a smooth flat face, but will not polish the crystal.
The final high polish is secured by using jeweler's rouge
and a piece of thin felt stretched taut over plate glass.
The felt is moistened with water, the rouge rubbed into
it and the polishing done by rubbing the crystal on the
felt. While the work entailed is not difficult, it is some-
what tedious, and may be avoided by turning the job over
to an optometrist. With his power tools he can turn out
a good polishing job in a fraction of the time required to
do it at home.
The polished crystals are now cemented together with
some of the same balsam that is vised to cement cover
glasses to slides. Cement the two halves together so that
they occupy the same relative positions as they did in the
170 MICROSCOPIC TECHNIQUE
whole crystal. Warm the halves slightly before applying
the balsam, but do not get them too hot or the expansion
will cleave the crystal. Cover the entire face with a thin
film of balsam and press the two together to assure perfect
adhesion at every point. The cemented nicols are now
set aside for the cement to harden after which the four
long sides are painted with black paint, India ink or
opaque water color.
While the nicols are setting the mounts may be pre-
pared. The polarizer prism is the most difficult to mount
because this must be located under the microscope stage.
The exact method of attaching it to the stage will depend
upon the microscope and the amount of space available.
The simplest mount is made of cardboard tubing large
enough to permit insertion of the nicol. A narrow flange
can be glued to one end of the tube. Thus the nicol may
be attached to the stage by spring clips, or, more simply
still, by strips of adhesive tape. If the microscope is
equipped with a substage condenser it is an easy matter
to design the nicol mount so that it slips over the con-
denser housing.
The analyzer may be conveniently mounted in the tube
of the microscope, either in the eyepiece or lower in the
tube. Whatever style of mounting is selected it is es-
sential that one of the nicols be arranged so that it can be
rotated about its long axis, for only in this way can the
phenomenon of polar rotation be studied, and the gor-
geous colors resulting from some materials with crossed
nicols be seen. Mounting the analyzer in the eyepiece of
the microscope makes this possible by simply rotating the
eyepiece. If it is more convenient to mount the polarizer
in a rotating mount do so, for it makes no difference which
one turns.
ACCESSORIES 171
The most gorgeous colors are produced with polarized
light when a mica or selenite plate is placed in the
optical path. Excellent results are obtained by using a
piece of clear mica. For this purpose there is nothing
better than a mica disc such as is used in a phonograph re-
producing head. This is clear and unsplit, of uniform
thickness, and the best mica obtainable. Discs may be
purchased for a few cents at music stores or from phono-
graph repairmen.
From the foregoing discussion it is evident that easily
procurable, low cost substitutes may be provided for the
more pretentious, expensive commercial items of equip-
ment for the pursuit of microscopy. A little thought and
study is required. After all, the end is what matters, and
workmen are not judged by their tools. The early in-
vestigators in microtechnique had little to work with, yet
they conducted researches and made the discoveries
which are the foundation of our present knowledge of sci-
ence. If satisfactory results can be obtained from the use
of any equipment, that equipment is serving its purpose,
regardless of its appearance or origin. The greatest pho-
tographer of snowflakes who ever lived did his work with
an old battle-scarred camera and an equally old lens, yet
his photographs were sought after by scientists from all
over the world. The point is, he knew how to get results,
regardless of, or in spite of, his equipment.
CHAPTER XII
Photomicrography
Ever since the microscope has been in use students
have made graphic records of the details it reveals. Be-
fore the invention of photography these records were
drawn by hand, a laborious and often inaccurate method.
The development of photography, however, placed at
the disposal of the student a rapid and accurate means of
preserving in exquisite detail every feature of the object
under observation.
Good photomicrographs depend upon several factors,
not the least of which is good slide preparation. The
camera records every detail of the slide, and if preparation
is slovenly, it will present a picture of similar slipshod
methods. The artist may alter his hand drawings of mic-
roscopical subjects but the camera does not have this
power of elimination, therefore a more severe burden is
placed upon the preparer. Unless the material has been
properly prepared and mounted it is useless to expect
good pictures. On the other hand, good preparations
yield passable results even with mediocre microscope and
camera equipment. The student should always aim to
produce the best results possible with the equipment at
his disposal. This will necessitate the keeping of accu-
rate records of every exposure made. Whether the nega-
tive is good or bad does not make the slightest difference
in the record, for from this record he can make the neces-
sary alterations in conditions to improve the succeeding
effort. The following table is arranged for the notation
of results of photomicrographic exposures.
172
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u
Q
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174 MICROSCOPIC TECHNIQUE
Fig. 62. Horizontal set-up for photomicrography, showing
polarizer and capping analyzer in place.
Reference to the notes contained in such a tabulation
will make the duplication of results more certain when
dealing with similar subjects, and will indicate the
changes necessary when undertaking work with other sub-
jects. You will note that spaces are provided in the table
for the name and catalog number and the stain used on
the preparation. This means that when the slide was
made a record was kept of each detail as noted in Chap-
ter VIII. It would be well to review the data that the
slide label and catalog should contain.
THE CAMERA Any camera can be used for taking
photomicrographs. This may seem like a broad state-
ment, but it is literally true. The only purpose served by
the camera is to hold the sensitive negative material and
provide a light-tight coupling between the microscope
and the negative. In its simplest form this purpose is
served by a wooden box with a hole at one end for inser-
tion of the microscope tube, and provision at the opposite
end for holding the film or plate. The arrangement may
be either vertical or horizontal, as best suits the conven-
ience of the student, since either produces identical re-
sults. General preference seems to be for the horizontal
PHOTOMICROGRAPHY
75
Fig. 63. Vertical set-up of camera
and microscope when using a small
camera.
set-up, since this permits simplified construction, greater
stability, easier alignment and more flexibility. Such an
arrangement is shown in Fig. 62.
It is very important that the plane of the sensitive ma-
terial be exactly parallel with the plane of the slide. If
this condition does not obtain, uniformly sharp negatives
will be impossible. If the camera has a ground glass back
for focusing, the image may be inspected visually and the
alignment altered until the entire field is sharp. Roll
film cameras and others not fitted with a ground glass can
be aligned by measuring from the stage of the microscope
to the back of the camera.
176 MICROSCOPIC TECHNIQUE
When only a few photomicrographs are to be taken at
infrequent intervals it is possible to secure satisfactory re-
sults by merely standing the camera on the eyepiece of
the microscope as shown in Fig. 63. This arrangement is
quite feasible if the construction of the camera is such that
it will rest firmly on the eyepiece. This condition is ful-
filled in most box cameras of the Brownie type, a great
many of the small folding cameras and some of the minia-
ture cameras. Those types which will not balance in the
position suggested must be supported in some way to pre-
vent falling. Lightweight cameras with a tripod socket
can be supported very nicely on a chemical ring-stand by
making an adapter with a projecting screw that engages
the tripod socket, while a clamp holds it to the vertical
rod. The microscope then stands between the legs of the
tripod. The ingenious student will be able to devise
some means of holding the camera in place on the micro-
scope.
Bear in mind three things. First, the lens mount of the
camera must be axially central with the microscope. Sec-
ondly, the front of the camera must be as close as possible
to the eyepiece of the microscope to reduce the possibility
of cutting off the edges of the circular field, and the joint
must be light-tight. Finally, the entire outfit must be set
up in a place reasonably free from vibration. If a con-
crete basement floor is available that is an excellent place
for the work. Be sure the table on which the outfit stands
is resting solidly on the floor. If one leg is short, block it
up with pieces of cardboard to insure a firm foundation.
The results attending such an arrangement are not the
best, naturally, but careful work will produce acceptable
results.
If the student wants to improve his work and get the
PHOTOMICROGRAPHY 177
best pictures his equipment is able to produce, his prepa-
rations will need to be somewhat more elaborate. For
this purpose, and especially for work at high powers, the
horizontal arrangement is far better. Great flexibility in
the disposal of equipment is one of its outstanding fea-
tures, as well as firm construction and ease of manipula-
tion. The floor space required is greater than in the
vertical type, but this disadvantage is more than offset by
the numerous advantages.
All professional and some of the amateur microscope
stands have a knee joint that permits inclination of the
tube to an angle of 90 degrees with the base, thus making
the tube horizontal. When used in this position the mi-
croscope is set on a bench or table, the camera attached
to the eyepiece and the illuminant placed behind the
stage, all in a straight line, thus making for the greatest
efficiency. Into the light path we can then introduce con-
densing lenses and diaphragms to parallelize the beam,
and filters to control its color.
ADJUSTING THE APPARATUS Set up the camera, mi-
croscope and light, fastening each rigidly in place. Place
the slide on the stage and focus it roughly, either with the
eyepiece or on the ground glass. If the microscope is
being used without the eyepiece you will probably see on
the ground glass a number of curious reflections. Ex-
amine the connection between camera and microscope.
If this is light-tight remove the ground glass and look
down the microscope tube. The cause will at once be
apparent. The inside of the tube is shiny. In this case
make a tube of black velvet to line the draw tube, and the
trouble will be corrected.
Remember that when making photomicrographs, er-
rors of focusing cannot be corrected by stopping down the
178 MICROSCOPIC TECHNIQUE
lens, as is sometimes done with photographic lenses. The
focus must be exactly right on the ground glass for critical
definition. To assist in securing the best possible focus,
the ground glass should have a clear spot. Rule fine di-
agonal lines from each corner of the glass with India ink
and a ruling pen. Over their intersection cement a cover
glass with Canada balsam. Exact focus can then be se-
cured by examining the image with a magnifier adjusted
so that it is in exact focus on the cross lines. When the
image and the lines are both in focus the definition is the
best possible.
In order to take full advantage of the resolving power
of the objective certain conditions of illumination must
be fulfilled. These are not difficult to attain at low
powers, but when lenses of wide numerical aperture are
used the conditions are more difficult to meet. Full re-
solving power is secured only when (i) the entire aper-
ture of the objective is filled with light, which may be
ascertained by looking down the tube with the eyepiece
removed, and (2) when critical illumination is used.
Critical illumination is obtained, theoretically, when
all the light waves reaching the slide and forming the
image at any one instant of time also leave the light source
at the same instant of time and come from the same point.
This condition is secured in practice by focusing the light
source on the object by means of condensing lenses.
These lenses, with a parallelizing diaphragm, are used be-
tween the light source and the substage condenser. The
flask shown on the lamp house (Fig. 56) serves as one
lens, auxiliary condensing lenses such as a plano-convex
or a double convex lens in an independent mount as
the other. Between the two lenses is placed a diaphragm,
either of the iris type or a plate with several holes of
PHOTOMICROGRAPHY 179
graded diameters. The focal length and placing of the
lenses determine the size of the image of the light source
projected on the substage condenser.
After the light source has been arranged in line, close
the iris of the substage condenser to its smallest diameter
and focus this opening accurately on the ground glass,
centering it carefully. Now open the substage iris and
cut down the iris on the outside condensing lens. Move
the parallelizer back and forth until its image is sharp
on the ground glass, then center it and lock in place. Close
the substage iris again and adjust the position of the light
source so that its image is sharply defined and falls cen-
trally on the substage iris. This light image should just
fill the working aperture of the objective.
One of the most frequent causes of poor images in
photomicrographs is the diffraction caused by the use of
too much uncontrolled light. Only that light that passes
through the objective is of any use in forming the image,
hence only that portion of the slide directly under the ob-
jective should be illuminated. To secure this result the
substage iris must be reduced to the absolute minimum
for the objective in use. Examine the image and while
watching it, reduce the iris opening until the scattered
light disappears and the image is sharp and clean. Do
not use an opening smaller than is necessary, for this too
results in degraded images.
With the object sharply focused examine it on the
ground glass to determine whether the size is correct.
This can be varied at will by moving the ground glass
toward or away from the eyepiece. The magnification
of microscope optical systems for photomicrography is
usually computed at a point eight or ten inches from the
eyepiece. Thus, if a X i oo objective is used with a X6 eye-
i8o MICROSCOPIC TECHNIQUE
piece the magnification represented by the photograph
will be loo x 6 or X6oo with a bellows extension of eight
inches. If the bellows is extended to sixteen inches the
magnification will be doubled and the photograph will
show a magnification of Xi^oo. 'The resolving power,
however, will not be increased. The apparatus is set up
and adjusted and we are now ready to make the negative.
Since the only reason for making photomicrographs is
to record the details of the object, the matter resolves itself
into a recording of contrasts. This is not always easy to
do, for the contrast of different subjects varies greatly.
The most difficult cases encountered are those arising
from a lack of contrast between the object and the back-
ground, as in lightly stained sections, in many aquatic
organisms, micro-organisms and the like. Sections that
are deeply stained with haematoxylin, eosin, indulin,
safranin, etc. usually present little difficulty when photo-
graphed as a whole on a clear ground. When only a small
area is to be photographed at high powers to show struc-
tural details, trouble may be encountered.
Dirt in the form of dust and grease on the lenses may
ruin otherwise contrasty images. Lenses should be care-
fully cleaned with lens paper, or the dust accumulation
will cover the image with scattered light and ruin the
contrast. Diffraction resulting from the passage of extra-
neous light through an unnecessarily large parallelizer or
substage iris will destroy contrast. Control of contrast
lies in the choice of the proper filter to use with color
sensitive material to record the image. Filters are used
to produce monochromatic light, by which the details of
microscopic structures may be intensified or reduced at
will.
In order to give the student a clearer understanding of
PHOTOMICROGRAPHY 181
the nature of light and color, let us examine these two
phenomena.
If a beam of light, either daylight or artificial, is ana-
lyzed in the spectroscope, it will be found to consist of
several bands of color, called the spectrum. There are
three major divisions of color, blue-violet, green and red,
with intermediate steps that form the transition bands.
According to the absorption theory of color formation,
color is the sensation produced when white light falls
upon an object which does not reflect all of the incident
white light. Thus, if white light falls upon an object that
absorbs the entire spectrum except the green portion, that
portion is reflected to the eye and we see green in the ob-
ject. In other words, a colored object is one which does
not absorb all the constituents of white light, but reflects
some, the reflected portion producing the sensation of
color.
Again using the spectroscope, examine the light re-
flected from a colored object or passed through a colored
filter. We find that instead of the continuous spectrum
of white light we now have a spectrum from which a part
of the band is missing. In the spectroscope the missing
portion appears as a black line or band which represents
the color that has been absorbed by the filter or reflected
by the colored object. This is known as the absorption
band. Since reflected light produces the sensation of color
in an object, it follows that the object is absorbing the
complementary or reverse color component of white light.
Turning the spectroscope upon objects of varied colors
we find the absorption band shifting with each change of
color. We find, for example, that a light blue object has
an absorption band in the red portion of the spectrum.
Since light blue is a mixture of green and blue-violet light,
i8s> MICROSCOPIC TECHNIQUE
of which red is the complementary color, the blue sensa-
tion is due to the absorption by the object of all the red
portion of the incident white light, and its reflection of
the green and blue-violet components. Similarly, a sensa-
tion of yellow color results when an object absorbs the
blue-violet portion of the spectrum and reflects a mixture
of green and red light.
Now the question arises how this color theory may be
used in photomicrography. The answer is that by intelli-
gent selection of a filter we can increase or decrease the
contrast of the object and its background or the various
details within the object itself. In order to make this
selection with precision it will be necessary to go deeper
into the scientific theory of color.
Light propagation is in the form of waves, each color
having a specific wavelength, measured from the crest of
one wave to that of the next. This distance is expressed
in Angstrom units, one unit being one ten-millionth of
a millimeter. By assigning each color to its proper place
and expressing this position in terms of Angstrom units
we are able to place the absorption band. With this in-
formation and a knowledge of the absorption band of the
filter we can accurately forecast the effect.
From the following diagram we see that the blue-violet
band is at the left of the spectrum with a wavelength of
4000 units and extends almost to the blue-green which
occurs at the 5000 unit division. Bright green then ex-
tends to about 5500 units, orange and yellow to Gooo units
BLUE-VIOLET
1
BLUE
GREEN
GREEN
1
ORANGE
YELLOW
RED
Fig. 64. Diagram of absorption band.
PHOTOMICROGRAPHY
,83
and red completes the visible spectrum, extending to 7000
units. Thus the position of an absorption band may be
accurately placed by referring to it in terms of Angstrom
units. Suppose we say that the absorption band of a fdter
lies in that portion of the scale from 6000 to 6400 units.
We know at once that this filter is absorbing the red waves
that lie in that portion of the spectrum and is passing the
blue-violet and green light.
We may also approach this subject by thinking in terms
of the colors remaining after the filter has absorbed some
of the components of white light. These are called
the residual colors. This method is best illustrated by
another diagram, in which the absorption bands are
shown with sharply defined edges. This condition does
not always exist in practice, especially with natural colors,
but most microscope stains and filter stains are rather
sharp, justifying our assumption of a sharp absorption
band for this purpose.
CHART OF RESIDUALS
ABSORPTION BANDS
PURPLE
VIOLET-BLUE
SKY BLUE
4000 5000^ 6000 TOOO
BLUE GREEN RED
Fig. 65. Diagram of the residual colors
left in the spectrum after certain bands
have been absorbed.
184
MICROSCOPIC TECHNIQUE
Fig. 66. Fresh water crustacean photographed for contrast
with background. Note absence of detail. X25
The diagram (Fig. 65) shows an absorption band ex-
tending from 4000 to 4400. The residual color here is
light yellow, since the filter is absorbing most of the blue-
violet light and is transmitting the green and the red, and
a little blue. As the absorption band shifts to the right
more violet and less blue is absorbed, and along with the
blue some of the green is included, so the residual color
is yellow of a deeper hue because of the presence of more
violet light. Again shifting the absorption band, we
eliminate practically all of the blue-green, leaving the
deep violet, bright green and red, which produces a resid-
ual color of orange. If we eliminate most of the green
PHOTOMICROGRAPHY
185
Fig. 67. Hie same crustacean photographed for de-
tail. The dark objects in the thorax are eggs.
by moving the absorption band from 5200 to 5600 we will
have a residual of violet, blue-green and red, which pro-
duces a magenta or red-purple. When the band shifts to
the region from 5600 to 6000 most of the green and
orange-yellow are absorbed, leaving a residual of violet-
blue. Another shift of the band to a position from 6400
to 6900 stops all red waves and allows the transmission of
the blue-green, the green and a few of the orange-red to
produce a light blue color.
We are now in a position to select a filter which we are
sure will produce the desired result. If we wish to make
a color as dark as possible we select a filter whose trans-
mission band completely covers the absorption band of
i86 MICROSCOPIC TECHNIQUE
Fig. 68. Blue cobalt mercuric thiocyanate crystals as
seen through the microscope. Xi25. Courtesy of
Eastman Kodak Co.
the color to be photographed. In other words, it must be
photographed by the light of the wavelengths that make
up its absorption band.
If we wish to secure the maximum amount of contrast
between a stained subject and the background we use
panchromatic negative material and a filter having the
same absorption band as the object itself. For example,
let us suppose we have a section stained with eosin to be
photographed with the maximum of contrast with the
background. Eosin absorbs light at from 4900 to 5300
units. If we use a filter that transmits light at from 5000
to 5400 units the resultant photograph will show the sec-
tion in high contrast, black and worthless because of
PHOTOMICROGRAPHY
187
Fig. 69. Same crystals photographed on Eastman
" M " plate with C filter. Courtesy of Eastman Ko-
dak Co.
blocked up detail. This filter is greenish-blue, the com-
plementary of red. Now going to the other extreme, let
us photograph our section by light of 6300 to 6500 wave-
lengths, the same as transmitted by eosin. The result will
be a thin, flat negative, containing little detail. Some-
where between these extremes must be a medium that
will give the desired result. This is to be found at the
border of the absorption band of eosin, so if we photo-
graph by light of wavelength 5700 we will get a negative
of normal contrast, full of detail and entirely satisfactory.
This filter is greenish-yellow in color.
This brings us to the next consideration, that of con-
trast in the object itself. It is necessary to record the
i88
MICROSCOPIC TECHNIQUE
Fig. 70.
Photographed with blue
filter for maximum con-
trast.
Whalebone section.
Photographed with red
filter for maximum de-
tail.
Courtesy of Eastman Kodak Co.
details of various structural elements in contrast with one
another in order that their distinguishing characteristics
may be delineated. To do this the usual procedure is to
photograph the object by the light it transmits. Thus, if
the beetle Sylvanus surinamensis (Fig. 46) , which is
brownish-yellow in color (wavelength 5800 to 6100)
were to be photographed for contrast within the object,
we would use light of the same wavelength, employing a
yellow or red filter and a panchromatic plate. If we de-
sired contrast with the background we would make the
photograph using a blue filter in the absorption band.
This would render the insect black and devoid of detail.
Filters may be used singly or in combination. Which
PHOTOMICROGRAPHY 189
filter or which group to be used in each case is decided by
the operator. One way to decide is to make the selec-
tion by the methods given above; the other is by visual
examination of the subject through the microscope with
different combinations of filters until one is found which
gives the desired results. Due to the great variation of
color sensitivity in different eyes the former method is
preferred since it is predicated on scientific facts. Manu-
facturers of reliable filters will provide technical data on
absorption bands and transmission bands. Correspond-
ing data for microscope stains may be secured from manu-
facturers of stains. For the student's convenience the
following table of stains and their absorption bands is in-
cluded. This contains only the stains used in making
preparations described in this book.
STAIN COLOR ABSORPTION BAND USED
BAND
Aniline blue Blue 5500-6200 5600-6000
Eosin Red 4900-5300 5100-5400
Fuchsin Red 5300-5700 5100-6000
Haematoxylin Dark blue Gradual through green 5100-6000
Methylcne blue Light blue 600062008065006800 64006800
Methyl violet Deep violet 58006000 56006000
Methyl green Blue-green 6200-6500 6100-6800
Picro-carmine Red 5 1 00-5300 & 5600-5700 5100-5400
Unstained sections, unstained whole objects of low con-
trast, such as aquatic forms, diatoms, colorless insects, etc.
should be photographed in polarized light with crossed
nicols (applicable only where objects are optically ac-
tive) , on a dark-field or with negative material of high
contrast. This is referred to by the manufacturers as
process material, and for greatest contrast should be de-
veloped in a caustic-hydroquinone developer.
Low-contrast objects may be advantageously photo-
igo MICROSCOPIC TECHNIQUE
graphed by using an oblique light. While this is some-
what analogous to dark-field illumination it differs in that
Fig. 71. Section of skin of man.
With dye injected blood ves- Wratten " M " plate and B
sels. Wratten "M" plate filter,
and F filter.
Courtesy of Eastman Kodak Co.
the object in a true dark-field illumination is uniformly
lighted from all sides by the dark-field condenser, while
in oblique lighting the specimen is lighted only from the
mirror side. This is often an advantage since it gives the
object a high-light side that seems to acid a third dimen-
sion, making it appear somewhat stereoscopic.
Illumination is achieved by swinging the mirror on its
arm until it is close to the underside of the stage, and
tilting it so that it projects a beam of light diagonally
across the underside of the slide, rather than vertically
through it. The contrast produced by this lighting can
be further increased by standing the microscope on a piece
of black velvet, or, better still, by arranging a piece of
black velvet under the stage in such a way that it does
not interfere with the light beam. This diagonal lighting
PHOTOMICROGRAPHY
Fig. 72. Sylvanus surinamensis, photographed with di-
agonal lighting to show high-light effect on partial dark-
field.
is reflected from the original light source, so that the in-
line system of illumination cannot be employed.
CALCULATING THE EXPOSURE The most satisfactory
method of arriving at the correct exposure is to make a
set of test negatives of an average slide under standard
conditions. Future exposures may then be calculated
with reasonable accuracy by referring to the record.
Variable elements must be reduced to a minimum to cut
down the chances of error. We standardize the light by
using the same bulb and by placing the microscope, con-
denser, parallelizing iris and lamp house in the same rela-
MICROSCOPIC TECHNIQUE
Fig. 73. Lead iodide precipitate. Xi25
On ordinary plate. On " M " plate with B filter.
Courtesy of Eastman Kodak Co.
tive position. Variables of magnification factors and
bellows extension affect the exposure in mathematically
exact ratios. Data on variables of filter factors and speed
of negative material are available from the manufacturer.
Let us first examine the effect of numerical aperture on
exposure. It is an optical rule that exposure varies as
..^ . This may be tabulated as follows:
TABLE OF NUMERICAL APERTURE FACTORS
Focal Length Average N. A. Approx. Exposure
Factor
inches
100 mm.
75 "
50 "
25 "
16 "
12 "
8 "
6 "
4 "
(Oil Itnra.) 2
.o8
.09
25
35
45
.50
.8
.85
9
1.3 (only i.o for photo.)
4
3
10
4
2
PHOTOMICROGRAPHY 193
This table is for professional microscopes and does not
apply to amateur equipment. If the student is working
with a microscope in which the optical system is desig-
nated in terms of magnification, the following table will
give the data necessary for computation of exposures.
Given the correct exposure without a filter on given nega-
tive material at a given magnification, the factor for any
other magnification may be found in the table, since ex-
posure varies directly as the square of the magnification.
TABLE OF MAGNIFICATION FACTORS
Magnification Exposure Factor
10 1/100
25 1/16
50 1/4
1OO 1
250 6
500 25
1000 100
Thus, if correct exposure with standard lighting is
found to be one second at Xioo, the correct exposure un-
der exactly similar conditions will be six and one quarter
seconds at X2r,o, since 250- = 6.25. Six seconds is close
enough for practical work.
Exposure varies with the bellows extension in the same
ratio as magnification, since it is, in effect, the same thing.
As the ground glass recedes from the eyepiece the image
grows larger and less bright, for the same amount of light
is now being used to cover an increased area. For each
doubling of the bellows extension the exposure must be
squared. For example, if the correct exposure with six
inches of bellows is two seconds, the exposure for twelve
inches of bellows is four seconds, for twenty-four inches,
sixteen seconds, etc.
i 9 4 MICROSCOPIC TECHNIQUE
We have stated that manufacturers of sensitive material
and filters will supply tables of comparative speeds and
multiplying factors for filters. Both of these sets of data
are necessary in the calculation of exposure. Suppose the
test negatives upon which future exposures are to be
based were made on commercial panchromatic film with-
out a filter. Using the same film we now want to know
how much the exposure must be increased if we use a red
filter. Reference to the table tells us that the exposure
must be increased ten times to secure a negative of the
same density. Now let us assume that the commercial
emulsion is not contrasty enough, so we decide to use
process panchromatic film with a red filter. Reference
to the table of comparative film speeds informs us that
process film is eight times slower than commercial pan-
chromatic film, the filter factor remaining the same. So,
with process film we multiply our first exposure by eight
and this result by ten to get the correct exposure.
Before any of this data can be used for computation of
subsequent exposures, test negatives must be made under
standard conditions. This can be clone by taking an ob-
jective, say N.A. 0.50, and making a photomicrograph at
Xioo without a filter. The correct way to expose the test
negative is to withdraw the dark slide and expose it for
a time estimated to be nearly right, say one second. Then
insert the slide a quarter of the way and make another
exposure of one second. Push the slide in to the half-
way mark and expose for two seconds, push it in three
quarters of the way and expose four seconds. The first
quarter of the negative will then have had one second
exposure, the second quarter two seconds, the third quar-
ter four seconds and the fourth quarter eight seconds.
Some effort should be made to estimate the correct ex-
PHOTOMICROGRAPHY 195
posure as a starting point. If the correct exposure is in
the neighborhood of 1/25 of a second, one second, the
lowest step in the test, would be too much.
Now let us compute the exposure from the beginning.
We have to photograph a section with a 16 mm objec-
tive, N.A. 0.25 at fifty diameters on commercial panchro-
matic material, using an orange and a green filter. The
following calculation, based on test negatives made under
standard conditions, will give the correct exposure.
Standard exposure x Factor for N.A. x Magnification
Factor x Filter Factor
Suppose our test negative made under standard condi-
tions shows an exposure of one-half second to be correct.
We then calculate as follows:
.5 seconds x 4 x i = 2 seconds
Filter Factors green 10 seconds
orange = 4 seconds
/. 10 x 4 40 seconds
Hence 2 sec. x 40 sec. = 80 seconds exposure required.
When using an amateur microscope for which the N.A.
is not known, proceed as above, omitting the second
(numerical aperture) factor.
As the negative material and bellows extension are al-
tered the proper multiplying factors must be included in
the calculation.
It is assumed that anyone sufficiently advanced in
microscopy to take up the making of photomicrographs
is also competent to do his own negative processing, so we
will omit the purely photographic aspects of the opera-
tion.
Since this book is intended for the novice as well as the
more advanced student, it might be well to amplify an
196 MICROSCOPIC TECHNIQUE
earlier statement, to the effect that any camera can be
used for taking photomicrographs.
The author recently saw a very clever device made by
a student using a No. 2 Brownie box camera with a micro-
scope. The camera did not balance safely on the eyepiece
of his amateur microscope, so he made a block of quarter
inch wood slightly larger than the front of the camera.
In this he cut a hole just large enough to fit over the eye-
piece and lined it with black velvet. To one face of the
block he glued another block of the same size with a hole
in it slightly smaller than the eyepiece housing. The two
blocks formed a rebated hole that fitted snugly over the
microscope. To the upper face of the block he tacked a
short tin tube of such diameter that when the outside was
covered with black velvet it fitted into the lens opening of
the camera. He thereby provided a solid support for the
camera and a light tight coupling to the microscope.
When using box cameras the lens must be left in place,
unless the camera is to be used for no other purpose than
photomicrography. The photographic lens has no effect
upon the image produced by the microscope. Removable
lenses should be screwed out of the shutter or barrel.
The usual amateur practice is to set the shutter for
time exposure, open it and make the exposure by turn-
ing the light on and off by a remote switch to prevent
vibration.
PHOTOMICROGRAPHS WITHOUT THE EYEPIECE When
photographing entire specimens or large sections it some-
time happens that the subject cannot be covered entirely
by a low power objective. Such cases may be dealt with
by removing the eyepiece from the tube and using the ob-
jective alone. This enables us to include a larger field,
although the magnification is not as great. Unless the
PHOTOMICROGRAPHY 197
objective is very good the edges of the field will not be
sharp. The marginal definition can be improved by in-
serting a black paper diaphragm with a small hole in its
center in the objective tube. The edges of the hole must
be clean and sharp, such as are made by small paper-
punches. Draw the outline of the disc with a compass to
locate the center, then cut it to fit the tube snugly and
punch the hole in the exact center. Push the diaphragm
into the tube, being careful not to scratch the lens.
For this kind of photography the light can be consider-
ably less than that required for regular work. It must be
just as carefully arranged, however, for the effect of dif-
fraction is increased in wide fields, because of the larger
area of illumination. The inside of the microscope tube
should be lined with black velvet to avoid reflections that
would cause flare spots. With the change in illumination
Fig. 74. Flea from cat. This photomicro-
graph shows the opacity resulting horn in-
complete dehydration. Irving L. Shaw.
198 MICROSCOPIC TECHNIQUE
the exposure will be altered so that a new test negative
will be needed.
PHOTOGRAPHING OPAQUE OBJECTS The technique
of the photography of opaque objects differs from that of
transparent objects principally in the lighting. Illumina-
tion for opaque objects must come from above the object.
For this purpose the large microscope light described in
Chapter XI is excellent, all of the illustrations of opaque
objects in this book having been made with it. The con-
densing flask on this light makes it possible to concentrate
Fig. 75. Photomicrograph of smooth
paper surface. The black lines are
ink lines drawn with a ruling pen.
Extremely oblique light used to give
relief. Xioo
the light in a spot that can be placed exactly where it is
needed. At the same time the lamp house itself is far
enough away from the object not to interfere with the
camera when working close to the subject.
When making enlarged photographs directly from the
PHOTOMICROGRAPHY ig g
Fig. 76. Eggs of Podisus spinosus.
Photographed to show markings on
caps. X75
subject several courses are open. The entire microscope
may be used, or the microscope stand and the objective
only. The objective may be removed from the stand and
used to replace the camera lens, or a short focus photo-
graphic objective may be used in a camera of long bellows
extension.
Subjects having practically all the desired detail in the
same plane may be satisfactorily photographed by the first
method. The depth of focus of microscopic objectives of
even medium power is very small, so that unless the sur-
face of the object is almost flat a great part of the detail
will be out of focus.
The photograph of the surface of a smooth pen-and-ink
board shown in Fig. 75 is an illustration.
The second method is used when not so much enlarge-
ment and more depth of focus is required. The illustra-
tion showing the markings on the caps of the eggs (Fig.
200 MICROSCOPIC TECHNIQUE
76) was made by this method. It will be observed that
the depth of focus is somewhat increased, including the
entire depth of the cap, which is about 1/100 of an inch,
with sufficient definition.
Method number three was used to make the negative
for Fig. 77.
The same eggs shown in the last figure were photo-
graphed with a 32 mm. objective held in the iris dia-
phragm of an old lens barrel fastened to the front of the
Fig. 77. Eggs of Podisus spinosus.
Photographed to show entire
depth of specimen. Xgo
camera. A diaphragm of black paper was inserted in the
tube to increase the marginal definition and depth of
focus. The bellows extension was 12 inches. You will
note that the depth is considerably greater, since both the
caps and the bottom of the open eggs are in focus. The
magnification is not as great. These eggs are about 1/64
of an inch high, which gives some idea of the depth of
focus.
Many subjects may be photographed only by the fourth
PHOTOMICROGRAPHY 201
Fig. 78. Head of spicier. Photographed to in-
clude as much depth as possible.
method. A short-focus photographic objective with an
iris diaphragm is used with a long bellows. The iris dia-
phragm increasing the depth of focus is an advantage.
The illustration (Fig. 78) was made with an $:. cine
lens held in an iris lens flange on the camera. The thirty-
six inch bellows could not be entirely vised because the
lens approached the subject too closely to permit proper
illumination.
The greatest difficulty encountered in photographing
opaque objects is the lighting. When using a long-focus
micro-objective in the draw tube, either with or without
an ocular, the separation between subject and objective
is usually great enough to permit projection of a spot from
the lamp onto the subject. When working with a photo-
graphic lens this is so close to the subject that proper light-
ing is difficult. Various expedients, some of them calling
202 MICROSCOPIC TECHNIQUE
for considerable ingenuity, are used to secure the desired
results.
The usual practice of the writer is to illuminate from
one side with the projector-lamp, focusing to a small spot.
This is placed so that it strikes the subject at an angle of
about 45 degrees from the top and front. An auxiliary
light is then directed at the shadow side by picking up the
light from a loo-watt clear bulb on a concave mirror,
focusing this in another spot on the subject. The prin-
ciple is exactly the same as that used in ordinary lighting
for portrait photography, and achieves the same result. If
a uniformly strong light were used on both sides of the
subject the modeling would be destroyed arid there would
be no sense of roundness. If a ventral light is required to
illuminate dark places on the under side of the subject it
is a good plan to place the subject on a sheet of glass, un-
der which is a light so controlled that only the immediate
area of the subject is illuminated. Otherwise there might
be too much light, resulting in flare or halo.
Some time ago the writer had to photograph a number
of opaque objects which required even lighting over the
entire area. Detail, not modeling, was important. The
depth of focus required the use of a photographic objec-
tive and iris diaphragm, so the cine lens was used. The
lighting problem was solved by making a square frame of
wood to fit over the frontboard of the camera, the lens
projecting through the frame. A 3 2 -candle-power auto
headlight bulb in a reflector made from a smooth gela-
tin dessert mould was placed at each corner of the frame.
The four bulbs were wired parallel and operated from a
toy train transformer. This arrangement provided a per-
fectly flat, even light that recorded details very well.
STEREOSCOPIC PHOTOMICROGRAPHY Stereoscopic pho-
204 MICROSCOPIC TECHNIQUE
tographs of microscopic subjects yield good results and
make excellent study material. The process is fully adapt-
able only to opaque material, such as is illustrated in
Fig. 79. Sections are so thin that little would be gained
by making them in stereo: insects, coins, leaves, fabrics,
crystals, etc. make excellent stereo pictures.
The easiest and most precise method is the one de-
scribed above, using a photographic lens on the camera
which must be mounted on a platform so that lateral mo-
tion of the entire camera is possible. The negative holder
is provided with an extra dark slide that uncovers only
half of the plate at one time. This may be made of heavy
cardboard cut to a si/e to fit the frame of the holder
tightly.
The subject is set up and lighted as described above,
and is then focused so that its center comes well off to the
side of the center of the ground glass, let us say the left
side. Its center should be about one and a half inches
from the center of the glass. Cover the right side of the
holder with the half-slide and make the exposure. Re-
move the holder and slide the camera to the right so that
the image now moves to the right side of the center, plac-
ing it in the same relation as the first image. Cover the
left side of the negative and expose again, giving it ex-
actly the same time as the first exposure.
The prints from these negatives must be transposed,
that is, the left hand negative, looked at from the emul-
sion side, right side up, must be printed on the right hand
side of the sheet, and the right hand image on the left side
of the sheet. The easiest way to do this is to make the
prints on separate pieces of paper, marking them left and
right, and then mount them in their proper places on a
card mount.
PHOTOMICROGRAPHY 205
Another method of making stereo negatives does not
involve moving the camera, but moves the subject, so that
the displacement on the ground glass is about three
inches. While this method is effective there is danger of
shifting the subject so that the two images will not coin-
cide when viewed through the stereoscope.
This treatise of photomicrography contains sufficient
information for the beginner to enable him to produce
satisfactory photomicrographs from the material he col-
lects. The author has endeavored to give information
and data to assist the student in shaping his course along
the approved lines of practice. If specific information
and data on any branch of the work are required they may
be secured by referring to the books mentioned in the
bibliography.
Bibliography
The literature of microscopy is so extensive that it would
be almost impossible to compile a complete list. The follow-
ing references will give the student any information he may
require for instruction in any special branch of the work.
BALFOUR, JOHN H., Class Book of Botany.
BENTON, B. S., Elements of Botany.
BEALE, LIONEL S., How to Work the Microscope.
CARPENTER, BENJAMIN W., The Microscope and its Revela-
tions.
CHAMBERLAIN, CHARLES }., Methods in Plant Histology.
CROSS and COLE, Modern Microscopy.
COOK, MELVILLE T., Applied Economic Botany.
COMSTOCK, JOHN H., Manual for the Study of Insects.
CORRINGTON, JULIAN D., Adventures with the Microscope.
DAVIS, GEO. E., Practical Microscopy.
DAVIES, THOMAS, The Preparation and Mounting of Micro-
scopic Objects.
FOLSOM, JUSTUS W., Entomology, Its Economic and Bio-
logical Aspects.
FERNALD, H. T., Applied Entomology.
GAGE, SIMON H., The Microscope, Dark Field Edition.
GALIGHER, ALBERT E., Essentials of Microtechnique.
GATENBY, J. B. and COWDRY, E. V., Lee's Microtomist's Vade-
Mecum.
GUYER, FREDRIC M., Animal Micrology and Zoological Micro-
technique.
HOGG, JABEZ, The Microscope, Its History, Construction and
Applications.
HANAUSEK, FRANZ T., Microscopy of Technical Products.
MCFARLAND, JOSEPH, Pathogenic Bacteria and Protozoa.
McCLUNG, C. E., Microscopical Technique.
Micrographic Dictionary.
MALLORY and WRIGHT, Pathological Technique.
PACKARD, ALPHEUS S., Guide to the Study of Insects.
SCHWARZ, ROBINSON E., Textiles and the Microscope.
STOKES, ALFRED C., Aquatic Microscopy.
WHIPPLE, GEO. C., Microscopy of Drinking Water.
206
Index
Absorption band
Accessories
Acetic acid
PACE
181
152
f)K
Celloidin .
PACE
KQ
Celloidin, Coating with . .
Chitin
72
Agents, Fixing
34
Chloral Hydrate .
.... 104
30
Alcohols, Graded Series of ....
Alcohol Methyl
1 12
y 1
Chlorox
Cilia .
.... 1 20
2 t
Alum-Haematoxylin
Amoeba
8l
20
|8 2
9*
103
4
104
103
186
160
117
105
1 08
108
206
120
95
93
87
160
4i
12
i()G
.64
129
28
120
128
*74
23
80
35
35
'43
Clearing
(Move Oil
46. 'A 72
..31, 1 36
.... 1OO
99
'<>/
15
6
133
1 86
.... ,87
70
5
82
125
1 25
1 21
Angstrom Units
Aniline Blue
Animal Material, Preparation
of
Coal, Cutting Sections of .
Coal, Grinding Sections of
Coleoptera
Collecting Net
Condenser
Aperture, Numerical
Arachnida
Arthropoda
Background Contrast
Balsam Bottle
Bard-Parker Scalpels
Beetle, Foot of
Beetle, Japanese
Beetle, Potato
Bibliography
Bleaching
Bone, Decalcified
Coniferous Leaves
Contrast, Background
Contrast, Object
Coplin Jars
Corrosion . .
Counter Staining
Clover Glass, Choice of . . .
Cover Glass, Placing
Cover Glass Support .
Critical Illumination .
i?
lf >3
1 O 1
Crustacea
Crustacean, Fresh Water
Bone, Grinding Sections of ....
Borax Carmine, Grenadier's
Alcoholic ....
Cuticles, Leal
Cyclops
Decalcified Bone
.... 104
.... 147
82
95
95
VI 8l
Bottle, Balsam
Bonin's Fluid
Decalcifying Reagent
Dehydration
Bourn's Fluid, Alcoholic
Box Cameras for Photomicrog-
raphy
Dela field's Haematoxylin
Depression Slide
136
Brown University Oven
Bulbs
Desmids
29
'7
Diatoms
Cage, Live
Calcium Hypochlorite
Cambium Layer
Camera for Photomicrography
Carchesium
Dicotyledonous Stems
Difflugia
Dipping Tube
Diptera
.... 17
.... 127
.... 21
.... 27
1()6
Dissecting Knives
1 f\A
Dissecting Microscope
Dissecting Needles
. ll-l. 152
4 a i f a
Carmine
Carnoy and Le Brim's Fluid . .
Carnoy's Fluid .
Dissection
IH
11 7
116
Dissection Dish
Dissection, Tools for
Castor Oil, Mounting in
207
2o8
INDEX
Dissociation
Dry Ice
Embryonic Seeds
Emery Saw
Endogenous Stems
Eosin 81
Eosin V
Epsom Salt
I'.-UiF.
43
75
97
127
9
3
165
75
127
8G x
'93
192
1 19
127
188
33
31
21
165
116
10
96
1 55
3
36
53
73
101
47
37
7
93
9
80
UQ
Hairs, Leaf
148
93
104
104
150
105
106
.67
,78
62
9
22
I '9
8l
108
8
37
33
3"
12
66
160
156
117
1 17
148
148
35
106
2
l8 2
28
23
47
46
193
i
92
74
14
Hard Objects, Preparing and
Mounting . .
Hemiptera
Hexapoda
Horny Tissues
House Fly, Wing of
Hymenopte'ia
Iceland Spa
Illumination, Critical
Imbedding
Erlenmeyer Flask
Ethyl Chloride
Exogenous Stems
Fxoskeleton
Indulin
Exposure, Calculating the ...
Exposures, Standard
Eye of Rat
Factors, Table of Magnification
Factors, Table of Numerical
Aperture
Infiltration, Paraffin
Infusoria
Insect Larvae
Insects, Preparation of
Iron -Haematoxylin
Japanese Beetle
Japanese Tissue
Karpenchenko's Fluid
Killing
Fibers, Textile
Fibro-vascular Bundles
Filters
Fixation
Fixing Agents
Flagella
Killing Agents
Killing Jar
Klcinenberg's Fluid
Knife for Section Cutting ....
Knives, Dissecting
Lamp, Spirit
Lamps
Lancet
Leaf Cuticles
Leaf Hairs
Leaf Scales
Leaves Coniferous
Flask, Erlenmeyer
Flea from Cat
Florence Flask
Focus a Microscope, How to . .
Foot of a Rat
Forceps 117,
Formalin
Formalin, Acetic-Alcohol
Free/ing
Freezing, Section Cutting by . .
Friable Objects, Grinding ....
Gage's Formalin Solution
Germinating Seeds ...
Gilson's Fluid
Glycerin Jelly
Grcenough Binocular Micro-
scope
Grenadier's Alcoholic Borax-
Carmine
Grinding Sections of Bone ....
Gum Acacia
Le Brim's Fluid, Carnoy and . .
Lcpidoptera
Light
Light Propagation
Live Cage
Loricae . . .
Macerating Agents
Maceration
Magnification Factors, Table of
Magnifying Power
Malachite Green
Haematoxylin
Hairs
Mass, Infiltrating
Material, Sources of
INDEX
209
PACE
Mayer's Albumen Fixative {9
Menthol 30
Mercuric Chloride 37
Metallic Silver, Crystals of .... 144
Methyl Alcohol 34
Mica Plate 171
Microscope, Care of 6
Microscope, Compound i
Microscope, Dissecting i i j, 152
Microscope, Greenough ttinocu-
lar 115
Microscope, How to Focus .... 10
Microscope, Optics of the i
Microscope, Polarizing 139
Microscope, Setting up the .... 12
Microscope, Simple i
Microtome 52, 162
Mitosis 84
Monocotyledonous Stems
Moth, Tomato Hawk
Mounting
Needles, Dissecting 153
Ncmatodcs 36
Net, Collecting 15
Nenroptera 107
Nicol Prisms 139
Nuclear Stains 79
Numerical Aperture 4
Numerical Aperture Factors,
Table of 192
Physiological Salt Solution .... 118
Picric Acid 40
Picro-Carmine 88
Plasma Stains 79
Podistis Spinosus 108, 199
Point of Extinction 140
Polari/ed Light 141
Polari/er i(5(5
Polarizing Microscope 139
Pollens 133
Potassium Cyanide 119
Preparing the Slides 69
Prisms, Nicol 139
Progressive Staining 82
Propagation, Light 182
Pseudopoda io(5
Pupal Stage 106
Radula of Snails 149
Ranvier's One-Third Alcohol 47
Ranvier's Picro-Carminc 89
Raphides 14(5
Record Sheet for Photomicro-
graphic Exposures 173
Regressive Staining 82
Residual Colors 183
Resolving Power 2
Rhizomes 129
Rhizopods 20
Rhubarb 91
Rose Chafer 108
Object Contrast 187
Oblique Light 190
Opaque Objects, Lighting .... 201
Opaque Objects, Photographing 198
Optics of the Microscope i
Orthoptcra 107
Osmotic Currents 122
Oven, Brown University 164
Oven, Paraffin 162
Paraffin 58
Paraffin Infiltration Go
Paraffin Oven 162
Paraffin, Removal of 71
Paramecium 26
Photomicrography 172
Photomicrography without the
Eyepiece 196
Safranin
Scales, Leaf
Scalpel
Scissors
Section Cutting
Section Cutting, Frceha
Section Lifter
Sections, Loose
Seeds, Germinating . .
Shaudinn's Solution . .
Shears
Silicious Skeletons
Skin of Man
Slide, Depression
Slides
Snails, Radula of
Spectrum
Spirit Lamp
81
148
117
117
52
66
156
76
1 35
3'
156
M7
190
29
159
H9
181
160
210 INDEX
I'.U.l. 1'ACF.
Stain, Transferring to 72 Teeth, Sections of 96
Staining 45 'Textile Fibers 149
Staining Counter 82 Tissues, Horny 150
Stains, Absorption Rands of . . 189 Turntable 98, 165
Stains, Nuclear 79
Stains, Plasma 79 Ulricuh ma 23
Standard Aluin-Haciiiatoxylin 83
Standard Exposures iq.i , r , , t .
t , . . . * ,' \ ei'e table Specimens, Preparing 127
Starch in situ i |(> ., l i .-> /
f, . \ ermes ior,
Starches 144 , , , ^ , ,
,, , , v olvox Globator 27
Stems, iMidogenous and Kxogen- \- n
ous 127 01 tKC <IC " :)
Stentor 2(>
Stereoscopic Photoiniciogiaphy 202 Whalebone Section 188
Stretching '. 69 Worcester's Fluid 39
Sylvanus'surina.nensis 107 Wright's Method 77
4 [ /enker's Fluid 39
AGFA ISOPAN FILM'S
fineness of grain and maximum free-
dom from halation It partieu
larly adapted to photomicrography.
Isopan is fully panchromatic. It
a remarkably high degree of
enlargement without loss of defini-
tion or detail, without showing
grain. And the delicacy of detail
registered with Isopan is held clean
and clear hy its special and unusually
efficient anti-halation coating.
Use Agfa Isopan Film , , . you'll
quickly see why it has a
reputation as the ideal film for Pho-
tomicrography, Isopaa is by
Agfa AUSCG Corporation in BIng
hamton. New York.
425-POWER
MICROSCOPE $ 1852
A good microscope gives life long
service, so be sure you select
yours wisely, then you- can look
forward to years of ever increas-
ing pleasure discovering thou-
sands of Nature's miracles.
Every Wollerisak microscope
is produced by lens special-
ists famous for optical ex-
cellence since 1899.
1 n this 42 5X model we offer
the highest magnification
you can buy anywhere
near this price. Magnifi-
cation from 100 to
425 diameters. Thor-
oughly corrected ach-
romatic optics with
excellent resolving
power. Ideally suited
for photomicrog-
raphy. Tilting
stand, detach-
able base. At
dealer's or direct
order or C.O.D.) postpaid,
k guarantee. Other models.
(check^
Moneyt
MODEL 236X Magnifies 110
to 236 diameters $16.50
MODEL 150X Magnifies 70
to 150 diameters 12.60
MODEL 100X Magnifies 100
diameters 5.00
SLIDE SET Make slides at
home 3.60
$3.50 m
Dissecting
Microscope
Useful for making careful dissections
and preparing subjects for microscope
slides. Equipped with 7-X adjustable
magnifier, reflecting mirror, handy
drawer for instruments.
FREE BOOK "Revealing Nature's
Wonders", complete catalog sent free.
WOLLENSAK OPTICAL COMPANY
713 Hudson Avenue, Rochester, N. Y.
TELESCOPtbl
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BARD-PARKER
Detachable Blade
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for
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Noted for the uniform
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work. Available in 8 pat-
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Blades, six of one size per
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Handles $1.00 each.
Ask Your Dealer
BARD-PARKER COMPANY, Inc.
DANBUKY, CONN.
MORE FOR YOUR MONEY
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Model R Micro-
scope 75 to 300
power. Complete
with walnut
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page book, Ad-
ventures with the
Microscope, $21.
Gem Science Kit, 49 piece
portable home laboratory. With
75 to 172 tower microscope,
$24. Kit alone, $9, 50.
Model HH,
standard size
professional mi-
croscope. All
standard optics^
and microscope
accessories can
be used with
it. As shown,
IN THE great universities of the
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Whether you want a simple in-
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micrography as an inexpensive
hobby, or the most advanced of
research equipment that permits
photomicrographs, you will find
one of the big family of Bausch &
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Precision in optical systems is a
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And it is this precision which
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made Bausch & Lomb micro-
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Write for detailed literature.
BAUSCH & LOMB
182 Bausch St.
Rochester, N. Y.
Lomara Microscopes
These small microscopes, in shape like a fountain pen, are handy pocket
instruments for the
I heir compact
form makes them
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vest pocket. Their
excellent optical quality
makes them suitable for
serious work in and out of the
laboratory.
STUDENT,
INDUSTRIAL,
MACHINIST,
EXPLORER,
PROSPECTOR
and in many other
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have to be
inspected.
Small Lomara with stand |
15 to 250 times
A well designed stand with fixed stage, two illuminating mirrors and, for
the Ultra-Lomara, an effective condenser system and the micro-adjustable
stage converts the pocket instrument into a laboratory tool which, for a
great variety of microscopic observations, compares favorably with more
expensive standard laboratory instruments.
Interchangeable objectives and oculars make possible combinations
giving magnifications from
15 to 250 times in the small Lomara
and 20 to 1400 times in the Ultra-Lomara
With the addition of the new Marks polarizing biplates observations in
polarized light are posssible.
For literature and prices write to the U. S. importers
C. P. GOERZ AMERICAN OPTICAL CO. Dept. M.
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Pamphlet No. 1180:
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Pamphlet No. 9:
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Other pamphlets on metal microscopes, mineralogical microscopes,
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60 East 10th St. New York, N. Y.
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Chicago, Illinois Los Angeles, Calif.
R,
LECOGNIZING the need of the student and ama-
teur nricroscopist for stains, reagents chemicals and
mounting mediums in small quantities, the author of
this volume wishes to call attention to the fact that he
is in a position to supply materials of high quality in
small quantities.
Stains, either dry or in solution
Canada balsam Glycerin Jelly
Gum da mar
Xylol Toluol
Benzol Beechwood creosote
Clove Oil
Fixing Solutions Alcohols
Microscope Slides Cover Glasses
Prepared Slides of Sections and Whole Mounts
Photomicrographs in black-and-white or color
Lantern Slides in black-and-white or color
Send for complete catalog-price list
J. CARROLL TOBIAS
534 MAIN STREET
BETHLEHEM PA.
